Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body (Advances in Anatomy, Embryology and Cell Biology, 237) 3031447565, 9783031447563

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Advances in Anatomy, Embryology and Cell Biology

Nikolai E. Lazarov Dimitrinka Y. Atanasova

Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body

Advances in Anatomy, Embryology and Cell Biology Volume 237

Editor-in-Chief Peter Sutovsky, Division of Animal Sciences and Department of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, MO, USA Series Editors Z. Kmiec, Department of Histology and Immunology, Medical University of Gdansk, Gdansk, Poland Michael J. Schmeisser, Institute of Microscopic Anatomy and Neurobiology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany Jean-Pierre Timmermans, Laboratory of Cell Biology and Histology/Core Facility Biomedical Microscopic Imaging, Department of Veterinary Sciences, University of Antwerp, Wilrijk, Belgium Sven Schumann, Inst, f. Mikroskop. Anatomie u. Neurobio, Johannes Gutenberg University of Mainz, Mainz, Rheinland-Pfalz, Germany

Advances in Anatomy, Embryology and Cell Biology publishes critical reviews and state-of-the-art research in the areas of anatomy, developmental and cellular biology. Founded in 1891, this book series has a long standing tradition of publishing focused and condensed information on a given topic with a special emphasis on biomedical and translational aspects. The series is open to both contributed volumes (each collecting 7 to 15 focused reviews written by leading experts) and single-authored or multi-authored monographs (providing a comprehensive overview of their topic of research). Advances in Anatomy, Embryology and Cell Biology is indexed in BIOSIS, Medline, SCImago, SCOPUS.

Nikolai E. Lazarov · Dimitrinka Y. Atanasova

Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body

With 46 figures

Nikolai E. Lazarov Department of Anatomy and Histology Faculty of Medicine Medical University of Sofia Sofia, Bulgaria

Dimitrinka Y. Atanasova Institute of Neurobiology, Bulgarian Academy of Sciences Sofia, Bulgaria

ISSN 0301-5556 ISSN 2192-7065 (electronic) Advances in Anatomy, Embryology and Cell Biology ISBN 978-3-031-44756-3 ISBN 978-3-031-44757-0 (eBook) https://doi.org/10.1007/978-3-031-44757-0 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

To my parents to whom I owe everything and to my wife Rummy and my son Vladdy without whom this book would never have been written. —Nikolai E. Lazarov I would like to dedicate this monograph to my first supervisor in histology, Assoc. Prof. Dimitar Itzev, inspired me to enter the world of histology and morphology. My favorite German supervisor Prof. Dr. Markus Kipp from the Institute of Anatomy, Rostock University Medical Center, in whose laboratory I learned many things. My irreplaceable and favorite laboratory assistant Sabina Mitova for her precision and professionalism. My parents, Yordan and Ivanka, taught me to be determined, believe in myself and always persevere. I am truly honored to have you as my parents. Thank you both for your unwavering love and for support. My brother Dimitar may not always be at my side, but he is always in my heart. My husband Nikolay for the patience, love and support so that I could walk all this way.

Words can hardly describe my thanks and appreciation to you. My little treasures, the twins Dimitar and Nicole who give me energy and inspire me every day with their mischief and childish smiles. —Dimitrinka Y. Atanasova

Preface

The current monograph summarizes our long-standing experience in the exploration of the morphology and neurochemistry of the carotid body and the latest progress in this field. During my stay as a research fellow at the Institute of Anatomy of the Ludwig Maximilian University in Munich, my host professor asked me to supervise the MD thesis of a medical student from the university. Thus, I started working on this tiny but important structure in the peripheral nervous system. After my return to Bulgaria, I directed one of my young research associates from the Bulgarian Academy of Science, Dimitrinka Atanasova, to conduct Ph.D. research on this subject under my guidance. In the course of elaborating the dissertation and after its defense, we continued our joint work on this remarkable anatomical structure and further focused on its morphofunctional and neurochemical changes in essential hypertension. We believe that expanding the knowledge about the morphological features and physiological processes that operate in it, and the pathophysiological mechanisms that alter its function, would certainly help facilitate the translational research on the carotid body. Sofia, Bulgaria June 2023

Nikolai E. Lazarov

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Acknowledgements

This study was funded in part by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01 and by the Bulgarian Ministry of Education and Science within the framework of the National Recovery and Resilience Plan of Bulgaria, Component “Innovative Bulgaria”, project No. BG-RRP-2.004-0006-C02 “Development of research and innovation at Trakia University in service of health and sustainable well-being”. We are indebted to our collaborators for their contributions to the experimental data in this book and for the constructive criticism of the manuscript. The authors are grateful to Dr. Anastassia Stoykova, Department of Molecular Cell Biology at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, for kindly providing the Pax6 mutant mice, Dr. Ruslan Hlushchuk, Institute of Anatomy, University of Bern, Switzerland, for the generation of microCT images for correlative morphology, and Dr. Andrey Ivanov, Department of Anatomy and Histology, Medical University of Sofia, Bulgaria, for his help with stereology and 3D-imaging. Special thanks to Dr. Angel Dandov for editing the English text. Finally, we would like to thank the staff at Springer, in particular Tanja Weyandt, Dr. Eliana Acosta and Rajesh Manohar, for their assistance and support to this monograph.

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Contents

1

Carotid Body: The Primary Peripheral Arterial Chemoreceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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History and Recent Progress in Carotid Body Studies . . . . . . . . . . . . .

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3

General Morphology of the Mammalian Carotid Body . . . . . . . . . . . .

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Structural Plasticity of the Carotid Body . . . . . . . . . . . . . . . . . . . . . . . .

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Mechanisms of Chemosensory Transduction in the Carotid Body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Neurochemical Anatomy of the Mammalian Carotid Body . . . . . . . .

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Neurochemical Plasticity of the Carotid Body . . . . . . . . . . . . . . . . . . . . 105

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Carotid Body Dysfunction and Mechanisms of Disease . . . . . . . . . . . . 123

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Stem Cell Niche in the Mammalian Carotid Body . . . . . . . . . . . . . . . . 139

10 Carotid Body and Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 11 The Carotid Body: A Tiny Structure with Many Roles . . . . . . . . . . . . 161 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

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Abbreviations

5-HT ACh AChE Ang II AP ATP ATPase BChE BDNF BrdU CA CaBPs CB CCA CG ChAT CHF cGMP CGRP CO CSE CSN DA DBH ECA ENK ET FGF G6PD GABA GAL

5-Hydroxytryptamine (Serotonin) Acetylcholine Acetylcholinesterase Angiotensin II Alkaline phosphatase Adenosine-5' -triphosphate Adenosine triphosphatase Butyrylcholinesterase Brain-derived neurotrophic factor Bromodeoxyuridine Catecholamine Calcium-binding proteins Carotid body Common carotid artery Carotid ganglion Choline acetyltransferase Chronic heart failure Cyclic guanosine monophosphate Calcitonin gene-related peptide Carbon monoxide Cystathionine-γ-lyase Carotid sinus nerve Dopamine Dopamine β-hydroxylase External carotid artery Enkephalin Endothelin Fibroblast growth factor Glucose-6-phosphate dehydrogenase Gamma-aminobytiric acid Galanin xiii

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GDH GDNF GFAP GLU GPN HDC HIF HIS HO H2 S ICA IDH IL IN LDH LEP MAO MKI67 NA NADPH NO NOS NPY OSA PACAP PG RAS ROS SCG SDH SHR SIDS SP TH TPP I VAH VIP VMAT

Abbreviations

Glutamate dehydrogenase Glial cell line-derived neurotrophic factor Glial fibrillary acidic protein Glutamate Glossopharyngeal nerve Histidine decarboxylase Hypoxia-inducible factor Histamine Heme oxygenase Hydrogen sulfide Internal carotid artery Isocitrate dehydrogenase Interleukin Insulin Lactate dehydrogenase Leptin Monoamine oxidase Marker of proliferation Ki-67 Noradrenaline (Norepinephrine) Nicotinamide adenine dinucleotide phosphate Nitric oxide Nitric oxide synthase Neuropeptide Y Obstructive sleep apnea Pituitary adenylate cyclase-activating polypeptide Petrosal ganglion Renin-angiotensin system Reactive oxygen species Superior cervical ganglion Succinate dehydrogenase Spontaneously hypertensive rat Sudden infant death syndrome Substance P Tyrosine hydroxylase Tripeptidyl aminopeptidase I Ventilatory acclimatization to hypoxia Vasoactive intestinal peptide Vesicular monoamine transporter

Chapter 1

Carotid Body: The Primary Peripheral Arterial Chemoreceptor

Abstract The carotid body (CB) is a polymodal chemosensory organ that plays an essential role in initiating respiratory and cardiovascular adjustments to maintain blood gas homeostasis. Much of the available evidence suggests that chronic hypoxia induces marked morphological and neurochemical changes within the CB, but the detailed molecular mechanisms by which these affect the hypoxic chemosensitivity still remain to be elucidated. Dysregulation of the CB function and altered oxygen saturation are implicated in various physiological and pathophysiological conditions. Knowledge of the morphological and functional aspects of the CB would improve our current understanding of respiratory and cardiovascular homeostasis in health and disease. Keywords Carotid body · Chemoreception · Hypoxia · Respiratory and cardiovascular homeostasis · Structural and neurochemical plasticity

The carotid body (CB) is a paired neural crest-derived small ovoid mass of tissue, which has long been the sensory receptor for chemical changes occurring in blood. It is purposefully situated in the vicinity of the common carotid artery bifurcation for monitoring blood chemicals just before they reach the brain, an organ that is extremely sensitive to oxygen and glucose deprivation. The CB is a polymodal peripheral chemoreceptor that registers the arterial blood levels of oxygen (O2 ), carbon dioxide (CO2 ) as well as the hydrogen ion concentration (pH) and reacts to their changes by the initiation of an appropriate respiratory and cardiovascular response to hypoxia, hypercapnia and acidosis, thus leading to the restoration of blood gas homeostasis. It is also believed to be sensitive to several other blood-borne stimuli such as potassium concentration, blood temperature and osmolarity (Kumar and Bin-Jaliah 2007), numerous humoral agents, circulating hormones (insulin and leptin) and blood flow (Ortega-Sáenz and López-Barneo 2020). However, its initial proposed potential role as a peripheral glucose sensor (López-Barneo 2003) is now being questioned since both in vivo and in vitro experiments do not support a direct CB response to hypoglycemia (reviewed by Kumar and Prabhakar 2012). Also, it has recently been revealed that the CB in Wistar rats © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_1

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1 Carotid Body: The Primary Peripheral Arterial Chemoreceptor

is not sensitive to lactate (Spiller et al. 2021). Nonetheless, according to the modern concept, it is thought to be a multimodal integrated metabolic sensor. The CB works in concert with the apposing afferent nerve endings of the petrosal ganglion (PG) cells, and they together form a functional unit, the CB chemosensory system. According to the current belief, the CB type I (also called glomus) cells are the initial transducers of the hypoxic stimuli. In response to hypoxia, these cells release a variety of neurotransmitters which activate chemoafferent nerve endings of PG neurons. The latter provide the afferent link between the CB chemoreceptors and respiratory nuclei in the brainstem, thus ensuring the transmission of chemosensory information from the chemotransductive cells to the central nervous system. The efferent limb of the chemoreceptor reflex arc is formed by solitary axons projecting to the respiratory centers, distributed in a ponto-medullary respiratory network. They control the coordinated contractions of the abdominal, thoracic and laryngeal respiratory muscles and upon hypoxia stimulate breathing (Dutschmann and Paton 2002). In addition, the chemosensors send signals through the afferent branches of the glossopharyngeal nerve to the cardiovascular regulatory center in the medulla oblongata to increase the oxygen supply to tissues via the autonomic nervous system, and thus to correct the condition. In the absence of these compensatory responses, systemic hypoxia would lead to tissue hypoxia, which may have potential harmful consequences throughout the organism. The discovery that the CB is the principal organ for sensing arterial blood O2 and CO2 changes has opened new perspectives in respiratory physiology and pathophysiology. Related to this role, the CB shows remarkable structural and neurochemical plasticity in hypoxic conditions. Much of the available evidence suggests that its dysfunction and altered oxygen homeostasis are involved in the pathogenesis of several human diseases, some of which are of a high incidence. Moreover, recent experimental data show that the mammalian CB is a neurogenic center, and its stem cells could be potentially useful for tissue repair after injury, or in developing stem cell-based therapy in human neurodegenerative disorders. Nevertheless, the physiology and pathology of the human CB remain unclear and controversial. Therefore, the present monograph aims to provide a complete and exhaustive overview of the morphofunctional organization of the mammalian CB and give an updated insight into the neurochemical anatomy of its cell populations with a special reference to the aspects of their structural and neurochemical plasticity. We believe that a better knowledge of the basic morphology and physiology of the CB in mammals will contribute to our current understanding of respiratory and cardiovascular homeostasis in health and disease. Compliance with Ethical Standards The authors declare no conflict of interest. This chapter is a review of previously published studies, and as such, no animal or human studies were performed.

References

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References Dutschmann M, Paton JF (2002) Inhibitory synaptic mechanisms regulating upper airway patency. Respir Physiol Neurobiol 131:57–63 Kumar P, Bin-Jaliah I (2007) Adequate stimuli of the carotid body: more than an oxygen sensor? Respir Physiol Neurobiol 157:12–21 Kumar P, Prabhakar NR (2012) Peripheral chemoreceptors: function and plasticity of the carotid body. Compr Physiol 2:141–219 López-Barneo J (2003) Oxygen and glucose sensing by carotid body glomus cells. Curr Opin Neurobiol 13:493–499 Ortega-Sáenz P, López-Barneo J (2020) Physiology of the carotid body: from molecules to disease. Annu Rev Physiol 82:127–149 Spiller PF, da Silva MP, Moraes DJA (2021) Lactate does not activate the carotid body of Wistar rat. Resp Physiol Neurobiol 285:103593

Chapter 2

History and Recent Progress in Carotid Body Studies

Abstract This chapter describes the history of the carotid body (CB) and the subsequent research on its structure and function. The chronological development of ideas about its anatomical structure as a ganglion, the first descriptions of its glandular nature as a ball of highly vascular tissue (glomus), the discovery of its neural crest origin and relevant embryological views as a true paraganglion toward a more conclusive understanding of its sensory nature as a chemoreceptor for chemical changes in blood have been consistently demonstrated. The knowledge of the CB neurochemistry, physiology and pathophysiology has progressed immensely in the past century and a large and compelling body of evidence for the presence of a neurogenic niche in the CB has accumulated over the last two decades, thus underlying its function and possibility for the development of cell replacement therapies. Keywords Carotid gland · Chemoreceptor · Ganglion · Glomus caroticum · Neurogenic niche · Paraganglion

The study of the CB has a long history (for a recent review, see Prabhakar and Peng 2017). The first anatomical report on its existence in the human body appeared in the doctoral theses of Hartwig Taube in 1742 and Matthias Berckelmann in 1744, although its discovery is attributed to the studies of their mentor, the great Swiss anatomist and physiologist Albrecht von Haller around the mid-eighteenth century (Pick 1959; Heath 1991). Since then, the interest of anatomists and physiologists in this tiny neurovascular structure lying in the carotid fork has continued to be strong, although over the past centuries numerous anatomical and physiological studies on the CB in mammals have been conducted.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_2

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2.1 Eighteenth Century: The Discovery of the Carotid Body Based on dissection studies, eighteenth-century anatomists discovered the CB, though they did not attempt to distinguish it from neighboring cervical ganglia with a similar gross appearance (Grimley and Glenner 1968). In his work, Taube mentioned the presence of a small ganglion-like mass of tissue located in the back of the carotid bifurcation which was referred to as “ganglion minutum” (Taube 1743). A few years later, Haller (1747, 1749) in his Disputationum anatomicarum republished the results of his pupils and later called the structure the “ganglion exiguum” (Haller 1762). During this period, Neubauer (1772), working independently of Haller and his group, also noted this small structure, naming it the “ganglion parvum”. Andersch (1797) claimed that his father, an ex-pupil of Haller, believed that he was the first to have seen the CB himself. He also stated that he had accurately described its blood supply and given it a proper name on account of its location, i.e., the “ganglion intercaroticum”. In the years to follow, the CB was forgotten until Mayer (1833), the creator of the term histology, rediscovered it in man and several other species and introduced, as he originally thought, the term ganglion intercaroticum. He and, very shortly afterward, Valentin (1833) were the first to recognize the sensory innervation of the CB by the glossopharyngeal nerve. Besides, the latter described a small blood vessel from the carotid bifurcation as its vascular supply.

2.2 Nineteenth Century: The Microscopic Description of the Carotid Body In the mid-nineteenth century, the improved techniques for microscopy gave a new impetus to the study of the CB. Beginning with the pioneering studies of the German anatomists Hubert von Luschka (1862), the histological structure of the CB in different animal species was repeatedly described and an increased knowledge of its anatomy accumulated. One of the greatest contributions of Luschka and later nineteenth-century histologists to the microscopic description of the CB was the recognition of its glandular nature. Luschka pointed out that its structure differed from that of ordinary ganglia though it was intimately related to the cervical sympathetic trunk. He provided the first accurate measurements of the CB and its anatomical variations in humans but erroneously considered sympathetic innervation as the only nerve supply to the CB. Luschka also observed that the organ was extremely well vascularized with a typical glandular structure and accordingly renamed it “glandula carotica”. Contrary to this belief, Julius Arnold, son of Friedrich Arnold, the renowned anatomy teacher of Luschka, supposed that the glandular tubules described by Luschka were convoluted blood vessels and therefore coined the name “glomeruli arteriosi intercarotici” (Arnold 1865). The vascular hypothesis of Arnold was widely accepted in the second half of the nineteenth century, and this terminology was adopted by the Basle Nomina Anatomica (BNA; 1895) where the organ

2.3 Twentieth Century: Toward the Sensory Nature of the Carotid Body

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was referred to as “glomus caroticum”. Near the end of the nineteenth century, Kohn (1900) described the general organization of the CB parenchyma as arranged in islets of cells, called clusters, glomeruli or glomoids. Around that time experimental embryology flourished and the study of the formation and development of the CB also received considerable attention. In support of the glandular nature of the CB, Luschka (1862) and his follower Stieda (1881) mistakenly believed that the CB has derived from the pharyngeal endoderm, confusing its anlage with that of the parathyroid gland. Arnold’s glomerular conception also received support from embryology since Kastchenko (1887) proposed a mesodermal origin of the CB from a condensation of mesenchymal cells around the internal carotid artery. Later, Kohn (1900) mentioned that these observations had nothing to do with the origin of the CB cells which actually arrive there along the sympathetic plexus growing in the carotid bifurcation (cited in González et al. 2014). He asked himself how a structure that was clearly neither a true ganglion nor a simple gland or a vascular glomus should be named and introduced the term “paraganglion intercaroticum” as the most appropriate one from an embryologic standpoint.

2.3 Twentieth Century: Toward the Sensory Nature of the Carotid Body In the early-twentieth century, the publications on the CB were largely engaged with a discussion of its true nature as a paraganglion. As already mentioned, Kohn in 1900 first reported that the organ arose along with the anlage of the sympathetic ganglia and originally defined it as being derived from the neuroectoderm. Nonetheless, the generally accepted nowadays neural crest origin of the CB was proposed later in the century. Kohn (1903) also claimed that some of its cells showed the chromaffin reaction and believed the organ to be similar to the adrenal medulla and other paraganglia. This interpretation was questioned by several of his contemporaries in the first third of the twentieth century because they had observed that chromaffin cells were found only isolated or in groups and that many of these did not stain yellow with dichromate salts (Mönckeberg 1905). They described the CB as a mixed paraganglion, composed of both chromaffin and non-chromaffin cells. This understanding continued to dominate until the eminent Spanish neurohistologist Fernando de Castro, one of the most brilliant pupils of Cajal, published the first accurate and detailed study of the structure, innervation and function of the CB in several mammals (De Castro 1926). He pointed out that in almost all studied species, the glomus caroticum comprises “a tangle of small blood vessels, sympathetic axons and glandular cells which may form small glomeruli within the CB as well as minuscule and complicated plexuses of glossopharyngeal fibers that surround these glomeruli”. Subsequently, he completed his experiments on the CB innervation and its physiological implications and published his findings in a second paper on this subject (De Castro 1928). In agreement with an earlier description of Hering (1927), De Castro described in detail the sensory

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innervation of the carotid region by a branch of the glossopharyngeal nerve to the carotid sinus, the carotid sinus nerve (now known as Hering’s nerve) which he called “nerve intercarotiden”. Accordingly, he deduced that if the terminals innervating the CB are of primary afferent origin, then it should function as a sensory organ. Despite the increased understanding of the CB morphology in the first quarter of the twentieth century, its detailed function remained either completely unknown to scientists or greatly misunderstood. In his 1928 paper, De Castro, based on the anatomical description of the CB location and its morphology, made his brilliant intuitive proposal that the CB functions as a chemoreceptor, hypothesizing that its principal cells would detect changes in the chemical composition of blood. These aspects of the CB physiology started to be developed almost immediately in the laboratory of the Belgian physiologist Corneille Heymans in the 1930s. The pioneering studies of Heymans and his collaborators, whose results were brought together in a book (Heymans et al. 1933), constituted the basis for accepting the CB as a sensory receptor for chemical changes occurring in blood. They revealed that the CB was activated by a fall in oxygen tension (hypoxia) in arterial blood and in case of abnormally elevated carbon dioxide levels (hypercapnia) or decreased pH (acidosis). In addition, his research group was looking for the anatomical basis of this respiratory reflex at the carotid sinus and found that hypoxia-induced stimulation of breathing was abolished after sectioning carotid sinus nerves. For the discovery that oxygen concentration and blood pressure were sensed peripherally with information transmitted to the brain, and his fundamental contribution to the knowledge of the regulatory effect on respiration of sensory organs associated with the carotid artery, Corneille-Jean-François Heymans was awarded the 1938 Nobel Prize in Physiology or Medicine. Realizing the significance of these discoveries, Watzka (1931) introduced the concept of the CB as a nonchromaffin paraganglion and parasympathetic homologue. The revised paraganglion theory became significant in the next three decades when the fluorescent methods for the histochemical demonstration of biogenic monoamines emerged and developed. The application of this technique to CB studies revealed that its principal cells must contain biogenic amines (reviewed in Biscoe 1971; Alfes et al. 1977). In addition, in the 1970s with the advance of electronmicroscopic techniques, various studies demonstrated numerous osmiophilic densecored vesicles in their cytoplasm (McDonald and Mitchell 1975; Verna 1979). Although smaller in size, they were quite similar to those in the adrenal chromaffin cells and ganglionic small intensely fluorescent (SIF) cells and thus closely resembled the granules of paraneurons belonging to the diffuse neuroendocrine system cell family. This similarity, together with findings from developmental studies, led some authors to consider the CB glomus cells as paraneurons (Le Douarin et al. 1972; Pearse et al. 1973). The morphological and functional aspects of the CB in different animal species have repeatedly been described in the years to come and a succession of researchers proceeded to refine the body of its morphofunctional knowledge, producing many excellent research papers, monographs and reviews (Biscoe 1971; Acker et al. 1977; Verna 1979; McDonald 1981; Eyzaguirre et al. 1983; González et al. 1994). The next major advance in our understanding has occurred in the second part of the twentieth

2.4 Twenty-First Century: Evidence for a Neurogenic Niche in the Carotid …

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century, with the first descriptions of the cellular responses and electrophysiology of isolated and cultured CB parenchymal cells (reviewed by Kumar and Prabhakar 2012). In addition to the morphofunctional descriptions, advances in immunohistochemistry and in situ hybridization histochemistry demonstrated the neurochemical anatomy of the mammalian CB. As a result, various kinds of neurotransmitters and their receptors as well as modulator substances have been identified in the glomus cells in normal and hypoxic conditions, and they have been proposed to play a role in the chemosensory function (for reviews, see Rey and Iturriaga 2004; Iturriaga and Alcayaga 2004; Nurse 2005, 2010; Kumar and Prabhakar 2012). Based on observations accumulated during the first half of the century, the so-called cholinergic and purinergic hypotheses for hypoxic chemosensitivity were introduced (see Fitzgerald 2009). On the other hand, based on the detection of high levels of dopamine in the glomus cells and its release upon hypoxia, the dopaminergic hypothesis on CB chemoreceptor function was proposed (see Fitzgerald 2009). Following these influential discoveries, research on CB anatomy and neurochemistry moved forward progressively through the twenty-first century, with many descriptions of morphological and neurochemical plasticity of the hypoxic CB (Heath et al. 1985; Wang and Bisgard 2002; Kusakabe et al. 2005).

2.4 Twenty-First Century: Evidence for a Neurogenic Niche in the Carotid Body During the last two decades, the stem cell research of the CB has received much attention. Because of their dopaminergic nature, attempts have been made to use glomus cell aggregates for therapeutic applications in Parkinson’s disease (ToledoAral et al. 2002; Arjona et al. 2003). Recent laboratory research of López-Barneo’s group revealed that the adult sustentacular cells are quiescent stem cells that can proliferate and differentiate into new glomus cells in response to physiologic hypoxia (Pardal et al. 2007, 2010; López-Barneo et al. 2009). A good understanding of the cellular interactions in the CB stem cell microenvironment (cell niche) and the molecular events responsible for its survival in hypoxic conditions will help us to correlate the basic knowledge of CB stem cells with the development of a stem cellbased replacement therapy in the treatment of neurological disorders. Thus, with the progress of stem cell research, the CB has entered the twenty-first century with its actual designation. Compliance with Ethical Standards The authors declare no conflict of interest. This chapter is a review of previously published research, and as such, no animal or human studies were performed.

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References Acker H, Fidone S, Pallot D, Eyzaguirre C, Lübbers DW, Torrance RW (1977) Chemoreception in the carotid body. Springer-Verlag, New York Alfes H, Kindler J, Knoche H, Matthiessen D, Möllmann H, Pagnucco R (1977) The biogenic amines in the carotid body. Prog Histochem Cytochem 10:1–69 Andersch CS (1797) Tractatio anatomico-physiologica de nervis corporis humani aliquibus, In: Andersch EP (ed). Pars altera, Regiomonti, August Fasch, pp 187 (cited in Pick J (1959) The discovery of the carotid body. J Hist Med Allied Sci 14:61–73) Arjona V, Mínguez-Castellanos A, Montoro RJ, Ortega A, Escamilla F, Toledo-Aral JJ, Pardal R, Méndez-Ferrer S, Martín JM, Pérez M, Katati MJ, Valencia E, García T, López-Barneo J (2003) Autotransplantation of human carotid body cell aggregates for treatment of Parkinson’s disease. Neurosurgery 53:321–328 Arnold J (1865) Über die Struktur des Ganglion intercaroticum. Virch Arch Path Anat 33:190–209 Biscoe TJ (1971) Carotid body: structure and function. Physiol Rev 51:437–495 De Castro F (1926) Sur la structure et l’innervation de la glande intercarotidienne (glomus caroticum) de l’homme et des mammifères, et sur un noveau système d’innervation autonome du nerf glossopharyngien. Trab Lab Invest Biol Univ Madrid 24:365–432 De Castro F (1928) Sur la structure et l’innervation du sinus carotidien de l’homme et des mammifères. Nouveaux faits sur l’innervation et la fonction du glomus caroticum. Trab Lab Rech Biol 25:331–380 Eyzaguirre C, Fitzgerald RS, Lahiri S, Zapata P (1983) Arterial chemoreceptors. In: Shepherd JT, Abboud FM (eds) Handbook of physiology, vol 3. Williams &Wilkins, Baltimore, pp 557–621 Fitzgerald RS (2009) Oxygen and carotid body chemotransduction: the cholinergic hypothesis—a brief history and new evaluation. Resp Physiol 120:89–104 González C, Almaraz L, Obeso A, Rigual R (1994) Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol Rev 74:829–898 González C, Conde SV, Gallego-Martín T, Olea E, González-Obeso E, Ramirez M, Yubero S, Agapito MT, Gomez-Niñno A, Obeso A, Rigual R, Rocher A (2014) Fernando de Castro and the discovery of the arterial chemoreceptors. Front Neuroanat 8:25 Grimley PM, Glenner GG (1968) Ultrastructure of the human carotid body: a perspective on the mode of chemoreception. Circulation 37:648–665 Haller AV (1747, 1749) In Disputationum Anatomicarum Selectorum, vols II and IV. Abram Vandenhoeck, Göttingen Haller AV (1762) Elementa Physiologiae Corporis Humani, vol 4. Grasset, Lausanne Heath D (1991) The human carotid body in health and disease. J Pathol 164:1–8 Heath D, Smith P, Fitch R, Harris P (1985) Comparative pathology of the enlarged carotid body. J Comp Pathol 95:259–271 Hering HE (1927) Die Karotissinusreflexe auf Herz und Gefässe vom normal-physiologischen, pathologisch-physiologischen und klinischen Standpunkt. Steinkopff, Dresden Heymans C, Bouckaert JJ, Regniers P (1933) Le sinus carotidien. G. Doin, Paris, p 334 Iturriaga R, Alcayaga J (2004) Neurotransmission in the carotid body: transmitters and modulators between glomus cells and petrosal ganglion nerve terminals. Brain Res Rev 47:46–53 Kastchenko N (1887) Das Schicksal der embryonalen Schlundspalten bei Säugetieren. Arch Mikrosk Anat 30:1–26 Kohn A (1900) Über den Bau und die Entwicklung der sogenanten Carotisdrüse. Arch Mikrosk Anat 56:81–148 Kohn A (1903) Die Paraganglien. Arch Mikrosk Anat 62:263–365 Kumar P, Prabhakar NR (2012) Peripheral chemoreceptors: function and plasticity of the carotid body. Compr Physiol 2:141–219 Kusakabe T, Matsuda H, Hayashida Y (2005) Hypoxic adaptation of the rat carotid body. Histol Histopathol 20:987–997

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Le Douarin N, Le Lievre C, Fontaine J (1972) Recherches experimentales sur l’origine embryologique du corps carotidien chez les oiseaux. C R Hebd Seances Acad Sci Ser D 275:583–586 López-Barneo J, Pardal R, Ortega-Sáenz P, Durán R, Villadiego J, Toledo-Aral JJ (2009) The neurogenic niche in the carotid body and its applicability to antiparkinsonian cell therapy. J Neural Transm 116:975–982 Luschka H (1862) Über die drüsenartige Natur des sogenannten Ganglion intercaroticum. Arch Anat Physiol Lpz 405:413–430 Mayer AFJK (1833) Über ein neuentdecktes Ganglion im Winkel der aussern und inner Carotis, beim Menschen und den Säugetieren (Ganglion intercaroticum). Notizen Geb Nat Heilk 36:8–9 McDonald DM (1981) Peripheral chemoreceptors. Structure-function relationships of the carotid body. In: Hornbein TF (ed) Regulation of breathing. Dekker, New York, pp 105–319 McDonald DM, Mitchell RA (1975) The innervation of glomus cells, ganglion cells and blood vessels in the rat carotid body: a quantitative ultrastructural study. J Neurocytol 4:177–230 Mönckeberg IG (1905) Die Tumoren der Glandula carotica. Beitr Pathol Anat Allg Pathol 38:1–66 Neubauer IE (1772) Descriptio anatomica nervorum cardiacorum. Fleischer, Frankfurt and Leipzig Nurse CA (2005) Neurotransmission and neuromodulation in the chemosensory carotid body. Auton Neurosci 120:1–9 Nurse CA (2010) Neurotransmitter and neuromodulatory mechanisms at peripheral arterial chemoreceptors. Exp Physiol 95:657–667 Pardal R, Ortega-Sáenz P, Durán R, López-Barneo J (2007) Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body. Cell 131:364–377 Pardal R, Ortega-Sáenz P, Durán R, Platero-Luengo A, López-Barneo J (2010) The carotid body, a neurogenic niche in the adult peripheral nervous system. Arch Ital Biol 148:95–105 Pearse AGE, Polak JM, Rost FWD, Fontaine J, Le Lievre C, Le Douarin N (1973) Demonstration of the neural crest origin of Type I (APUD) cells in the avian carotid body, using a cytochemical marker system. Histochemie 34:191–203 Pick J (1959) The discovery of the carotid body. J Hist Med Allied Sci 14:61–73 Prabhakar NR, Peng YJ (2017) Oxygen sensing by the carotid body: past and present. In: Halpern H, LaManna J, Harrison D, Epel B (eds) Oxygen transport to tissue XXXIX. vol 977. Springer, Cham, pp 3–8 Rey S, Iturriaga R (2004) Endothelins and nitric oxide: vasoactive modulators of carotid body chemoreception. Curr Neurovasc Res 1:465–473 Stieda L (1881) Untersuchungen über die Entwicklung der Glandula thymus, Glandula thyroidea und Glandula carotica. Engelmann, Leipzig Taube H (1743) De Vera Nervi Intercostali Origine. Abram Vandenhoeck, Goettingen Toledo-Aral JJ, Méndez-Ferrer S, Pardal R, López-Barneo J (2002) Dopaminergic cells of the carotid body: physiological significance and possible therapeutic applications in Parkinson’s disease. Brain Res Bull 57:847–853 Valentin G (1833) Über das Ganglion intercaroticum. Wiss Ann Ges Heilk 26:398–407 Verna A (1979) Ultrastructure of the carotid body in the mammals. Int Rev Cytol 60:271–330 Wang Z-Y, Bisgard GE (2002) Chronic hypoxia-induced morphological and neurochemical changes in the carotid body. Microsc Res Tech 59:168–177 Watzka M (1931) Über die Verbindungen in Kretorischer und neurogenen Organe. Anat Anz Erganz Z 71:185–190

Chapter 3

General Morphology of the Mammalian Carotid Body

Abstract The carotid body (CB) is the main peripheral arterial chemoreceptor that registers the levels of pO2 , pCO2 and pH in the blood and responds to their changes by regulating breathing. It is strategically located in the bifurcation of each common carotid artery. The organ consists of “glomera” composed of two cell types, glomus and sustentacular cells, interspersed by blood vessels and nerve bundles and separated by connective tissue. The neuron-like glomus or type I cells are considered as the chemosensory cells of the CB. They contain numerous cytoplasmic organelles and dense-cored vesicles that store and release neurotransmitters. They also form both conventional chemical and electrical synapses between each other and are contacted by peripheral nerve endings of petrosal ganglion neurons. The glomus cells are dually innervated by both sensory nerve fibers through the carotid sinus nerve and autonomic fibers of sympathetic origin via the ganglioglomerular nerve. The parasympathetic efferent innervation is relayed by vasomotor fibers of ganglion cells located around or inside the CB. The glial-like sustentacular or type II cells are regarded to be supporting cells although they sustain physiologic neurogenesis in the adult CB and are thus supposed to be progenitor cells as well. The CB is a highly vascularized organ and its intraorgan hemodynamics possibly plays a role in the process of chemoreception. Keywords Carotid sinus nerve · Ganglioglomerular nerve · Glomeruli · Glomus cells · Petrosal ganglion · Superior cervical ganglion · Sustentacular cells

Dating from the classic pioneering studies in the early-twentieth century of Kohn (1900), the structural organization of the CB in different animal species has repeatedly been described. Much of the available evidence came from studies performed on lower mammals, in particular rodents (Atanasova et al. 2011). Here, we will address only certain morphological features of the mammalian CB, focusing on some structural characteristics of the rat and human CB.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_3

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3.1 Size, Weight and Anatomical Location of the Carotid Body In mammalian species, the CB is bilaterally located at the bifurcation area of the common carotid artery (CCA), though this may vary between species (Clarke and de Burgh Daly 1981). In humans, its location may vary among individuals, and even between the right and left CBs of the same subject (Arias-Stella and Valcarcel 1976; Ortega-Sáenz et al. 2013). A double CB is found on one side in about 7% of autopsied subjects (Heath 1991). It is a reddish-brown or tan in color with a typically oval body, around 2–5 mm in diameter in humans (Wang and Bisgard 2002; Schulz et al. 2016) and less than 1 mm in rats (McDonald 1981). Anatomical variations in its form, such as a bilobed, duplicated or leaf-shaped CB, have been reported in the human (Khan et al. 1988). The weight of the CB may be correlated to the body size, particularly to that of the left ventricle of the heart (Heath 1991) and its mean wet weight ranges from about 60 μg in the rat (McDonald 1981) and less than 2 mg in the cat (Clarke et al. 1986) to 13 mg in the adult human (Heath et al. 1970). In the latter, its average volume is estimated to be around 20 mm3 without significant differences with respect to sex or age (Ortega-Sáenz et al. 2013). Our stereological analysis has revealed that the average estimated volume of the adult rat CB is 5 mm3 . In rats, the organ has an intra-adventitial location and it is situated between the external carotid artery (ECA) and the internal carotid artery (ICA) (Fig. 3.1). Significant variations regarding its location and syntopy in humans have recently been reported (Schulz et al. 2016). Specifically, the CB is associated with CCA (42%), ECA (28%) and ICA (30%) lying on the medial or lateral surface (82% or 13%, respectively) or exactly in the middle (5%) of the carotid bifurcation (Schulz et al. 2016). Thus, such a position is strategic for monitoring blood chemicals before they reach the brain.

3.2 Structure of the Carotid Body The general organization of the CB was originally described by Kohn (1900). The organ is structurally complex, still its basic plan is quite similar in all mammals. As seen in the light microscope, the CB is composed of four principal components: cell clusters, blood vessels, connective tissue and nerve fibers (Fig. 3.2). The small ovoid clusters, known as glomeruli or glomoids, are the basic morphofunctional units of the CB. They are separated by septa of dense connective tissue, which converge on the surface to form a fibrous capsule for the whole structure. Generally, there is relatively little connective tissue in the majority of young animals, and its amount increases with age, constituting 50–60% of the total volume of the adult rat CB. Its amount also varies between species being more compact in rats and cats or with a more diffuse appearance in the rabbit and sheep CB (McDonald 1981). In humans, the CB is best developed in children and young people, in whom the characteristic cell clusters are more compact and separated by thin septa of connective tissue

3.2 Structure of the Carotid Body

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Fig. 3.1 Location of the carotid body (CB) of a rat. a Low-magnification Nissl-stained section showing the bifurcation area of the common carotid artery (CCA). The glomus organ is strategically positioned between the external carotid artery (ECA) and the internal carotid artery (ICA). Note the closely located superior cervical ganglion (SCG). b Hematoxylin and eosin (H&E) staining of the rat CB at higher power illustrating its location in the tunica adventitia (TA) at the bifurcation of the internal (ICA) and external carotid arteries (ECA). Scale bars = 500 μm in a and 200 μm in b. Panel A reproduced from Atanasova et al. (2018) with permission from Acta morphologica et anthropologica

(see Chap. 4). In contrast, an apparent increase of the interlobular and intralobular connective tissue is observed in elderly individuals (Lazarov et al. 2009), in people with chronic obstructive pulmonary disease (Heath et al. 1970; Habeck 1986) and in opiate-addicted patients (Porzionato et al. 2005). The stroma around the lobules contains relatively large blood vessels and nerve bundles.

Fig. 3.2 Structural organization of the rat carotid body (CB). a Low-power photomicrograph of a representative H&E-stained section showing that the CB consists of lobules divided by vascularized septa and is surrounded by adipose and connective tissue (arrows) and penetrated by the carotid sinus nerve (CSN). b The higher magnification of the boxed area in a demonstrates that the glomic lobules are organized in ovoid cell clusters (CC) that are separated by dense connective tissue (CT) intermingled with a profuse capillary network. Scale bars = 200 μm in a, 50 μm in b. Panel A reproduced from Lazarov and Atanasova (2019) with permission from Trakia Journal of Sciences

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3.2.1 Cell Types As initially described by Gomez (1908), the cellular parenchyma of the CB consists of islets of cells, interspersed by fine blood vessels, and richly innervated by bundles of nerve fibers. The glomerulus is a conglomerate of two juxtaposed cell types: neuronlike type I or glomus cells and glial-like type II, also called sustentacular cells, in a ratio of approximately 4:1 (Biscoe 1971; McDonald 1981; Kondo 2002). By a 3Dbased counting method, we have calculated that the total number of parenchymal cells in the adult rat CB is approximately 11,700. Both parenchymal cell types can be clearly distinguished from one another, even at the light microscope level. In rats, the glomerulus contains 2–12 glomus cells, four cells on the average, each of them incompletely invested by long cytoplasmic processes of 1–3 sustentacular cells (Fig. 3.3). This arrangement is ideally suited for both autocrine and paracrine regulations of the glomus cell function.

3.2.1.1

Type I (Glomus) Cells

The glomus cells, the principal cell type, are the most common in the CB comprising up to 80% of the cell population. They are regarded the chemosensory cells of the CB (González et al. 1994). By stereological methods, Laidler and Kay (1975) determined that the CB of the adult rat contains 11,500 ± 2500 glomus cells (mean ± SE), while in cats their total number is up to 60,000 (Kumar and Prabhakar 2012). Like sympathetic neurons and chromaffin cells of the adrenal medulla, they are derived from the neural crest cells (Le Douarin 1982) and structurally are very similar to

Fig. 3.3 General structure of the rat carotid body (CB). a Schematic representation of an adult CB glomerulus illustrating the most relevant cell types interspersed by blood vessels (BV). b Routine H&E staining showing a cell cluster composed of centrally located oval-shaped type I (glomus) cells (arrows) and peripherally displaced elongated type II (sustentacular) cells (arrowheads). Note that neuron-like glomus cells are partially enveloped by glial-like sustentacular cells. Scale bar = 50 μm. Panel B reproduced with permission from Atanasova and Lazarov (2015). This article was published in Acta Histochemica, Volume 117, Issue 2, March 2015, Pages 219–222, Copyright Elsevier

3.2 Structure of the Carotid Body

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them albeit they show cellular heterogeneity. Glomus cells are usually located in the center of a CB glomerulus (Fig. 3.3). They are generally round to oval in shape, and their size usually varies between 8 and 16 μm in rats. The cells contain a centrally placed, single spherical nucleus and a copious and distinctly granular cytoplasm filled with widely distributed mitochondria and numerous small clear or larger dense-cored vesicles, suggesting the storage of various neurotransmitters. Initially, on the basis of the electron density of their cytoplasm, two subtypes of glomus cells, light and dark glomus cells, were distinguished in the CB of rats (Hellström 1975) and humans (Grimley and Glenner 1968). Based on the size and morphology of their granules, A and B types of glomus cells in nearly equal proportions were described in the rat CB (McDonald and Mitchell 1975). However, later observations stated that the intensity of cytoplasm staining is an unreliable criterion for distinguishing subtypes of glomus cells in different animal species (McDonald 1981). Other authors ascribed these structural differences to technical artifacts either due to the surgical removal of the organ or to its fixation (see Kumar and Prabhakar 2012 for a review). Nonetheless, this distinction is still held for the human CB, where a third more descriptive variant, “pyknotic” glomus cells, has been described as well (Heath et al. 1970; Heath and Smith 1985).

3.2.1.2

Type II (Sustentacular) Cells

The sustentacular cells (~ 15–20% of all cells) are typically located at the periphery of the glomerulus and they wrap around glomus cells (Fig. 3.3). They are glial-like cells of mesenchymal origin possessing long-shaped bodies (~ 10 μm in diameter) with elongated hyperchromic nuclei, a thin cytoplasmic layer with intermediate filaments and extended processes that ensheath or penetrate the groups of glomus cells and nerve fibers (Pallot 1987). Ultrastructural studies later revealed that sustentacular cells do not completely envelop glomus cells, so that adjacent glomus cells, capillaries or nerve endings come in direct contact with their cell bodies (McDonald 1981; González et al. 1994). Classically, the sustentacular cells are supporting cells which play a role in metabolic support. In addition, they have recently been proposed to be the CB stem cells that behave as glomus cell precursors (Pardal et al. 2007, 2010). The sustentacular cells lack significant inward sodium or calcium currents and are, therefore, unexcitable (Pardal et al. 2007; Leonard et al. 2018). Nonetheless, emerging evidence suggests that they may play a key role in the sensory signaling process and serve as paracrine modulators of CB chemoreception (Tse et al. 2012; Nurse 2014; Nurse et al. 2018; Leonard et al. 2018).

3.2.1.3

Minor Cell Types

Autonomic (both sympathetic and parasympathetic) ganglion cells in the CB were originally described by De Castro (1926), and they are present in most mammals with

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a varying number and location in the organ between the different animal species and breeding strains (Pallot 1987). In the rat CB, they are embedded within or situated at the periphery of the organ (see Fig. 3.6), and their number varies from 10 to 20 (McDonald and Mitchell 1975). Some microganglion cells in rats are situated within the organ capsule or in close contact to glomus cells (Kondo 1976). Their morphology is rather similar to neurons in autonomic ganglia. They have large, round vesicular nuclei and finely granular cytoplasm. These cells mainly provide innervation for the blood vessels (De Castro 1926) but may also innervate the glomus cells (McDonald and Mitchell 1975). Numerous Schwann cells are dispersed in the interlobular septa where they are associated with bundles of myelinated nerve fibers (Heath and Smith 1985). Their morphological features are identical to those of sustentacular cells. Mast cells are also numerous in the CB of humans (Heath et al. 1987) and rats (Atanasova et al. 2018). They are confined to the interlobular stroma in close association with small blood vessels (Fig. 3.4a, b). Such cells are not found in the cell clusters though a few are tightly packed to the covering shell of sustentacular cells. They are easily recognized by their rounded bodies and the presence of numerous cytoplasmic granules (Fig. 3.4c). Some elongated cells resembling fibroblasts are also observed within the connective tissue and are detected in the adventitia of CB blood vessels as well (Fig. 3.4d). The wall of the abundant glomic capillaries is formed by a fenestrated endothelium covered by pericytes. Abundance of macrophages is also found in the rat CB. Their presence is associated with an immunomodulatory function (Conde et al. 2020) and its role as a major source of cytochrome b558, a potential oxygen sensor candidate (Dvorakova et al. 2000).

3.2.2 Vascularization 3.2.2.1

Blood Supply

The CB is made up of a ball of highly vascular tissue; hence, the Latin term “glomus” is used to denote the structure. Consistent with its ability to sense blood-borne chemicals, the CB as a single entity is one of the most irrigated organs per unit weight of any tissue in the body (McDonald 1981; González et al. 1994). In fact, its vascularization is five–six-fold higher than that of the brain. This rich vascularization attributes to its pink-colored appearance (Fig. 3.5a). Stereological analysis of the tissue components in the cat CB reveals that nearly 25% of its total volume is occupied by blood vessels (Ballard et al. 1982). Our microangioCT data verify that the same is also true for the vascular supply of the rat CB (Fig. 3.5b–d). The origin of the arterial blood supply to the CB varies between species (Seidl 1975). In the rat, it receives blood through a single short branch, called the “CB artery”, arising from either the ECA or the occipital artery (Fig. 3.5c) (McDonald 1981; McDonald and Lurue 1983; Verna 1997). In humans, the CB receives its arterial

3.2 Structure of the Carotid Body

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Fig. 3.4 Minor cell types in the rat carotid body (CB). a A toluidine-blue stained section from the adult CB indicating a few mast cells (arrows) situated in the connective tissue around the cell clusters and often associated with blood vessels. b Higher magnification of the rectangle in a showing two mast cells (arrows) with their typical stromal location. c Methylene blue-basic fuchsin stain clearly demonstrates the dense granular appearance of mast cells (arrow) located in close proximity to blood vessels (BV). d Bismarck brown staining and hematoxylin counterstaining selectively validate two mast cells (arrows) and an elongated fibroblast (arrowhead) observed in the interlobular septa. Scale bars = 100 μm in a, 50 μm in b–d

feed from a small artery known as “the glomic artery” with a varying origin across individuals (Schaper 1892) and this difference might be related to ethnicity (Khan et al. 1988). The entire blood supply of the CB is derived by third- or fourth-order branches of this artery that intralobularly give rise to terminal arterioles (10–15 μm thick) which terminate into a dense vascular bed of fenestrated capillaries within the CB (McDonald and Haskell 1984). It has recently been suggested that arterioles within the CB are located predominantly to one side, making the “vascular side” more sensitive to sympathetic innervation (Gold et al. 2022). The capillaries in the center of the glomoids are convoluted, while those at the periphery of the organ are straight (McDonald 1981). It has been found that terminal arterioles and capillaries have precapillary sphincters. Therefore, the vasculature appears to form separate circuits inside the organ. Such a vascular system is capable of regulating CB blood flow through its parenchyma. In addition, a profuse capillary network travels in the walls of the connective tissue surrounding the CB glomeruli. Also, the capillaries are arising from the CB anastomose with venules of variable diameters that form a

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Fig. 3.5 Blood vascular architecture of the rat carotid body (CB). a H&E staining illustrates structural tissue details of the glomus organ which is highly irrigated by blood vessels (arrows) that are filled with erythrocytes. Note the intimate contact of capillaries with the cell clusters (CC). CT, interlobular connective tissue. b Low-power microcomputed tomography (microCT) angiogram of the common carotid artery bifurcations (rectangles) highlighting the blood supply of the CB. c Highresolution microCT imaging of the right carotid bifurcation with the encircled CB showing the CB artery (arrow) arising from the external carotid artery (ECA). d High-resolution microCT imaging of the right carotid bifurcation depicting the venous drainage through the homonymous veins into the internal jugular vein (arrowhead). ICA, internal carotid artery; OA, occipital artery. Scale bar = 100 μm in a. Panel A reproduced from Atanasova et al. (2011) with permission from Biomedical Reviews. Panels B–D courtesy of Dr. Ruslan Hlushchuk, Institute of Anatomy, University of Bern, Switzerland

diffuse venous plexus on the surface of the organ. The venous drainage of the CB is via one or two small veins, emerging from this superficial plexus, which empty into the internal jugular vein or one of its tributaries (Fig. 3.5d) (González et al. 1994). Arteriovenous anastomoses have also been described in the organ in rats (McDonald and Haskell 1984) and cats (Schäfer et al. 1973) but not in humans (Heath et al. 1983). These could act as shunt vessels that control the blood flow and/or its distribution through the organ (Habeck et al. 1984). Altogether, the total blood flow through the CB is regulated by sphincters in the arterial and venous vessels.

3.2 Structure of the Carotid Body

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Fig. 3.6 Schematic drawing of the rat carotid body (CB) innervation. a The sensory nerve supply of the chemosensory glomus cells is provided by petrosal ganglion neurons through the carotid sinus nerve. The sympathetic innervation is performed by postganglionic neurons from the closely located superior cervical ganglion via the ganglioglomerular nerve and by postganglionic fibers of intrinsic sympathetic ganglion cells. They mostly supply the blood vessels (BV) although some of them may also innervate type I (glomus) cells. b Photograph showing the nerves associated with the CB (arrow) labeled during the preparation by loops of differently colored threads. The glossopharyngeal nerve and its branches are marked by colored thread loops in green, the vagus nerve in red, the sympathetic trunk in blue and the hypoglossal nerve in black. (Courtesy of Prof. Dr. Doychin N. Angelov, Department of Anatomy, University of Cologne, Germany)

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3.2.2.2

3 General Morphology of the Mammalian Carotid Body

Blood Flow and Oxygen Consumption

In addition to the precapillary sphincters, another structure contributes to the control of blood flow within the CB known as the “intimal cushion” formed by circumferential smooth muscle cells, collagen fibers, basal and elastic laminae and also by components of the extracellular matrix (McDonald and Lurue 1983). Physiologists have shown that the total blood flow, measured in the middle zone of the cat CB at mean blood pressure of 120–130 mm Hg, was about 2000 ml/min/100 g (Barnett et al. 1988) and it decreases from the arterial inflow toward the periphery (Keller and Lübbers 1972). Given the low wet weight of tissue, it can be easily calculated that this flow is the highest for any organ and is 10–15 times larger than that observed per unit mass in the human cerebral circulation (Kumar and Prabhakar 2012). It has also been found that autoregulation of CB blood flow exists and the sympathetic stimulation and neurotransmitters such as noradrenaline reduce the level of tissue flow without abolishing its autoregulation (McCloskey and Torrance 1971). Despite the high blood supply to the CB for its volume, the oxygen utilization is very low and comprises less than 3% of the total metabolic demand (De Burgh Daly et al. 1954). The authors also claim that the oxygen consumption of the CB is extraordinarily rapid, estimating it to be 9 ml/min per 100 g wet weight, which is about 4 times greater than that of the adjacent superior cervical ganglion (SCG) and threefold more than that in the brain (De Burgh Daly et al. 1954). Later experiments however have shown that the oxygen consumption is, at most, 20% of that reported previously, calculated an average of 1.5 ml O2 /min/100 g wet weight in the cat CB (Fay 1970). On the other hand, it has been demonstrated that individual rabbit glomus cells have a very high metabolic rate, with an oxygen consumption at rest approaching the maximum, and that it is significantly higher than that of cells from other tissues, for example of the dorsal root ganglion or adrenal medulla (Duchen and Biscoe 1992). This very high oxygen consumption makes glomus cells very sensitive to decrease in the partial pressure of oxygen. To sum up, the data on the extensive vascularization show that the CB receives an adequate blood supply to support its active metabolism even during severe hypoxia, one that is far beyond its nutritional requirements (González et al. 2004). However, there is evidence that blood flow to the CB is diminished in disease characterized by a hyper-reflexic sympathetic response such as heart failure and hypertension (Ding et al. 2011).

3.2.3 Innervation Since the pioneering work of Fernando de Castro (1926) is showing the detailed CB innervation and vasculature, it has been known that this highly vascular complex is dually innervated by both sensory and autonomic nerve fibers (Figs. 3.6 and 3.7).

3.2 Structure of the Carotid Body

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Fig. 3.7 Innervation of the rat carotid body (CB). a Afferent fibers from the petrosal ganglion innervating the glomus cells of the CB via the carotid sinus nerve (CSN). b Postganglionic sympathetic fibers from the adjacent superior cervical ganglion (SCG) mostly supplying blood vessels but also some glomus cells in the CB. Single retrogradely Fast Blue-labeled ganglion cell profiles scattered in the caudal portions of the petrosal ganglion (PG) (c) and superior cervical ganglion (SCG) (d). Scale bars = 200 μm in a, b, 100 μm in c and 50 μm in d. Panel B reprinted from Atanasova et al. (2011) with permission from Biomedical Reviews

For more details about the innervation of the mammalian CB, the reader is to refer to the review by Ichikawa (2002).

3.2.3.1

Afferent Innervation

The careful preparation of the CB using microsurgical instruments and operation microscope reveals numerous nerves associated with it in humans. Most of them are found on the medial aspect of the carotid bifurcation forming a dense network (Fig. 3.6b). The sensory nerve fibers which convey chemosensory impulses from the CB into the brainstem are mainly supplied by the carotid sinus nerve (CSN; also known as Hering’s nerve), and their cell bodies are located in the petrosal ganglion (PG) of the glossopharyngeal nerve (GPN; Hess and Zapata 1972) (Figs. 3.7a, c). Each afferent fiber of the CSN innervates up to 30 glomus cells in the human CB. In addition, the CB in the rat receives sensory innervation from the superior (jugular)

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ganglion and inferior (nodose) ganglion of the vagus nerve (Hess and Zapata 1972; McDonald and Mitchell 1975). In rats, there are about 450–750 axons in the CSN and most of them are unmyelinated fibers (McDonald 1983). Entering the cell cluster, each of them branches to innervate more than 20 glomus cells. In the cat and human CB, both myelinated (A-) and unmyelinated (C-) fibers are found in the CSN (De Castro 1926; Eyzaguirre and Uchizono 1961), where about two-thirds of the A-fiber population and just 15% of the C-fibers are of chemoafferent origin (Fidone and Sato 1969). In summary, studies based on retrograde neuronal tracing have shown that in rats 94.5% of afferent neurons supplying the CB are localized in the PG, 5.2% are located in the jugular ganglion and 0.3% are in the nodose ganglion (Ichikawa et al. 1993). Moreover, the nerve fibers in the CB are characterized by a relatively high degree of neurochemical differentiation (cf. Chap. 6).

3.2.3.2

Efferent Innervation

In addition to sensory input, the CB receives a rich autonomic innervation by postganglionic fibers, representing ~ 5% of fibers in the CSN, of both sympathetic and parasympathetic origins which plays a pivotal role in the modulation of blood flow (De Caro et al. 2013). The sympathetic nerve supply is provided by postganglionic neurons from the closely located SCG via the ganglioglomerular nerve (Figs. 3.6 and 3.7b, d) (Verna et al. 1984; Kummer et al. 1990; De Burgh Daly 1997). Most sympathetic nerve fibers are thought to supply blood vessels (Biscoe and Stehbens 1967) and a few of them may also innervate glomus cells (McDonald and Mitchell 1975; Verna 1997). In addition to the existence of the ganglioglomerular nerve pathway, it has also been suggested that sympathetic efferents originating from the SCG course down the CSN toward the CB in cats (Eyzaguirre and Uchizono 1961; Biscoe and Sampson 1968). Finally, it has been proposed that scarce postganglionic fibers arise within the CB from a few intrinsic sympathetic ganglion cells (McDonald and Mitchell 1975). These authors propose that sympathetic innervation might have an excitatory effect on CB afferent discharge caused by vasoconstriction and reduced blood flow and mediated by the release of noradrenaline acting on vascular α1 -adrenergic receptors. The second mechanism is produced by the co-release of neuropeptides as well as an inhibitory response mediated by an evoked release of dopamine via the innervation of glomus cells (McDonald and Mitchell 1975). The parasympathetic innervation of the CB is scarce or absent. It is relayed by vasomotor fibers of ganglion cells located around or inside the CB and the latter are collectively referred to as the (internal) carotid ganglion (CG; McDonald and Mitchell 1975; Kondo 1976; Ichikawa 2002). These fibers are thought to innervate smooth muscle cells of CB arteries or arterioles, and according to De Castro and Rubio (1968), they are the efferent pathway of a reflex which tends to maintain a constant blood flow. There is also evidence that CSN efferents of parasympathetic nature may affect the glomus cell transmitter content and release (for a review, see Verna 1975). This parallel efferent pathway to CB chemoreceptors includes

3.3 Ultrastructure of the Carotid Body

25

two populations of autonomic neurons, presumed to be of parasympathetic origin, which contribute to CB inhibition. The neuronal somata of them are embedded in paraganglia or microganglia, proximally located near the branch point of the CSN and GPN or concentrated more distally along the GPN (Campanucci and Nurse 2007). The presence of such an inhibitory efferent innervation, mediated by the release of nitric oxide, provides additional control of the afferent discharge. On the overall, it is now believed that the autonomic nerves subserving the CB provide a rapid mechanism to tune the gain of peripheral chemoreflex sensitivity based on alterations in blood flow and oxygen delivery, and they might provide future therapeutic targets (Brognara et al. 2021).

3.3 Ultrastructure of the Carotid Body As early as 1957, Lever and coworkers identified the presence of osmiophilic membrane-bound vesicles in the glomus cells of the rabbit CB, and in nerve endings on them (Lever and Boyd 1957; Lever et al. 1959). Since then, the CB ultrastructure has received much attention, and it has repeatedly been demonstrated in different animal species.

3.3.1 Ultrastructure of the Parenchymal Cells Electron-microscope studies have shown no remarkable structural differences regarding the ultrastructure of parenchymal cells among animal species. In general, the glomus cells have the morphological characteristics of actively synthesizing cells (McDonald 1981). As seen with the transmission electron microscope, their cell bodies contain a large round, euchromatic nucleus surrounded by a nuclear envelope with multiple pores and an abundant pale cytoplasm with numerous organelles (Fig. 3.8a, b). Among them, most notable are the abundant free ribosomes and polysomes, the flattened cisternae of rough endoplasmic reticulum, the welldeveloped Golgi apparatus and a large number of compact mitochondria. Another striking ultrastructural feature of these cells is the presence of osmiophilic densecored vesicles in their cytoplasm, where they are not randomly distributed. Indeed, they are rare in the Golgi region and occur in large groups tending to accumulate in the periphery of the cells (Fig. 3.8b). The size of the dense-cored vesicles in the rat ranges from 50 to 200 nm, with a mean diameter about 100 nm (Verna 1979). Although smaller in size, they closely resemble the granules of paraneurons belonging to the diffuse neuroendocrine system cell family. In particular, the glomus cells look alike cytologically to the adrenal chromaffin cells and ganglionic SIF cells. Similar to them, the glomus cells contain various biogenic amines and neuropeptides in the dense-cored granules (González et al. 1994; Nurse 2005). This similarity, together with findings from developmental studies as already noted, has historically led to the

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concept of classical “Paraganglion” (Kohn 1903; Watzka 1943) and more recently to that of “Paraneuron” (Fujita 1977; Fujita et al. 1988). Therefore, all these are in favor of the proposal that the CB can be regarded as a secretory organ. On the basis of the size and staining properties of their dense-cored vesicles, McDonald and Mitchell (1975) and independently Hellström (1975) have categorized two subtypes of glomus cells: type A or large vesicle cells (mean vesicle diameter 116 nm) and type B or small vesicle cells (mean vesicle diameter 90 nm). The authors estimate that in rats type A cells comprise 51 ± 10% (mean ± S.D.) of the glomus cells. Besides, the density and population of these granules vary between glomus cells, and this may be indicative of differing states of secretory activity within the CB. Smaller in number and size (about 40 nm in diameter) clear vesicles also occur

Fig. 3.8 Ultrastructure of the parenchymal cells in the rat carotid body (CB). a An ultrathin section of a rat CB glomerulus indicating at a lower magnification a typical tightly packed cell cluster of glomus cells (G) and sustentacular cells (S) adjacent to blood vessels (BV). A glomus cell is partially invested by a sustentacular cell. b Electron micrograph of a glomus cell (G) with a round, euchromatic nucleus (N) and an abundant cytoplasm with numerous dense-cored vesicles (arrows) in its periphery. c Fragment of a CB glomerulus showing a couple of glomus cells (G) with the accumulation of their large dense-cored and small clear vesicles and one surrounding sustentacular cell (S) with an elongated hyperchromatic nucleus, vesicle-free cytoplasm and long processes. Note the nerve ending (ne) in direct contact with a glomus cell. d Electron micrograph of a section through the peripheral region of a glomerulus demonstrating the close somato-somatic appositions between two glomus cells (G). Scale bars = 0.5 in a, c and 1 μm in b, d. Panels A, B reproduced from Atanasova et al. (2011) with permission from Biomedical Reviews

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in rat glomus cells (Verna 1979). They tend to accumulate in the cell processes and occasionally in the regions apposing the nerve endings (Fig. 3.8c). The sustentacular cells contain a paucity of organelles in their cell bodies. The most distinguishing feature of them is the absence of secretory granules in their cytoplasm suggesting that they do not synthesize and store neurotransmitters. Therefore, despite their location in close proximity to the blood in the capillaries, they do not play a role in chemosensory function. Nonetheless, Golgi apparatus, ribosomes, scattered endoplasmic reticulum and occasional mitochondria are present, though developed to a lesser extent than in glomus cells (Fig. 3.8a, c). They also contain abundant, intermediate vimentin filaments and possess glial-like traits necessary to support and influence the behavior of glomus cells (Kameda 1996). Indeed, the sustentacular cells express glial markers such as the S100 protein and glial fibrillary acidic protein (Kondo et al. 1982; Pardal et al. 2007). A common trait of these cells is their possession of long cytoplasmic processes that extend away, partially envelop chemoreceptor cells and collectively form a protective network around them. Like Schwann cells, they may completely ensheath single or small groups of unmyelinated nerve fibers in the CB, thus guiding the axons to the glomus cells in the space between the Schwann cells and the cell clusters.

3.3.2 Synaptic Organization and Communications The synaptic connections of the CB have been characterized in the greatest details in the rat wherein both chemical (including reciprocal) and electrical synapses have been detected between adjacent glomus cells. The advent of the conventional electron microscopy has indicated that many adjacent glomus cells make “synaptic”-like somato-somatic contacts (Fig. 3.8d), thus explaining the characteristic morphological picture of cell clustering in the CB (McDonald 1981; González et al. 1994; Verna 1997). The intercellular space between the contacting cells is about 20 nm. Notably, both large dense-cored vesicles and small clear vesicles accumulate at this synapticlike junction (Figs. 3.8d and 3.9a). Subsequent examination by freeze-fracture electron microscopy and immunocytochemistry for connexins has additionally revealed the existence of gap junctions between some glomus cells in the CB which have been designated as electrical synapses (reviewed in Kondo 2002). Interestingly, gap junctions may also occur between glomus and sustentacular cells (Platero-Luengo et al. 2014) and such junctional specializations are observed between glomus cells and afferent nerve endings as well (Kondo and Iwasa 1996). The electrotonic coupling allows intercellular exchange of ions and small molecules and the passage of currents (Eyzaguirre 2000) that are unaffected by blockers (Eyzaguirre and Abudara 1996). Besides, it has been found that the glomus cells are contacted by peripheral nerve endings of PG afferent neurons (for references see McDonald 1981; Verna 1997). Sensory nerve endings on glomus cells may also appear as boutons “en passant”, making multiple synaptic contacts (Morgan et al. 1975). The presynaptic terminal contains a large number of mitochondria, numerous small (about 60 nm in diameter

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in the rat) clear vesicles and a few large (usually 70–150 nm in diameter) dense-cored vesicles (Fig. 3.9b, c). Some larger boutons apposed to chemoreceptor cells and typically seen as axonal varicosities are filled with abundant densely packed small clear vesicles, large dense-cored vesicles and mitochondria (Fig. 3.9d). They are considered preganglionic sympathetic efferent nerve endings, thus favoring the concept of the dual sensory and autonomic innervation of the CB (McDonald and Mitchell 1975; Kondo 1976, 1977). Such synaptic connections have also been shown on some ganglionic SIF cells (Kondo 1977). Sometimes, i.e., in about 10% of the cases, the “afferent” and “efferent” synapses are adjacent to each other forming reciprocal synapses in the CB of different animal species (Smith and Mills 1976; McDonald 1981; González et al. 1994; Verna 1997). The synaptic contacts on glomus cells are with both symmetric and asymmetric membrane morphology and have functionally been described as bidirectional (McDonald 1981). It is likely that in response to natural stimuli, peripheral processes of PG neurons release chemical substances at synapses triggering the exocytosis of one (or more) neurotransmitter(s) from the glomus cells (Iturriaga and Alcayaga 2004). The released transmitter, acting on specific postsynaptic receptors, increases the rate of chemosensory discharge in nerve fibers of PG neurons projecting to the CB (Eyzaguirre et al. 1983; González et al. 1994). Furthermore, auto- and paracrine signaling adds an extra level of complexity to fine-tune the CB output. In addition to nerve-glomus cell contacts, nerve endings are occasionally observed to make synaptic contact with another nerve or nerve ending (Fig. 3.10). The presynaptic profile possesses a few mitochondria and always contains groups of clear vesicles as well as occasional dense-cored vesicles (Fig. 3.10a–c). The same structures, i.e., mitochondria, clear vesicles and a few dense-cored vesicles are also present in the postsynaptic nerve ending. Usually these axo-axonic synaptic contacts have a symmetrical appearance. Interestingly, a presynaptic dense body is sometimes seen denoting this synaptic contact as a ribbon synapse (Fig. 3.10d). This type of synapse typically links some sensory receptor cells. The ribbon has been proposed to shuttle synaptic vesicles to exocytotic sites, promote their release at the synapse and thus enable a rapid information processing.

3.3.3 Microvasculature Ultrastructure Most blood vessels in the CB are capillaries, which are abundant and closely packed. According to their morphological features and size, two types of capillaries have been identified in the CB (De Castro and Rubio 1968; McDonald and Lurue 1983; McDonald and Haskell 1984). Type I capillaries are the prevailing type, comprising about 60% of the total. They are convoluted, larger in size (8–20 μm in diameter) and have a thin wall, formed by a fenestrated endothelium with short microvilli, a basal lamina (50–100 nm in thickness) beneath and an incomplete covering of pericytes (Fig. 3.11a). Endothelial cytoplasm contains scant mitochondria, numerous micropinocytotic vesicles and occasional Weibel–Palade bodies (Fig. 3.11b–d).

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Fig. 3.9 Synaptic organization of the rat carotid body (CB). a Detail of the cell junction between two glomus cells illustrating zones of close membrane appositions that represent gap junctions (arrowheads) and the synaptic-like somato-somatic contacts (arrows). Note the numerous small clear vesicles and a few large dense-cored vesicles at the periphery of the contacting cells. b Electron micrograph showing a sensory nerve ending (ne) making a synaptic contact (arrows) with a glomus cell (G). Note numerous mitochondria (M) in both glomus cell cytoplasm and nerve ending. N, cell nucleus. c An axon terminal (At) forming a symmetrical synaptic contact (arrow) with a glomus cell body containing the nucleus (N), flattened cisternae of rough endoplasmic reticulum (rER) and a large number of mitochondria (M). d An autonomic nerve fiber with characteristic varicosities (V) containing numerous dense-cored vesicles and mitochondria in the vicinity of a glomus cell (G). Scale bars = 1 μm. Panels A, C and D reproduced from Atanasova et al. (2011) with permission from Biomedical Reviews

These capillaries are closely associated with the cell clusters. Despite their morphological similarity, the fenestrated capillaries of the CB are not true sinusoids. They rather resemble the fenestrated capillaries of the adrenal medulla and other endocrine glands, mediating the characteristic hyperpermeability state in the CB. Type II capillaries are mostly straight, typically thinner (6–12 μm in diameter) and continuous. They are covered by pericytes and do not make contacts with cell clusters (Fig. 3.11e, f). The glomus cells are separated from the capillary endothelium by a “perisinusoidal” space containing collagen and are lined on both sides by a basement membrane applied to the contiguous walls of glomus and endothelial cells (Morgan et al. 1975). Collagen fibers are present not only in the “perisinusoidal” space but are

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Fig. 3.10 Nerve–nerve ending relationships in the rat carotid body (CB). a Electron micrograph showing typical axo-axonic synapses (arrows) in the rat CB. b, c Axo-axonic synaptic contacts (arrows). Note that the presynaptic profile contains a few mitochondria and groups of both small clear and occasional large dense-cored vesicles. d Ribbon synapses with the characteristic arrangement of electron dense structures (arrows) called synaptic body or ribbon in the presynaptic bouton apposing the postsynaptic terminal. Scale bars = 1 μm. Panels A, C, D reproduced from Atanasova et al. (2011) with permission from Biomedical Reviews

also found to a variable extent between the glomus cells. Bundles of myelinated and unmyelinated axons are frequently seen in “perisinusoidal” and intercellular spaces. Compliance with Ethical Standards This study was partially funded by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01. The authors declare no conflict of interest. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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Fig. 3.11 Ultrastructure of capillaries in the rat carotid body (CB). a A type I capillary closely associated with the cell clusters of glomus cells (G). Its thin wall is formed by a fenestrated endothelium containing the nucleus (N) and an attenuated part with short microvilli, lies on a basal lamina and is partially covered by a pericyte (P). b The endothelial cytoplasm of a fenestrated capillary which is closely associated with glomus cells (G) contains scant mitochondria, a few Weibel– Palade bodies and numerous micropinocytotic vesicles. c The tiny pores (arrow) of the fenestrated capillary endothelium are spanned by a diaphragm. Er, erythrocyte. d The capillary is separated by a continuous basal lamina (BL) from the glomus cell (G) clusters. e, f Cross-section profiles of continuous type II capillaries showing the thick portion of the endothelial cytoplasm (E) and pericytes (P) investing them. Note the “perisinusoidal” space filled with collagen fibers that isolates the capillary endothelium from glomus cells. Scale bars = 1 μm. Panels A, B, E and F reproduced from Atanasova et al. (2011) with permission from Biomedical Reviews

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Pardal R, Ortega-Sáenz P, Durán R, Platero-Luengo A, López-Barneo J (2010) The carotid body, a neurogenic niche in the adult peripheral nervous system. Arch Ital Biol 148:95–105 Platero-Luengo A, González-Granero S, Durán R, Díaz-Castro B, Piruat JI, García-Verdugo JM, Pardal R, López-Barneo J (2014) An O2 -sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia. Cell 156:291–303 Porzionato A, Macchi V, Guidolin D, Parenti A, Ferrara SD, De Caro R (2005) Histopathology of carotid body in heroin addiction. Possible chemosensitive Impairment. . Histopathology 46:296–306 Schäfer D, Seidl E, Acker H, Keller HP, Lübbers DW (1973) Arteriovenous anastomoses in the cat carotid body. Z Zellforsch Mikrosk Anat 142:515–524 Schaper A (1892) Beiträge zur Histologie der Glandula carotica. Arch Mikrosk Anat 40:287–320 Schulz SA, Wöhler A, Beutner D, Angelov DN (2016) Microsurgical anatomy of the human carotid body (glomus caroticum): features of its detailed topography, syntopy and morphology. Ann Anat 204:106–113 Seidl E (1975) On the morphology of the vascular system of the carotid body of cat and rabbit and its relations to type I cells. In: Purves MJ (ed) The peripheral arterial chemoreceptors. Cambridge University Press, London, pp 293–329 Smith PG, Mills E (1976) Autoradiographic identification of the terminations of petrosal ganglion neurons in the cat carotid body. Brain Res 113:174–178 Tse A, Yan L, Lee AK, Tse FW (2012) Autocrine and paracrine actions of ATP in rat carotid body. Can J Physiol Pharmacol 90:705–711 Verna A (1975) Observations on the innervation of the carotid body of the rabbit. In: Purves MJ (ed) Peripheral arterial chemoreceptors. Cambridge University Press, Oxford, pp 75–97 Verna A (1979) Ultrastructure of the carotid body in the mammals. Int Rev Cytol 60:271–330 Verna A (1997) The mammalian carotid body: morphological data. In: González C (ed) The carotid body chemoreceptors. Landes Bioscience, Austin, pp 1–29 Verna A, Barets A, Salat C (1984) Distribution of sympathetic nerve endings within the rabbit carotid body: a histochemical and ultrastructural study. J Neurocytol 13:849–865 Wang Z-Y, Bisgard GE (2002) Chronic hypoxia-induced morphological and neurochemical changes in the carotid body. Microsc Res Tech 59:168–177 Watzka M (1943) Die Paraganglien. Handbuch der mikroskopischen Anatomie, Band VI. Berlin, Springer

Chapter 4

Structural Plasticity of the Carotid Body

Abstract The mammalian carotid body (CB) exhibits considerable plasticity of its structure during development and aging and as a consequence of environmental, metabolic and inflammatory stimuli. The structural changes during maturation include an enlargement of the total and vascular volume of the CB. Conversely, aging results in a reduction in the number and volume of glomus cells with progressive cellular degeneration and an apparent increase in the surrounding connective tissue. Age-related structural alterations are similar to those during chronic hypoxia. Longterm hypoxic exposure and sodium nitrate treatment enlarge several-fold the size of the rat CB causing glomus cell hypertrophy and hyperplasia, and evoke changes in its vascular structure, inducing marked vasodilation and neovascularization. In humans, such structural CB adaptation responses to prolonged hypoxia occur during acclimatization to high altitudes. On the other hand, the hyperoxic CB is significantly smaller than those of age-matched normoxic controls. Morphological alterations in the CB in both hypertensive animals and humans are characterized by a slightly enlarged parenchyma without apparent vascular expansion and/or dilation. The CB structural plasticity depends on the existence of a population of multipotent neural crest-derived stem cells, which are activated during hypoxia to proliferate and differentiate into new both neuronal (glomus) and vascular cell types. Keywords Aging · Cell hypertrophy · Hyperplasia · Glomus cell volume · Neovascularization · Neural crest-derived stem cells · Structural plasticity · Vasodilation

The CB shows a remarkable structural plasticity, undergoing morphological and functional modifications during development, aging and as a consequence of environmental stimuli. Functional maturation of the CB in the postnatal period partially relies on structural alterations which include developmental changes in morphometric parameters such as glomic tissue volume, number of cells, proportion of different cell types, vascular compartment and synaptic organization (De Caro et al. 2013). In addition, a gradual increase in hypoxic chemosensitivity develops during maturation. There is a large body of evidence suggesting that the chemosensory transduction and © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_4

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transmission of the hypoxic stimulus evoke considerable plasticity of the CB structure and function in response to environmental, metabolic and inflammatory stimuli (reviewed in Kumar and Prabhakar 2012). Nonetheless, the identity of the signal that triggers CB morphological changes upon hypoxia remains elusive. A recent major breakthrough in CB physiology is the realization that autocrine and paracrine interactions between glomus and other cell types mediate CB plasticity but the detailed cellular and molecular mechanisms by which these affect the hypoxic chemosensitivity still remain to be elucidated. It has lately been revealed that the CB structural plasticity depends on the existence of a population of multipotent adult neural crestderived stem cells, which are quiescent in normoxia and activated during hypoxia to proliferate and differentiate into new glomus cells, as well as vascular smooth muscle and endothelial cells (discussed in Chap. 9).

4.1 Age-Related Morphological Changes of the Carotid Body Knowledge of the mechanisms of CB maturation is essential to understand how various environmental stimuli such as hypoxia or hyperoxia may disrupt this process. The main structural changes during maturation include an increased total and vascular volume of the CB in cats and human infants, variations in the relative proportions of glomus and sustentacular cell populations and elevated numbers of dense-cored granules in glomus cells in newborn rats, as well as an increase in afferent (mitochondriarich) and a decrease in efferent (vesicle-rich) nerve endings and an increased number of synapses between them per unit area (reviewed by De Caro et al. 2013). On the other hand, the aging process causes natural changes in the body and is characterized by a decline in several physiological functions resulting in a reduced capability to maintain homeostasis. In line with this, while aging, the mammalian CB undergoes substantial morphological and physiological changes leading to a reduction in homeostatic capacity (reviewed in Di Giulio 2018; Di Giulio et al. 2023). In older animals, the CB is enlarged mostly due to a significant increase in the extracellular matrix and a progressive proliferation of sustentacular cells while the chemoreceptor glomus cells show a concomitant decrease (Fig. 4.1) with fewer synaptic junctions among them (Di Giulio et al. 2003, 2009, 2023). In humans, aging results in a reduction in the number and volume of glomus cells with progressive cellular degeneration and an apparent increase in the surrounding connective tissue (Fig. 4.2) (Di Giulio et al. 2003; Lazarov et al. 2009). CBs from human males aged over 80 years showed a large amount of fibrous tissue replacing lobule tissue, lymphocyte infiltration and occasional grouping of autolytic glomus cells (Hurst et al. 1985). In subjects over the age of 50, a chronic carotid glomitis appears, in which aggregates of lymphocytes and fibrosis of the lobules occur throughout CB tissue, glomus cells are dehydrated with a profound vacuolization, a shrinking nucleus and lipofuscin accumulation (Hurst et al. 1985). In fact, all these age-related structural

4.2 Morphological Changes in the Hypoxic and Hyperoxic Carotid Body

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Fig. 4.1 Morphology of the aging rat carotid body (CB). Representative photomicrographs of AZAN-stained sections from the CB of a one-month-old rat (a), three-month-old rat (b), 12-monthold rat (c) and 18-month-old rat (d). Note that the glomic lobules at juvenile age are compact and separated by thin septa of connective tissue filled with a large number of fine blood vessels and nerve fibers. In the adult CB, the parenchyma is enlarged due to an increase in the extracellular matrix, dilated blood vessels and a progressive proliferation of sustentacular cells while the glomus cells in older animals diminish in number with increasing age. Scale bars = 50 μm

alterations are consistent with and similar to those during chronic hypoxia. However, the reduced cellularity and number of vesicles containing neurotransmitters suggest that the aged CB becomes less responsive to hypoxia. Nonetheless, it is likely that hypoxia and aging share a common background consisting of deficient oxygen tissue supply, mitochondrial dysfunction and a limited ability to deal with increased cellular oxidative stress (Di Giulio et al. 2023).

4.2 Morphological Changes in the Hypoxic and Hyperoxic Carotid Body The functional role of the CB in homeostasis is almost entirely defined by its response to hypoxia (Kumar and Prabhakar 2012). Hypoxia evokes both acute and chronic physiological responses. Acute responses to hypoxia occur within seconds

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Fig. 4.2 Morphology of the aging human carotid body (CB). H&E-stained histological images of the CB in an infant (a, b), an adult (c, d) and in elderly (e, f) individuals. Note that the infantile CB possesses more compact lobules that are surrounded by less prominent connective tissue stroma. In the adult CB, the glomeruli are less compact and the interlobular connective tissue is more intensely vascularized. Progressive degenerative changes with a lower number of glomus cells and an apparent increase of the surrounding connective tissue are observed in the aging CB. Scale bars = 200 μm in a, c, e, and 100 μm in b, d, f

and involve the modification of preexisting proteins leading to depolarization of glomus cells (described in Chap. 5). On the other hand, chronic hypoxia induces changes in gene expression, leading to profound morphological alterations in the CB. Hollinshead (1945) was the first to describe cytological modifications of CB cells after a severe and sustained hypoxia, and similar observations were also made by

4.2 Morphological Changes in the Hypoxic and Hyperoxic Carotid Body

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means of electron microscopy as early as the end of the fifties by Hoffman and Birrel (1958). A number of subsequent studies have described the structural alterations in the mammalian CB upon exposure to sustained hypoxia. Generally, the long-term hypoxic exposure enlarges several-fold the size of the rat CB (Lahiri et al. 2000; Kusakabe et al. 2005) causing glomus cell hypertrophy and hyperplasia (Laidler and Kay 1975; McGregor et al. 1984; Wang and Bisgard 2002; Schamel et al. 2016). Conversely, no definite evidence concerning alterations in glomus cell volume in rats exposed to short-term (for up to 24 h) hypoxia has been provided so far (Kato et al. 2010). On the other hand, no change or even a decrease in the covering of glomus cells by the sustentacular cells has been described in the CB of chronically hypoxic rats (Kusakabe et al. 1993). This alteration appears to increase the potential area available for gap junction connections between the glomus cells, which have been shown to enhance glomus cell sensitivity (Eyzaguirre and Abudara 1996). Although no structural modifications of the sustentacular cells have been observed under such conditions, it has been suggested that hypoxic adaptation of the rat CB involves proliferation of these cells as well (McGregor et al. 1984; Wang and Bisgard 2002). In addition, systemic hypoxia changes the CB vascular structure, inducing marked (tenfold in rats) vasodilation (Kusakabe et al. 2005; Schamel et al. 2016) and the growth of new blood vessels (Laidler and Kay 1975; Wang and Bisgard 2002). On the other hand, no variations of distribution of arterioles and capillaries within the tissue of hypoxic CB have been identified (Schamel et al. 2016). It is likely that the vascular changes occurring during chronic hypoxia are due to a vascular remodeling and proliferation of endothelial cells of existing blood vessels (Chen et al. 2001). The increase in vascularity of the hypoxic CB may be a mechanism to increase blood flow and thus of oxygen transport to a hypoxic organ with increased metabolic activity (Laidler and Kay 1975). In fact, the profuse angiogenesis facilitates irrigation of the newly generated chemoreceptor elements. However, the effects of prolonged hypoxia on CB morphology in rats are reversible after reoxygenation (Kusakabe et al. 2004). This phenomenon would be an argument in favor of increasing the volume of CB by dilation rather than proliferation of glomus cells during the period of the chronic hypoxia. Remarkably, the ability of the CB to grow in response to sustained hypoxia is a property that makes it unique among other organs of the adult peripheral nervous system. Lately, we have examined the effect of sodium nitrite-induced hypoxia on the CB morphology and have found that the structural changes following such treatment are similar to these detected in rats in the first days of chronic hypoxia (Atanasova and Lazarov 2016). We observe a prominent increase in the CB size mostly due to glomus cell hypertrophy and initial vasodilation and also, to some extent, to extracellular matrix expansion (Fig. 4.3). It seems, however, that acute nitrite infusion does not alter the number of blood vessels, and the pronounced neovascularization typical of chronic hypoxia CB adaptation is not a common trait in hypoxic stress conditions. In humans, such structural CB adaptation responses to prolonged hypoxia occur during long-term acclimatization to high altitudes (Arias-Stella and Valcarcel 1976) or pathologically in sea-level patients with emphysema (Arias-Stella and Valcarcel

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Fig. 4.3 Morphological changes in the rat carotid body (CB) after sodium nitrite-induced hypoxia. a Conventional H&E-stained section from a control normoxic (N) CB. b CB morphology 1 h after sodium nitrite treatment. Note the slight enlargement of its size and the compact glomic clusters which are surrounded by slightly distended blood vessels (BV). c 5 h following hypoxic exposure, the CB size is significantly enlarged and a marked vasodilation is observed in injected animals. It persists one day later (d) and is highest five days after the substance administration (e). f 20 days following the treatment, the expanded vasculature in the recovered CB decreases to that of untreated controls, but the parenchyma is increased to some extent with persistent glomus cells hypertrophy. Scale bars = 50 μm

4.3 Structural Alterations of Carotid Bodies in Hypertension

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1973) as well as in patients suffering from systemic hypertension and/or cardiopulmonary diseases with concomitant hypoxemia (reviewed in Heath and Smith 1985; Lazarov and Atanasova 2022). These include intense vacuolization and proliferation of the glomus cells, and hypertrophy of the CB parenchymal tissue with concomitant, progressive chemoreceptor insensitivity. On the other hand, a substantial body of evidence shows that chronic intermittent hypoxia has no significant effect either on the number and volume of glomus cells or the total volume of the CB compared to controls (Peng et al. 2003). The increased size of blood vessels found by Del Rio et al. (2011) has been associated with an upregulation of the vascular endothelial growth factor. Collectively, these studies suggest that changes in the number of glomus cells or the morphology of the CB do not account for the effects of intermittent hypoxia on the CB function (reviewed by Prabhakar et al. 2015). Prolonged exposure to hyperoxia during development also alters the CB morphology and developing respiratory control system (Bavis et al. 2013). Indeed, the CBs of hyperoxia-treated rats are significantly smaller than those of age-matched controls due to decreased cell division (Erickson et al. 1998; Wang and Bisgard 2005), and moreover, fewer unmyelinated axons are observed in the carotid sinus nerve (Erickson et al. 1998).

4.3 Structural Alterations of Carotid Bodies in Hypertension In both hypertensive animals and humans, the volume of the CB parenchyma and its histological structure are influenced by chronically elevated systemic arterial blood pressure (Smith et al. 1984a, b). In particular, in spontaneously hypertensive rats (SHR) of the Okamoto strain, the CB is 4–5 times larger in size than in age-matched normotensive rats of a common Wistar strain (Habeck et al. 1981; Honig et al. 1981). In addition, intimal damage and proliferation are seen in the CB arteries in hypertensive rats (Smith et al. 1984b). Our morphometric and stereological analyses have also shown striking differences in the CB structural plasticity in control and hypertensive rats. The data reconfirm prior findings that the CB parenchyma is larger in size in SHR than in normotensive Wistar rats (NWR; Atanasova and Lazarov 2014). In fact, we have observed that the hypertensive CB in rats could slightly enlarge its parenchyma with spreading vasodilation, but no apparent vascular expansion and an increase in extracellular matrix (Fig. 4.4). Of the tested CB, an almost twofold increase in their total volume is found in hypertensive animals when compared to normotensive controls (Fig. 4.5a). In addition, the mean cell number in the normotensive CB is estimated to be 11,527 ± 172.7 cells while the hypertensive CB is estimated to contain 23,900 ± 132.8 cells (Fig. 4.5b). Furthermore, the total length of the CB capillary network in SHR is 10.671 m ± 0.189 versus 5.357 m ± 0.16 in NWR (Fig. 4.5c). We have also found a statistically significant difference (p < 0.001) in the globally

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average cross-sectional area of a capillary in the CB of NWR and SHR (57.80 μm ± 0.4472 vs. 21 μm ± 0.5774) (Fig. 4.5d). In contrast, no difference in size is observed between New Zealand genetically hypertensive and normotensive Wistar rats (Bee et al. 1989). This discrepancy could be explained in the context of CB size differences between different rat strains, or it might be due to random sampling variability. The observed CB morphology in SHRs is quite similar to that in rats exposed to chronic hypercapnic hypoxia (Kusakabe et al. 2005). Nonetheless, no changes in the CB size of renal hypertensive rabbits have been registered (Angell-James et al. 1985). Initial evidence suggests a link between systemic hypertension and CB enlargement in humans as well although its morphological alterations are observed at later stages of the disease. Indeed, CB hyperplasia has been described in patients with primary hypertension (Smith et al. 1984a; Habeck 1986). However, Habeck (1991) subsequently has shown that the CB is normal in size in some young patients with essential hypertension and, moreover, he does not find CB enlargement in renal hypertensive patients. In our opinion, such changes in the CB general morphology could be considered to be a manner of its adaptation to hypertension. The structural

Fig. 4.4 Structural alterations of the hypertensive rat carotid body (CB). Light micrographs showing the CB morphology of control normotensive Wistar rats (NWR) at a low (a) and higher magnification (b). Note that the glomic clusters are separated by thin vascularized septa of connective tissue. c Overview of the CB in spontaneously hypertensive rats (SHR). d Higher magnification of the boxed area in c demonstrates the slightly enlarged CB parenchyma with dilated blood vessels (BV) and increased extracellular matrix. Scale bars = 100 μm in a, c, 50 μm in b, d

4.3 Structural Alterations of Carotid Bodies in Hypertension

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Fig. 4.5 Statistical comparison of the morphometric parameters of the carotid body (CB) in normotensive Wistar rats (NWR) and spontaneously hypertensive rats (SHR). a Box plots show that the total CB volume in hypertensive animals is statistically larger when compared to that in the normotensive control group (median, 0.015 mm3 vs. 0.005 mm3 , **p = 0.008). b Vertical bar graph represents the significantly increased total cell number in the hypertensive CB in comparison with normotensive CB (23,900 ± 132.8 vs. 11,527 ± 172.7, ***p < 0.001). Morphometric data are compared using the Shapiro–Wilk test. c Bar chart demonstrates that the length of the capillary network in SHR is twice the length in NWR (10.671 m ± 0.189 vs. 5.357 m ± 0.16, ***p < 0.001). d Bar graph showing that the sectional area occupied by blood vessels in NWR (57.80 μm ± 0.4472) is significantly increased (p < 0.001) in comparison with the value (21 μm ± 0.5774) in SHR. Mean ± S.E.M. values are shown; n = 5 per each group

changes in the CB are probably caused by another factor rather than hypertension per se because it is unlikely that blood pressure would remain high in a structure whose microvasculature is mostly composed of fenestrated capillaries closely associated with cell clusters (McDonald and Lurue 1983). In this respect, the recent results of Kato et al. (2012) suggest that the morphology of the CB is altered by the effect of sympathetic nerves, and thus, these changes could be ascribed to the increased sympathetic vasomotor tone under hypertensive conditions as in the case of hypercapnic hypoxic rats (Kumar and Prabhakar 2012). The hyperactivity and increased growth of the CB may be also consequent to atherosclerotic damage of its vasculature (Kumar 2012). More recently, it has been hypothesized that CB hyperexcitability is driven by its own sympathetic innervation (Felippe et al. 2023). Notwithstanding, the possibility that a hyperactive CB is the attributing cause of hypertension remains

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unconfirmed. In brief, it seems more likely that the morphological alterations in the CB are secondary to hypertension and, therefore, the connection between CB structure and hypertension needs further elucidation. In conclusion, it is evident from the above studies that the adult CB exhibits an extraordinary structural and functional plasticity, which is critical for its physiological adaptation to changing environments and pathological conditions. Compliance with Ethical Standards This study was financed in part by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01 and by the Bulgarian Ministry of Education and Science within the framework of the National Recovery and Resilience Plan of Bulgaria, Component “Innovative Bulgaria”, project No. BG-RRP-2.004-0006-C02 “Development of research and innovation at Trakia University in service of health and sustainable well-being”. The authors declare no conflict of interest. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

References Angell-James JE, Clarke JA, Daly MD, Taton A (1985) Respiratory and cardiovascular responses to hyperoxia, hypoxia and hypercapnia in renal hypertensive rabbit; role of carotid body chemoreceptors. J Hypertens 3:213–223 Arias-Stella J, Valcarcel J (1973) The human carotid body at high altitudes. Pathol Microbiol (Basel) 39:292–297 Arias-Stella J, Valcarcel J (1976) Chief cell hyperplasia in the human carotid body at high altitudes; physiologic and pathologic significance. Hum Pathol 7:361–373 Atanasova DY, Lazarov NE (2014) Expression of neurotrophic factors and their receptors in the carotid body of spontaneously hypertensive rats. Respir Physiol Neurobiol 202:6–15 Atanasova DY, Lazarov NE (2016) Morphological changes in the rat carotid body following acute sodium nitrite treatment. Respir Physiol Neurobiol 221:11–18 Bavis RW, Fallon SC, Dmitrieff EF (2013) Chronic hyperoxia and the development of the carotid body. Respir Physiol Neurobiol 185:94–104 Bee D, Barer G, Wach R, Pallot D, Emery C, Jones S (1989) Structure and function of the carotid body in New Zealand genetically hypertensive rats. Q J Exp Physiol 74:691–701 Chen J, Dinger B, Stensaas L, Fidone S (2001) Involvement of vascular endothelial growth factor (VEGF) in carotid body vascular remodeling induced by chronic hypoxia. FASEB J 15:A153 De Caro R, Macchi V, Sfriso MM, Porzionato A (2013) Structural and neurochemical changes in the maturation of the carotid body. Resp Physiol Neurobiol 185:9–19 Del Rio R, Munoz C, Arias P, Court FA, Moya EA, Iturriaga R (2011) Chronic intermittent hypoxiainduced vascular enlargement and VEGF upregulation in the rat carotid body is not prevented by antioxidant treatment. Am J Physiol Lung Cell Mol Physiol 301:L702–L711 Di Giulio C (2018) Ageing of the carotid body. J Physiol 596:3021–3027

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Di Giulio C, Cacchio M, Bianchi G, Rapino C, Di Ilio C (2003) Selected contribution: carotid body as a model for aging studies: is there a link between oxygen and aging? J Appl Physiol 95:1755–1758 Di Giulio C, Antosiewicz J, Walski M, Petruccelli G, Verratti V, Bianchi G, Pokorski M (2009) Physiological carotid body denervation during aging. Adv Exp Med Biol 648:257–263 Di Giulio C, Zara S, Mazzatenta A, Verratti V, Porzionato A, Cataldi A, Pokorski M (2023) Aging and the carotid body: a scoping review. Respir Physiol Neurobiol 313:104063 Erickson JT, Mayer C, Jawa A, Ling L, Olson EB Jr, Vidruk EH, Mitchell GS, Katz DM (1998) Chemoafferent degeneration and carotid body hypoplasia following chronic hyperoxia in newborn rats. J Physiol 509:519–552 Eyzaguirre C, Abudara V (1996) Reflection on the carotid nerve sensory discharge and coupling between glomus cells. Adv Exp Med Biol 10:159–167 Felippe ISA, Zera T, da Silva MP, Moraes DJA, McBryde F, Paton JFR (2023) The sympathetic nervous system exacerbates carotid body sensitivity in hypertension. Cardiovasc Res 119:316– 331 Habeck JO (1986) Morphological findings at the carotid bodies of humans suffering from different types of systemic hypertension or severe lung diseases. Anat Anz 162:17–27 Habeck JO (1991) Peripheral arterial chemoreceptors and hypertension. J Auton Nerv Syst 34:1–7 Habeck JO, Honig A, Pfeiffer C, Schmidt M (1981) The carotid bodies in spontaneously hypertensive (SHR) and normotensive rats—a study concerning size, location and blood supply. Anat Anz 150:374–384 Heath D, Smith P (1985) The pathology of the carotid body and sinus. Edward Arnold, London Hoffman H, Birrel JHW (1958) The carotid body in normal and anoxic states: an electron microscopic study. Acta Anat (Basel) 32:297–311 Hollinshead WH (1945) Effects of anoxia upon carotid body morphology. Anat Rec 92:255–261 Honig A, Habeck JO, Pfeiffer C, Schmidt M, Huckstorf C, Rotter H, Eckermann P (1981) The carotid bodies of spontaneously hypertensive rats (SHR): a functional and morphologic study. Acta Biol Med Ger 40:1021–1030 Hurst G, Heath D, Smith P (1985) Histological changes associated with ageing of the human carotid body. J Pathol 147:181–187 Kato K, Yamaguchi-Yamada M, Yamamoto Y (2010) Short-term hypoxia increases tyrosine hydroxylase immunoreactivity in rat carotid body. J Histochem Cytochem 58:839–846 Kato K, Wakai J, Matsuda H, Kusakabe T, Yamamoto Y (2012) Increased total volume and dopamine β-hydroxylase immunoreactivity of carotid body in spontaneously hypertensive rats. Auton Neurosci 169:49–55 Kumar P (2012) The carotid body in cardiovascular disease: more chicken and egg than horse and cart? J Physiol 590:4123 Kumar P, Prabhakar NR (2012) Peripheral chemoreceptors: function and plasticity of the carotid body. Compr Physiol 2:141–219 Kusakabe T, Powell FL, Ellisman MH (1993) Ultrastructure of the glomus cells in the carotid body of chronically hypoxic rats: with a special reference to the similarity of the amphibian glomus cells. Anat Rec 237:220–227 Kusakabe T, Hirakawa H, Oikawa S, Matsuda H, Kawakami T, Takenaka T, Hayashida Y (2004) Morphological changes in the rat carotid body 1, 2, 4, and 8 weeks after the termination of chronically hypocapnic hypoxia. Histol Histopathol 19:1133–1140 Kusakabe T, Matsuda H, Hayashida Y (2005) Hypoxic adaptation of the rat carotid body. Histol Histopathol 20:987–997 Lahiri S, Rozanov C, Cherniack NS (2000) Altered structure and function of the carotid body at high altitude and associated chemoreflexes. High Altitude Med Biol 1:63–74 Laidler P, Kay JM (1975) A quantitative morphological study of the carotid bodies of rats living at a stimulated altitude of 4300 metres. J Pathol 117:183–191 Lazarov N, Atanasova D (2022) The human carotid body and its role in ventilatory acclimatization to hypoxia. Acta Morphol Anthropol 29:63–68

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Lazarov N, Reindl S, Fischer F, Gratzl M (2009) Histaminergic and dopaminergic traits in the human carotid body. Respir Physiol Neurobiol 165:131–136 McDonald DM, Lurue DT (1983) The ultrastructure and connections of blood vessels supplying the rat carotid body and carotid sinus. J Neurocytol 12:117–153 McGregor KH, Gil J, Lahiri S (1984) A morphometric study of the carotid body in chronically hypoxic rats. J Appl Physiol 57:1430–1438 Peng YJ, Overholt JL, Kline D, Kumar GK, Prabhakar NR (2003) Induction of sensory long-term facilitation in the carotid body by intermittent hypoxia: implications for recurrent apneas. Proc Natl Acad Sci USA 100:10073–10078 Prabhakar NR, Peng YJ, Kumar GK, Nanduri J (2015) Peripheral chemoreception and arterial pressure responses to intermittent hypoxia. Compr Physiol 5:561–577 Schamel A, Chaouti A, Douma M, Sabour B (2016) Morphological and neurochemical plasticity of the carotid body after long-term hypoxia: vascular and cellular involvement, morphometric study in Meriones shawi rats. Der Pharma Chem 8:82–98 Smith P, Jago R, Heath D (1984a) Anatomical variation and quantitative histology of the normal and enlarged carotid body. J Physiol 137:287–304 Smith P, Jago R, Heath D (1984b) Glomic cells and blood vessels in the hyperplastic carotid bodies of spontaneously hypertensive rats. Cardiovasc Res 18:471–482 Wang Z-Y, Bisgard GE (2002) Chronic hypoxia-induced morphological and neurochemical changes in the carotid body. Microsc Res Tech 59:168–177 Wang Z-Y, Bisgard GE (2005) Postnatal growth of carotid body. Respir Physiol Neurobiol 149:181– 190

Chapter 5

Mechanisms of Chemosensory Transduction in the Carotid Body

Abstract The mammalian carotid body (CB) is a polymodal chemoreceptor, which is activated by blood-borne stimuli, most notably hypoxia, hypercapnia and acidosis, thus ensuring an appropriate cellular response to changes in physical and chemical parameters of the blood. The glomus cells are considered the CB chemosensory cells and the initial site of chemoreceptor transduction. However, the molecular mechanisms by which they detect changes in blood chemical levels and how these changes lead to transmitter release are not yet well understood. Chemotransduction mechanisms are by far best described for oxygen and acid/carbon dioxide sensing. A few testable hypotheses have been postulated including a direct interaction of oxygen with ion channels in the glomus cells (membrane hypothesis), an indirect interface by a reversible ligand like a heme (metabolic hypothesis), or even a functional interaction between putative oxygen sensors (chemosome hypothesis) or the interaction of lactate with a highly expressed in the CB atypical olfactory receptor, Olfr78, (endocrine model). It is also suggested that sensory transduction in the CB is uniquely dependent on the actions and interactions of gaseous transmitters. Apparently, oxygen sensing does not utilize a single mechanism, and later observations have given strong support to a unified membrane model of chemotransduction. Keywords Acidosis · Chemotransduction · Glomus cells · Glucose sensing · Hypercapnia · Hypoxia · Ion channels · Oxygen sensing · pH sensing

It is now generally accepted that glomus cells are the initial site of chemoreceptor transduction as they are electrically excitable neurosecretory cells with various oxygen-regulated ion channels in their plasmalemma. Due to their size, glomus cells possess high membrane resistance and low capacitance that enable them to function as powerful amplifiers, capable of converting small currents (transmembrane ion channels fluxes) into large and rapid changes in the membrane potential necessary to open voltage-gated ion channels (López-Barneo 2022). Besides, they contain numerous cytosolic granules with a rich diversity of neurotransmitters released upon exposure to chemostimuli. The mechanisms by which glomus cells detect changes in blood chemical levels and how these changes lead to transmitter release have remained obscure © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_5

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for several decades until technological advances facilitated physiological measurements of these. After the first report on the identification of oxygen-sensitive potassium channels in rabbit glomus cells (López-Barneo et al. 1988), subsequent studies on isolated and cultured glomus cells by single-cell microfluorimetry, patch-clamp and amperometric recording techniques have revealed that they express a broad range of specific voltage- and ligand-gated ion channels, as well as background potassium channels (Peers 1990; Stea and Nurse 1991; Wyatt and Peers 1995; Buckler 1997; López-López et al. 1997; Pardal et al. 2000). On the other hand, some researchers believe that the oxygen-sensitive membrane electrical events in glomus cells are not directly involved in chemotransduction. Therefore, although the cellular responses to low oxygen (hypoxia) are relatively well characterized, the molecular mechanism(s) underlying chemosensory transduction are not well understood (for recent reviews, refer to Peers et al. 2010; Kumar and Prabhakar 2012). Most likely acute oxygen sensing does not utilize a single mechanism, although the latter would not be too many given the limited manners of physico-chemical interactions between oxygen and biomolecules. A number of testable hypotheses and several possible mechanisms have been postulated, including a direct interaction of oxygen with ion channels in the glomus cells (membrane hypothesis), an indirect one by a reversible ligand like a heme or a related protein (either cytosolic or of a mitochondrial origin) as oxygen sensors (metabolic hypothesis), or even a functional interaction between putative oxygen sensors (chemosome hypothesis) or the interaction of lactate with Olfr78, an atypical olfactory receptor highly expressed in the CB (endocrine model). Biochemical or biophysical events then trigger the transmitter release for a subsequent activation of the afferent nerve ending. Furthermore, emerging evidence suggests that sensory transduction in the CB is uniquely dependent on the actions and interactions of gaseous transmitters, which act in concert with key effector molecules, in particular potassium ion channels (Prabhakar and Peers 2014). Notwithstanding this, pivotal questions regarding the nature of the currently proposed oxygen sensor molecules, and the mechanisms of interaction between the sensors and the downstream effectors still remain unanswered.

5.1 Ion Channels and Calcium Response A crucial objective in elucidating the complex mechanisms of chemotransduction is to determine the specific ion channels involved in glomus cell electric signaling. As noted above, glomus cells express two principal types of Ca2+ channels (lowand high-threshold) and various subtypes of membrane K+ channels such as the classical voltage-dependent K+ channels, noninactivating Ca2+ -dependent K+ channels, particularly HERG, BK, KATP and Kv channels, and subunits of the voltageindependent TASK channel family, though their proportion largely varies among cells in different animal species (Peers and Buckler 1995; Buckler 1997; Buckler et al. 2000; Overholt et al. 2000; López-Barneo et al. 2001, 2004; Kim et al. 2011). Even though these channels operate in different ranges of membrane potential, they

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may act in concert within a single cell to both induce and maintain the depolarization. Thus, it is probable that all of them contribute to the sensitivity of CB cells to acute hypoxia (López-Barneo et al. 2016a) though it has recently been revealed that the enhanced membrane response to it is not due to an increase in surface TASK channels (Matsuoka et al. 2022). In addition, electrophysiological studies have identified a few other oxygen-sensitive or nonselective ion channel classes in glomus cells in few species, perhaps rabbit only, including L- and N-type voltage-gated Ca2+ channels, and voltage-gated Na+ and Cl− currents (Stea and Nurse 1989; e Silva and Lewis 1995; Carpenter and Peers 1997, 2001) although their functional role is not necessarily well understood. These observations have given strong support to a unified membrane model of chemotransduction that explains the main sensory function of the entire CB. According to the current belief, in response to low oxygen or increased CO2 /H+ levels, K+ channels in the plasma membrane of glomus cells close, which leads to membrane depolarization, opening of voltage-gated calcium channels and an influx of calcium ions through them. This results in the elevation of the intracellular calcium concentration, an enhanced secretion of excitatory neurotransmitter(s) from granules within the glomus cells and a consequent activation of the adjacent afferent nerve terminals of the carotid sinus nerve (CSN) which, in turn, relay the information to the cardiorespiratory center in the brainstem that helps restore the condition (for references see González et al. 1994). This model of chemoreceptor activation, studied preferentially in rodents, has been shown to be also applicable to the human CB (López-Barneo et al. 2016b). Interestingly, the aforementioned responses are totally abolished by Ca2+ channel blockers or the removal of extracellular Ca2+ (for review, see López-Barneo 1996). The mechanisms of calcium signaling in glomus cells remain to be determined. It is known that glomus cells do have intracellular calcium stores from which its release may be evoked by receptor-activated signaling pathways. If so, then the modulation of membrane currents only serves a secondary role. Alternatively, there is evidence that the sustentacular cells which are nonexcitable; i.e., they lack voltagegated Na+ and Ca2+ channels and exhibit only a small high-threshold outward K+ current (Duchen et al. 1988), are activated by ATP released from glomus cells through P2Y metabotropic receptors, which in turn induces calcium release from internal stores (Xu et al. 2003; Murali and Nurse 2016). However, it is thought that calcium release from the internal stores does not contribute significantly to the neurosecretory response to chemostimuli (Vicario et al. 2000). It is possible that these play an important role in calcium signaling in vivo where the glomus cells are exposed to a variety of autocrine, paracrine and neurocrine factors (Nurse 2014). It is presumed that the primary mechanism of calcium ion extrusion from the cell is by a plasma membrane Ca2+ /H+ ATPase (Buckler 2015) although this remains to be confirmed. The role of mitochondria in calcium buffering has not yet been studied either.

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5.2 Sensing of Chemicals in Arterial Blood by Glomus Cells Glomus cells are polymodal arterial chemoreceptors, activated not only by hypoxia but also by other blood-borne stimuli, most notably hypercapnia, extracellular acidosis and hypoglycemia (reviewed by González et al. 1994; López-Barneo et al. 2008; Kumar and Prabhakar 2012). Chemotransduction mechanisms are best described for oxygen sensing and acid/carbon dioxide sensing.

5.2.1 Oxygen Sensing Oxygen (O2 ) sensing is of a paramount importance for the survival and adaptation of living organisms, particularly mammals, to changing environmental or physiological conditions (López-Barneo et al. 2004; Lahiri et al. 2006). It is well established that sensory discharge under normoxia is incredibly low but increases dramatically in response to hypoxia. Although oxygen-sensing cells and tissues have been known for decades, the nature of the basic O2 -sensing mechanisms in the CB has remained obscure until recently. As mentioned earlier, several appealing hypotheses (the lactate–Olfr 78 hypothesis, the AMP-activated protein kinase hypothesis, mitochondrial energy metabolism, mitochondria-generated thermal transients in microdomains, redox signaling, gasotransmitters, etc.) have been proposed lately to explain how changes in blood pO2 lead to modulation of K+ channel function. Besides, an earlier concept, the so-called metabolic hypothesis, states that the oxygen sensing is linked to oxidative phosphorylation metabolism in glomus cells before the detection that they are excitable and contain oxygen-sensitive membrane ion channels. However, recent advances in the field have given different support to all these concepts (for updated reviews, see López-Barneo et al. 2016a; Rakoczy and Wyatt 2018). Another major advance in the field of oxygen sensing has been the discovery of O2 -regulated transcription factors on which the expression of hypoxia-sensitive genes depends (see Chap. 9, and López-Barneo et al. 2001).

5.2.1.1

The Membrane Hypothesis

According to this long-established hypothesis, ion protein channels are central to the oxygen-sensing capabilities of glomus cells. The current model of chemoreception states that hypoxia induces the inhibition of K+ channels, mainly of the TASK subfamily, consequently leading to or facilitating glomus cell membrane depolarization, voltage-gated Ca2+ influx through L-type Ca2+ channels, an elevation in the cytosolic Ca2+ concentration and release of excitatory transmitters, which in turn increase the discharge frequency of the nerve endings of chemosensory neurons (González et al. 1994; Peers and Buckler 1995; López-Barneo 2003; Prabhakar 2006). These steps are a key requirement in the chemosensory transduction process

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and find a broad experimental support in the rabbit (Duchen et al. 1988; LópezBarneo et al. 1988; Hescheler et al. 1989), rat (Peers 1990; Buckler 1997), cat (Chou and Shirahata 1996), and mouse (Yamaguchi et al. 2004). Although this model explains the basic sensory function of the CB, it is difficult to test experimentally with biochemical methods given the small size of the CB and the fact that responsiveness to hypoxia is easily lost in dialyzed patch-clamped glomus cells (López-Barneo and Ortega-Sáenz 2022). In addition, since the process of hypoxic inhibition of ion channels cannot be considered as confined to the plasma membrane, and given the diversity of channel subtypes reported to be oxygen-sensitive, the idea that K+ channels may themselves act as sensors for molecular oxygen does not receive much support at present. Instead, the attention has recently turned back to the resurgence of the long-standing concept in CB transduction, the so-called mitochondrial hypothesis.

5.2.1.2

The Mitochondrial Hypothesis

This hypothesis suggests that mitochondria might be the primary site for oxygen sensing in glomus cells because these organelles consume almost all the available oxygen and since hypoxia depolarizes them and affects mitochondrial cytochromes (Biscoe et al. 1989). It is known that glomus cells have mitochondria with specialized metabolic and electron transport chain properties. Most likely the mitochondrial electron transport chain also plays a key role since its inhibitors such as rotenone mimic the actions of hypoxia and occlude its further effects (Cabello-Rivera et al. 2022). These changes in mitochondrial function induced by hypoxia lead to calcium release from the complex of mitochondria-endoplasmic reticulum (Duchen and Biscoe 1992). The mitochondrial model of CB oxygen sensing has lost much support after the discovery of oxygen-regulated K+ channels and the experimental demonstration that Ca2+ ions needed for glomus cell secretion in hypoxia enter the cell via plasmalemmal voltage-gated Ca2+ channels, as indicated in the previous section. On the other hand, in support of the mitochondrial hypothesis is the existence of oxygen-sensitive background K+ channels, which appear to be modulated by mitochondrial uncouplers (i.e., inhibitors of the electron transport chain) and ATP (López-Barneo et al. 2008). Accordingly, hypoxia leads to Ca2+ release from mitochondria (Duchen and Biscoe 1992) and, moreover, they generate signals that alter membrane ion conductances and increase the afferent activity of the CSN (Mills and Jöbsis 1972). It has also been suggested that mitochondrial dysfunctions might result in metabolic alterations leading to changes in membrane ion channels that could modulate glomus cell activity. In search of an upstream sensor(s), the CB researchers assume that a heme and/or a redox-sensitive protein in the glomus cells is the oxygen sensor, and a biochemical event associated with the heme protein triggers the transduction cascade (reviewed in Prabhakar and Overholt 2000). However, even in rats, there are still insufficient data to support strongly the concept that heme oxygenase-2 is a physiologically important oxygen sensor coupled to K+ channel inhibition and increased ventilation (reviewed by Peers et al. 2010).

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In addition to mitochondrial cytochromes, nonmitochondrial enzymes located in glomus cells such as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, NO synthases and heme oxygenases have recently been implicated in oxygen sensing via the regulation of ion channels. In fact, these proteins could contribute to transduction by the generation of gaseous molecules like nitric oxide, carbon monoxide and/or hydrogen sulfide that chemically resemble reactive oxygen species, which are metabolites of oxygen (Prabhakar and Overholt 2000; Prabhakar and Peers 2014). The latter, along with NADH, are mitochondrial signals generated by hypoxia. More recently, a novel mitochondrial protein, hypoxia-inducible gene domain factor isoform (HIGD1C), has been identified in the mouse glomus cells (TimónGómez et al. 2022). The authors have found that HIGD1C enhances the mitochondrial electron transport chain complex IV sensitivity to hypoxia and is therefore essential for CB oxygen sensing.

5.2.1.3

The Redox Hypothesis

Another plausible mechanism of CB oxygen sensing provides the so-called redox model based on the conversion of oxygen into reactive oxygen species (ROS), which would in turn alter the redox status of signaling molecules and the function of the effectors (reviewed in Gao et al. 2022). Indeed, hypoxic inhibition of mitochondrial electron transport results in increased production of ROS and reduced pyridine nucleotides from glomus cell mitochondria (Ortega-Sáenz et al. 2003). A variety of ROS may be thus produced, but the most likely to be functional in signaling are the superoxide anion (O2 − ) and its diffusible product, hydrogen peroxide (H2 O2 ), produced by the action of superoxide dismutase located at both intramitochondrial and cytosolic sites. An ROS-producing site postulated as an oxygen sensor are the mitochondria complexes I and III. Another source of ROS localized close to plasmalemmal K+ channels of glomus cells is the extramitochondrial membrane-bound NADPH oxidase complex, which produces O2 − and H2 O2 , though its action on glomus cell K+ channel inactivation has not been proved so far (Kumar and Prabhakar 2012). Presently, there is also an assumption that in glomus cells, NADPH-derived ROS may even be involved in the recovery from hypoxia rather than in its sensing (see in Kumar and Prabhakar 2007). Besides, the entire concept of redox-based oxygen sensing in glomus cells is challenged by the finding that the reduced/oxidized glutathione ratio in the CB remains unchanged during exposure to hypoxia (LópezBarneo et al. 2008). Therefore, most of the data available suggest that mitochondria do not directly contribute to the primary steps in CB oxygen sensing. Nonetheless, very recent data demonstrate that ablation of mitochondrial Ndufs2 gene selectively abolishes sensitivity of glomus cells to hypoxia, maintaining responsiveness to hypercapnia or hypoglycemia (reviewed in Gao et al. 2017). Likewise, it has lately been reported that genetic ablation of the gene encoding the Rieske iron-sulfur protein

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(RISP), an essential subunit of the mitochondria complex III, in CB catecholaminergic chemoreceptor cells abolishes acute oxygen sensing and acclimatization to hypoxia with normal responses to other stimuli (Gao et al. 2022).

5.2.1.4

The “Mitochondria–Membrane” Model

To unify the membrane and mitochondrial hypotheses, an integrated mitochondriato-membrane signaling model of CB acute oxygen sensing has recently been proposed (Ortega-Sáenz and López-Barneo 2020; Iturriaga et al. 2021; López-Barneo and Ortega-Sáenz 2022; Cabello-Rivera et al. 2022). In fact, it integrates membrane electrical events and mitochondrial function in glomus cells. Specifically, this model suggests that the structural substrates for acute oxygen sensing in CB glomus cells are unique “O2 -sensing microdomains” formed by mitochondria and neighboring K+ channels in the plasma membrane (Gao et al. 2017). Disarrangement of the oxygensensing microdomains in cells exposed to enzymatic or mechanical stress could also result in the loss of acute cellular activation during hypoxia (Ortega-Sáenz and López-Barneo 2020). In addition, it has newly been suggested that the hypoxic inhibition of mitochondrial thermogenesis in TASK-channel-containing microdomains may play an important role in oxygen chemotransduction in the CB (Rakoczy et al. 2022). This novel hypothesis is particularly compelling as it provides a unifying link between the mitochondrial and K+ channel responses to hypoxia without a need to necessarily invoke mitochondrial specialization or decreases in the concentration of ATP. However, no specialized morphological structures supporting this concept have been described yet. Nevertheless, it is likely that the large nuclei of glomus cells would restrict the mitochondria to a thin rim of their cytoplasm just beneath the plasma membrane, an ideal location for them to interact with ion channels. Another mechanism that provides the basis for unifying the two hypotheses is the lately reported activation of the energy-sensing enzyme AMP-activated protein kinase (AMPK) through the inhibition of oxidative phosphorylation as being central for hypoxic chemotransduction (Peers et al. 2010). Seemingly, AMPK may act as the primary metabolic sensor and may also play a key role in the generation of the acute hypoxic ventilatory response (Wyatt and Evans 2007; Wyatt et al. 2008). In fact, AMPK can provide an exquisitely sensitive coupling mechanism enabling the hypoxic inhibition of oxidative phosphorylation to be transduced into K+ channel inhibition and excitation in glomus cells. Nonetheless, its role in normal CB chemosensitivity particularly in the oxygen sensitivity in species other than rat has yet to be proved. In summary, although the cellular localization of oxygen sensor candidates has been made known (see in López-Barneo et al. 2001; Kummer and Yamamoto 2002), to date no definitive conclusion can be drawn regarding the molecular mechanisms of CB oxygen sensing. There are several attractive hypotheses and interesting proposals under debate, but the clarification of this important physiological process must await future experimental work (for reviews, see López-Barneo et al. 2008; Prabhakar and Peng 2017). It is likely that novel mitochondrial and extramitochondrial signaling

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mechanisms will emerge in the future as we move toward a more complete understanding (Holmes et al. 2022; Pak et al. 2022). Undoubtedly, the special metabolic properties of glomus cells must be studied in detail as well to elucidate their role in responsiveness to hypoxia. Thus, a unified theory of oxygen sensing will perhaps be discovered and confirmed in the years to come.

5.2.2 Hypercapnia Sensing In addition to hypoxia response, the CB increases its activity linearly as the partial pressure of carbon dioxide (CO2 ) in arterial blood raises, a condition called hypercapnia. Similar to hypoxia, hypercapnia and low pH depolarize the glomus cell membrane and induce transmitter release in an external Ca2+ -dependent manner (Zhang and Nurse 2004). Unlike oxygen sensing however, the CB response to elevated levels of CO2 differs in a number of ways. It is generally faster and has both a dynamic and a steady state component, and its amplitude is smaller than the response to physiological levels of hypoxia. The initial or transient response of glomus cells to hypercapnia depends on the activity of intracellular carbonic anhydrase, which catalyzes the hydration of CO2 (González et al. 1994; Nurse 2005). It is now generally accepted that stimulation by hypercapnia is subsequent to concurrent acidification following hydration of CO2 (Kumar and Bin-Jaliah 2007). In fact, an increase in PCO2 causes CO2 to move into the glomus cell, thereby generating H+ that leads to a virtually prompt fall of intracellular pH, and such an acidic shift causes or facilitates membrane depolarization (Peers and Buckler 1995).

5.2.3 Sensing of Acidic Stimuli As mentioned above, in addition to its role in respiratory gas sensing, the CB detects pH status too. In fact, changes in blood pH can alter the chemoreceptor discharge (Eyzaguirre and Koyano 1965) and the augmented activity produces, albeit more slowly, a stimulus–response profile like that in response to hypercapnia. The molecular mechanism of pH sensing by chemoreceptors in the CB is not clear, although it has been proposed to be mediated by a drop in intracellular pH of glomus cells, which inhibits one or more K+ channel(s), leading to membrane depolarization (Buckler et al. 2000; Prabhakar and Peng 2004). Later, results provided the first evidence that extracellular acidosis directly activates pH-sensitive ion channels on CB glomus cells through both ASIC and TASK channels (Tan et al. 2007). Transduction of acidosis in glomus cells may also be induced by the inhibition of Ca2+ -activated voltage-gated BK K+ channels (Buckler 2015).

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5.2.4 Glucose Sensing The CB may critically participate in glucose homeostasis, possibly giving it a key role in sensing body energy. Maintaining blood glucose within appropriate levels is vital because neurons utilize almost exclusively glucose as an energy source, and therefore, their functions largely depend on a steady glucose supply. The first evidence linking the CB with glucose metabolism was reported 35 years ago (Alvarez-Buylla and de Alvarez-Buylla 1988), and a direct molecular proof of the CB as a glucose sensor was provided using CB thin slice preparation and amperometry techniques (Pardal and López-Barneo 2002). The responsiveness of mammalian CB cells to low glucose (hypoglycaemia) was further confirmed in the in vitro CB–PG preparation (Zhang et al. 2007) and in dispersed cat glomus cells (Fitzgerald et al. 2009). Human glomus cells are also shown to be responsive to hypoxia and hypoglycaemia, both of which induce inhibition of K+ channels, an increase in cytosolic Ca2+ and transmitter release in an external Ca2+ -dependent manner (Ortega-Sáenz et al. 2013). Although hypoxia and hypoglycemia operate via different signaling pathways, oxygen and low-glucose responses share a common final pathway involving the inhibition of voltage-dependent (but Ca2+ -independent) K+ channels leading to membrane depolarization, extracellular calcium influx, an increase in cytosolic calcium concentration and neurotransmitter secretion, which stimulates afferent sensory fibers to evoke sympathoadrenal activation (López-Barneo 2003; Gao et al. 2014). Nonetheless, other authors have failed to find any responsiveness of explanted whole CB preparations to low glucose (Bin-Jaliah et al. 2004; Conde et al. 2007) or any significant changes in cytosolic Ca2+ levels in dispersed glomus cells in response to rapid glucose removal (Gallego-Martin et al. 2012). Therefore, the initially proposed potential role of glomus cells as low-glucose detectors and contributors to insulin resistance that help to prevent neuronal damage by acute hypoglycemia (López-Barneo 2003) is questioned since further experiments, both in vivo and in vitro, did not support a direct CB response to hypoglycemia (reviewed by Kumar and Prabhakar 2012).

5.3 Polymodal Sensor As described above, the CB can respond to a lot of adequate stimuli in the circulation and is involved in glucose, hormonal and immune regulation. Taken together, the above-mentioned findings show accumulating evidence supporting the concept that the CB is a multimodal arterial chemoreceptor on purpose (Kumar and Bin-Jaliah 2007), activated not only by hypoxia and accumulated during its exposure metabolite lactate (Chang et al. 2015; Torres-Torrelo et al. 2021; Leonard and Nurse 2023) but also by other interactive stimuli, including hypercapnia, extracellular acidosis (Eyzaguirre and Koyano 1965; González et al. 1994; Kumar and Bin-Jaliah 2007), hypoglycemia (López-Barneo 2003) and even by changes in physical parameters of the blood such as blood temperature and osmolality (González et al. 1994) or

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reductions in blood flow (Schultz et al. 2013). However, the underlying mechanisms of the sensory response and physiological impact of the last two have not been studied in detail and, therefore, are not fully understood. In addition, it is suggested that other circulatory modulating substances such as catecholamines, hormones like angiotensin II and vasopressin (ADH) and various endogenous substances including adenosine and endothelin which are released in conditions like systemic hypoxia may increase the chemosensitivity of the CB (Kumar and Bin-Jaliah 2007). Other hormones which contribute to increased sympathetic tone such as insulin (Ribeiro et al. 2013; Barbosa et al. 2018) and leptin (Porzionato et al. 2011) also induce the CB activation. The question remains as to whether one transduction process is sensitive to multiple stimuli or whether the CB has evolved distinct sensors that may utilize a common transduction pathway for the purpose of polymodal sensing. Since CB transcriptomic studies have shown that the CB of model organisms closely parallel that of humans, a strategy to better understand the mechanisms of multimodal sensing would be to adopt a powerful hypothesis-free, high-throughput transcriptomic approach, as recently suggested by Pauza et al. (2023). Compliance with Ethical Standards The authors declare no conflict of interest. This chapter is a review of previously published research, and as such, no animal or human studies were performed.

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López-Barneo J (1996) Oxygen-sensing by ion channels and the regulation of cellular functions. Trends Neurosci 19:435–440 López-Barneo J (2003) Oxygen and glucose sensing by carotid body glomus cells. Curr Opin Neurobiol 13:493–499 López-Barneo J (2022) Neurobiology of the carotid body. Handb Clin Neurol 188:73–102 López-Barneo J, Ortega-Sáenz P (2022) Mitochondrial acute oxygen sensing and signaling. Crit Rev Biochem Mol Biol 57:205–225 López-Barneo J, López-López JR, Urena J, González C (1988) Chemotransduction in the carotid body: K+ current modulated by PO2 in type I chemoreceptor cells. Science 241:580–582 López-Barneo J, Pardal R, Ortega-Sáenz P (2001) Cellular mechanism of oxygen sensing. Annu Rev Physiol 63:259–287 López-Barneo J, del Toro R, Levitsky KL, Chiara MD, Ortega-Sáenz P (2004) Regulation of oxygen sensing by ion channels. J Appl Physiol 96:1187–1195 López-Barneo J, Ortega-Sáenz P, Pardal R, Pascual A, Piruat JI (2008) Carotid body oxygen sensing. Eur Respir J 32:1386–1398 López-Barneo J, González-Rodriguez P, Gao L, Fernandez-Aguera MC, Pardal R, Ortega-Sáenz P (2016a) Oxygen sensing by the carotid body: mechanisms and role in adaptation to hypoxia. Am J Physiol Cell Physiol 310:C629–C642 López-Barneo J, Ortega-Sáenz P, González-Rodriguez P, Fernandez-Aguera MC, Macías D, Pardal R, Gao L (2016b) Oxygen sensing by arterial chemoreceptors: mechanisms and medical translation. Mol Aspects Med 47–48:90–108 López-López JR, González C, Pérez-García MT (1997) Properties of ionic currents from isolated adult rat carotid body chemoreceptor cells: effect of hypoxia. J Physiol 499(Pt 2):429–441 Matsuoka H, Pokorski M, Takeda K, Okada Y, Harada K, Inoue M (2022) Expression of p11 and TASK1 channels in rat carotid body glomus cells subjected to chronic intermittent hypoxia. J UOEH 44:249–255 Mills E, Jöbsis FF (1972) Mitochondrial respiratory chain of carotid body and chemoreceptor response to changes in oxygen tension. J Neurophysiol 35:405–428 Murali S, Nurse CA (2016) Purinergic signalling mediates bidirectional crosstalk between chemoreceptor type I and glial-like type II cells of the rat carotid body. J Physiol 594:391–406 Nurse CA (2005) Neurotransmission and neuromodulation in the chemosensory carotid body. Auton Neurosci 120:1–9 Nurse CA (2014) Synaptic and paracrine mechanisms at carotid body arterial chemoreceptors. J Physiol 592:3419–3426 Ortega-Sáenz P, López-Barneo J (2020) Physiology of the carotid body: from molecules to disease. Annu Rev Physiol 82:127–149 Ortega-Sáenz P, Pardal R, García-Fernandez M, López-Barneo J (2003) Rotenone selectively occludes sensitivity to hypoxia in rat carotid body glomus cells. J Physiol 548:789–800 Ortega-Sáenz P, Pardal R, Levitsky K, Villadiego J, Muñoz-Manchado AB, Durán R, BonillaHenao V, Arias-Mayenco I, Sobrino V, Ordóñez A, Oliver M, Toledo-Aral JJ, López-Barneo J (2013) Cellular properties and chemosensory responses of the human carotid body. J Physiol 591:6157–6173 Overholt JL, Ficker E, Yang T, Shams H, Bright GR, Prabhakar NR (2000) Chemosensing at the carotid body. Involvement of a HERG-like potassium current in glomus cells. Adv Exp Med Biol 475:241–248 Pak O, Nolte A, Knoepp F, Giordano L, Pecina P, Hüttemann M, Grossman LI, Weissmann N, Sommer N (2022) Mitochondrial oxygen sensing of acute hypoxia in specialized cells—is there a unifying mechanism? Biochim Biophys Acta Bioenerg 1863:148911 Pardal R, López-Barneo J (2002) Low glucose-sensing cells in the carotid body. Nature Neurosci 5:197–198 Pardal R, Ludewig U, Garcia-Hirschfeld J, López-Barneo J (2000) Secretory responses of intact glomus cells in thin slices of rat carotid body to hypoxia and tetraethylammonium. Proc Natl Acad Sci USA 97:2361–2366

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Pauza AG, Murphy D, Paton JFR (2023) Transcriptomics of the carotid body. In: Conde SV, Iturriaga R, del Rio R, Gauda E, Monteiro EC (eds) Arterial chemoreceptors, ISAC XXI 2022, vol 1427. Springer, Cham, pp 1–11 Peers C (1990) Hypoxic suppression of K+ currents in type I carotid body cells: selective effect on the Ca2+ -activated K+ current. Neurosci Lett 119:253–256 Peers C, Buckler KJ (1995) Transduction of chemostimuli by the type I carotid body cell. J Membr Biol 144:1–9 Peers C, Wyatt CN, Evans AM (2010) Mechanisms for acute oxygen sensing in the carotid body. Respir Physiol Neurobiol 174:292–298 Porzionato A, Rucinski M, Macchi V, Stecco C, Castagliuolo I, Malendowicz LK, De Caro R (2011) Expression of leptin and leptin receptor isoforms in the rat and human carotid body. Brain Res 1385:56–67 Prabhakar NR (2006) O2 sensing at the mammalian carotid body: why multiple O2 sensors and multiple transmitters? Exp Physiol 91:17–23 Prabhakar NR, Overholt JL (2000) Cellular mechanisms of oxygen sensing at the carotid body: heme proteins and ion channels. Respir Physiol 122:209–221 Prabhakar NR, Peers C (2014) Gasotransmitter regulation of ion channels: a key step in O2 sensing by the carotid body. Physiology (Bethesda) 29:49–57 Prabhakar NR, Peng YJ (2004) Peripheral chemoreceptors in health and disease. J Appl Physiol 96:359–366 Prabhakar NR, Peng YJ (2017) Oxygen sensing by the carotid body: past and present. In: Halpern H, LaManna J, Harrison D, Epel B (eds) Oxygen transport to tissue XXXIX, vol 977. Springer, Cham, pp 3–8 Rakoczy RJ, Wyatt CN (2018) Acute oxygen sensing by the carotid body: a rattlebag of molecular mechanisms. J Physiol 596:2969–2976 Rakoczy RJ, Schiebrel CM, Wyatt CN (2022) Acute oxygen-sensing via mitochondria-generated temperature transients in rat carotid body type I cells. Front Physiol 13:874039 Ribeiro MJ, Sacramento JF, González C, Guarino MP, Monteiro EC, Conde SV (2013) Carotid body denervation prevents the development of insulin resistance and hypertension induced by hypercaloric diets. Diabetes 62:2905–2916 Schultz HD, Marcus NJ, Del Rio R (2013) Role of the carotid body in the pathophysiology of heart failure. Curr Hypertens Rep 15:356–362 Stea A, Nurse CA (1989) Chloride channels in cultured glomus cells of the rat carotid body. Am J Physiol 257:C174–C181 Stea A, Nurse CA (1991) Whole-cell and perforated-patch recordings from O2 -sensitive rat carotid body cells grown in short- and long-term culture. Pflügers Arch 418:93–101 Tan ZY, Lu Y, Whiteis CA, Benson CJ, Chapleau MW, Abboud FM (2007) Acid-sensing ion channels contribute to transduction of extracellular acidosis in rat carotid body glomus cells. Circ Res 101:1009–1019 Timón-Gómez A, Scharr AL, Wong NY, Ni E, Roy A, Liu M, Chau J, Lampert JL, Hireed H, Kim NS, Jan M, Gupta AR, Day RW, Gardner JM, Wilson RJA, Barrientos A, Chang AJ (2022) Tissuespecific mitochondrial HIGD1C promotes oxygen sensitivity in carotid body chemoreceptors. eLife 11:e78915 Torres-Torrelo H, Ortega-Sáenz P, Gao L, López-Barneo J (2021) Lactate sensing mechanisms in arterial chemoreceptor cells. Nat Commun 12:4166 Vicario I, Obeso A, Rocher A, López-Lopez JR, González C (2000) Intracellular Ca2+ stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception. Am J Physiol Cell Physiol 279:C51–C61 Wyatt CN, Evans AM (2007) AMP-activated protein kinase and chemotransduction in the carotid body. Resp Physiol Neurobiol 157:22–29 Wyatt CN, Peers C (1995) Ca2+ -activated K+ channels in isolated type I cells of the neonatal rat carotid body. J Physiol 483(Pt 3):559–565

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Wyatt CN, Pearson SA, Kumar P, Peers C, Hardie DG, Evans AM (2008) Key roles for AMPactivated protein kinase in the function of the carotid body? Adv Exp Med Biol 605:63–68 Xu J, Tse FW, Tse A (2003) ATP triggers intracellular Ca2+ release in type II cells of the rat carotid body. J Physiol 549:739–747 Yamaguchi S, Lande B, Kitajima T, Hori Y, Shirahata M (2004) Patch clamp study of mouse glomus cells using a whole carotid body. Neurosci Lett 357:155–157 Zhang M, Nurse CA (2004) CO2 /pH chemosensory signaling in co-cultures of rat carotid body receptors and petrosal neurons: role of ATP and ACh. J Neurophysiol 92:3433–3445 Zhang M, Buttigieg J, Nurse CA (2007) Neurotransmitter mechanisms mediating low-glucose signalling in cocultures and fresh tissue slices of rat carotid body. J Physiol 578:735–750

Chapter 6

Neurochemical Anatomy of the Mammalian Carotid Body

Abstract Carotid body (CB) glomus cells in most mammals, including humans, contain a broad diversity of classical neurotransmitters, neuropeptides and gaseous signaling molecules as well as their cognate receptors. Among them, acetylcholine, adenosine triphosphate and dopamine have been proposed to be the main excitatory transmitters in the mammalian CB, although subsequently dopamine has been considered an inhibitory neuromodulator in almost all mammalian species except the rabbit. In addition, co-existence of biogenic amines and neuropeptides has been reported in the glomus cells, thus suggesting that they store and release more than one transmitter in response to natural stimuli. Furthermore, certain metabolic and transmitterdegrading enzymes are involved in the chemotransduction and chemotransmission in various mammals. However, the presence of the corresponding biosynthetic enzyme for some transmitter candidates has not been confirmed, and neuroactive substances like serotonin, gamma-aminobutyric acid and adenosine, neuropeptides including opioids, substance P and endothelin, and gaseous molecules such as nitric oxide have been shown to modulate the chemosensory process through direct actions on glomus cells and/or by producing tonic effects on CB blood vessels. It is likely that the fine balance between excitatory and inhibitory transmitters and their complex interactions might play a more important than suggested role in CB plasticity. Keywords Amino acids · Biogenic amines · Calcium-binding proteins · Gaseous transmitters · Metabolic and transmitter-degrading enzymes · Neuromodulators · Neuropeptides · Peptide hormones · Purines · Receptors

The mammalian CB transduces arterial blood gas levels into action potential activity on carotid sinus nerve (CSN) afferents. As mentioned earlier, in response to a natural stimulation, voltage-gated Ca2+ influx into glomus cells initiates neurosecretion and release of neurotransmitters. The glomus cells synthesize a variety of neurotransmitters and neuromodulators, both excitatory and inhibitory, and express even a broader range of corresponding ionotropic and metabotropic receptors (reviewed in Fidone et al. 1988).

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_6

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The chemoreceptor function of the CB and the nerve impulse conduction need an intensive molecular and cation exchange, and energy supply. In line with this, the glomus cells, the most active cation-exchange zone in the CB, possess enzymatic properties necessary for the generation of an action potential and for the secretory process of various putative neurotransmitter substances, whose precise functional significance is not yet known. Among several molecules present in the glomus cells, acetylcholine, adenosine triphosphate and dopamine have been proposed to be the main excitatory transmitters in the mammalian CB (Iturriaga and Alcayaga 2004; Kumar et al. 2003; Nurse 2005). Besides these putative excitatory transmitters, other molecules such as nitric oxide modulate the chemosensory process through direct actions on glomus cells and/or by producing tonic effects on CB blood vessels (Iturriaga and Alcayaga 2004).

6.1 Enzyme Content There is considerable evidence for the involvement of some metabolic and transmitter-degrading enzymes in the chemotransduction and chemotransmission in various mammals. These include certain hydrolytic enzymes such as alkaline phosphatase, acetylcholinesterase, butyrylcholinesterase and adenosine triphosphatase as well as oxidoreductases including oxidases like monoamine oxidase and dehydrogenases like succinate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase and nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase (NADPH-diaphorase).

6.1.1 Hydrolases By means of histochemical techniques, we have been able to show a positive reaction for alkaline phosphatase (AP) in a few glomus cells, the majority of the sustentacular cells and in the blood vessel walls in the CB of rats, guinea pigs and rabbits (Lazarov et al. 2013). AP activity is used to test pluripotency and detect undifferentiated stem cells, and its presence in the type II cells can be considered a strong indicator of their neurogenic nature (Pardal et al. 2007). The localization of acetylcholinesterase (AChE), the ACh-degrading enzyme, has often been interpreted as an indication of sites at which cholinergic mechanisms may act. Early studies by Koelle (1950, 1951) have revealed AChE and much higher butyrylcholinesterase (BChE) activities in the CB glomus and sustentacular cells as well as in some of the nerve fibers therein. However, their cellular distribution differs as the AChE reaction product is seen in the glomus cells, whereas some BChE activity is visualized on the sustentacular cell membrane. Consequently, the distribution of cholinesterases in the CB of a variety of animals has repeatedly been studied (Ballard and Jones 1971; Jones 1975; Nurse

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1987) and both specific AChE and nonspecific BChE reactivities have been found solely in postganglionic adrenergic axons and plexuses around the blood vessels in the cat CB (Biscoe and Silver 1966). Similarly, we have not found an intracellular reaction product, and the AChE and BChE reaction is exclusively seen in thick, often parallel nerve bundles throughout the organ in rabbits, some of which are associated with blood vessels (Lazarov et al. 2013). Therefore, it seems likely that a cholinergic pathway controls the blood vessels of the CB.

6.1.2 Peptidases It is generally assumed that both biogenic amines and peptide hormones are stored within small electron-dense granules in APUD cells. It has been reported that multiple intermediate metabolites of a locally generated in the CB angiotensin system (Lam and Leung 2002) and their corresponding receptors can influence the glomus cell function and afferent chemoreceptor activity (reviewed in Schultz 2011). Previous studies have revealed that the neutral endopeptidases are some of the major peptidases involved in the inactivation of neuropeptides in the mammalian CB (Kumar 1997). Enzyme histochemical experiments at our laboratory have indicated that tripeptidyl aminopeptidase I (TPP I), a lysosomal exopeptidase, is probably involved in the general turnover of collagen and certain hormone peptides in the CB. Specifically, the localization patterns of TPP I demonstrate that under physiological conditions both the parenchymal cells of the rat CB express the enzyme, albeit with a different intensity (Fig. 6.1) (Atanasova and Lazarov 2015). It seems likely that the glomus cells possess enzymatic equipment necessary for the neuropeptide intracellular and collagen extracellular initial degradation. Within their specific granules, glomus cells also contain large amounts of adenine nucleotides like adenosine triphosphate (ATP) and an intermediary of its metabolism, adenosine, which is released when exocytosis takes place (Böck 1980). The release of neurotransmitters requires energy provided by the breakdown of ATP, and its hydrolysis is assisted by adenosine triphosphatase (ATPase) that might also be linked directly to Ca2+ . Accordingly, ATPase activity is found on the surface of the CB glomus cells and capillary bed in cats and rats (Nada and Ulano 1972; Böck 1980). With respect to enzymatic degradation of ATP, ATPase activity is detected in the CB (Starlinger 1982) and localized in the cell membranes of glomus and sustentacular cells and nerve fibers (Nada and Ulano 1972). In line with these findings, we have also observed dense deposits of an ATPase reaction product in the periphery of the glomus cells in the rabbit CB (Lazarov et al. 2013). Thus, the changing levels of ATP within CB tissues and the chemosensory excitatory effects observed upon its administration have initially been considered part of the ATP metabolic role; later its possible role as a transmitter acting between glomus cells and sensory nerve endings has also been suggested (González et al. 1994; Iturriaga and Alcayaga 2004; Nurse 2005).

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Fig. 6.1 Histochemical localization of TPP I in the rat carotid body (CB). a Demonstration of TPP I activity in the CB using the fluorogenic substrate GPM-HHNI. b Higher magnification of the boxed area in a showing orange-red to bright red granules of the final reaction product in glomus cells (arrows) and a few sustentacular cells (arrowhead). c Visualization of TPPI activity in the CB using the chromogenic substrate GMP-CAH. d High-power view of the rectangle in c indicating the strong granular staining in the glomus cells (arrows) and a slightly weaker reaction in a subset of peripherally located sustentacular cells (arrowhead). Note the typical appearance of glomus cells with large nuclei and a thin layer of brown-stained cytoplasm. Scale bars = 100 μm in a, c, 50 μm in b, d

6.1.3 Oxidoreductases Light microscope histochemistry has revealed the presence of certain oxidoreductases in the mammalian CB. In particular, MAO staining is attributed mainly to the sustentacular cells though glomus cells also stain in the rat and rabbit CB (Woods 1967, 1975; Thybusch 1968). Conversely, we have observed only a trace MAO activity in both the CB parenchymal cells in rabbits, guinea pigs and rats (Lazarov et al. 2013). However, the glomus cells display a strong dehydrogenase enzyme activity. The enzymes exhibited in them include succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH), glucose-6-phosphate dehydrogenase (G6PD), glutamate dehydrogenase (GDH) and NADH dehydrogenase (NADH-DH). The sustentacular cells also exhibit a faint staining for LDH, G6PD, GDH and DH. Some SDH activity is seen in the CB microvasculature as well.

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Fig. 6.2 Histochemical staining for NADPH-d in the rat carotid body (CB). a Photomicrograph showing NADPH-d-reactive nerve fiber arborizations that are associated with negative glomus cells (arrows) and blood vessels (arrowhead), and also surround clearly delineated glomeruli. b Sparse NADPH-d-stained varicosities are also observed perivascularly in the CB. Scale bars = 50 μm

On the other hand, the CB is recognized as a site of synthesis of the gaseous neuromessenger nitric oxide. Its generation from the amino acid L-arginine is catalyzed by the enzyme nitric oxide synthase (NOS) which requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) to be enzymatically active. Accordingly, NADPH dehydrogenase commonly known as NADPH-diaphorase (NADPH-d) is now considered a selective histochemical marker for neurons producing NOS (Dawson et al. 1991; Hope et al. 1991). By means of NADPHd histochemistry, we have revealed that negative glomus cells are surrounded by fine pericellular arborizations of varicose nerve fibers and such diaphorase reactivity is also observed in thin varicosities around blood vessels in the rat CB (Fig. 6.2) (Atanasova et al. 2016a).

6.2 Neurotransmitters and Their Receptors In the last 75 years, CB research has focused on the establishment of transmitters that translate the receptor potential into an increase in afferent discharge and in understanding neurotransmitter and neuromodulatory mechanisms operating during CB chemoexcitation. There is convincing evidence that glomus cells in most mammals and man contain a broad diversity of endogenous neuroactive ligands, including the classical neurotransmitters, neuropeptides and gaseous signaling molecules (reviewed by González et al. 1994; Iturriaga and Alcayaga 2004; Nurse 2005; Prabhakar and Semenza 2012). These are candidates that possibly act as transmitters at the junctions between them and petrosal ganglion (PG) nerve terminals, wherein they are linked to both ionotropic and metabotropic receptors. However, the presence of the corresponding biosynthetic enzymes for some of transmitter candidates has not been confirmed, and therefore, they are defined as putative neuromodulators. In

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addition, co-existence of biogenic amines and neuropeptides has been reported in the glomus cells of the cat CB (Wang et al. 1992), thus suggesting that glomus cells store and release more than one transmitter in response to natural stimuli. In fact, the CB, despite its small size, expresses as many types of transmitters as the brain, emphasizing the complexity of its information processing. The reason for such a plethora of neurochemicals in such a tiny organ remains obscure. Besides, despite the significant recent progress in elucidating the functional significance of several neurochemicals, the role of endogenous CB neurotransmitters and neuromodulators in shaping the afferent response is still debatable. Moreover, this task has become more challenging given the growing evidence that the glial-like sustentacular cells may not be silent partners during sensory transmission but probably participate actively in paracrine signaling during chemotransduction (Nurse and Piskuric 2013; Nurse 2014; Leonard et al. 2018; Leonard and Nurse 2023). Small-molecule transmitters in the CB include the amino acids, biogenic amines and purines. The amino acids and biogenic amines are considered classical or conventional transmitters that are stored in synaptic vesicles and mediate their actions via activation of specific, mostly G-protein-coupled receptors. The nontraditional or unconventional transmitters belong the endogenous gaseous neuromessengers that are not stored in synaptic vesicles and exert their biological actions either by interacting with cytosolic enzymes or by direct modification of proteins.

6.2.1 Amino Acids CBs express both excitatory amino acid transmitters like glutamate and inhibitory transmitters such as gamma-aminobutyric acid.

6.2.1.1

Glutamate

Glutamate (GLU) is the major excitatory neurotransmitter in the nervous system acting on GLU receptors. Although it has been proposed that the hypoxia-evoked activity of the CSN is modulated by GLU (Vardhan et al. 1993), it has not been examined in depth in the CB. Initially, it has been shown by immunohistochemistry that GLU is associated with glomus cells in the cat CB (Torrealba 1990), but the same author later has revealed that superfusion with high potassium solution fails to cause GLU release from in vitro CB (Torrealba et al. 1996). This finding has led to the speculation that GLU is a metabolite rather than a neurotransmitter within the glomus cells. However, our immunohistochemical experiments have also demonstrated that GLU is expressed in a few glomus cells and in intraglomerular nerve fibers around immunonegative glomus cells in the rat CB while the periglomerular nerve fibers show a weaker immunoreactivity for this excitatory amino acid (Fig. 6.3a, b). Moreover, it has subsequently been found that ionotropic NMDA, AMPA and kainate receptors (Liu et al. 2009, 2018) as well as metabotropic GLU receptors (Li et al.

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2021; Zhao et al. 2022) are widely expressed in both rat and human glomus cells, thus suggesting that the CB chemoreceptor response to hypoxia might be mediated by glutamatergic signaling. Glutamatergic neurotransmission, however, is not only dependent on GLU and the corresponding receptors, but also on its transporters, including vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs). Accordingly, multiple VGLUTs and EAATs have been detected in the rat and human CB (Liu et al. 2018; Li et al. 2020) although VGLUT2 labeling is not observed only within nerve endings that are immunoreactive to P2X3 (Yokoyama et al. 2014). Taken together these findings suggest that all the molecular components of a functional glutamatergic signaling system exist in the CB, and therefore, GLU is more than simply a metabolic substrate and may actually be a transmitter in the CB. It could be released from afferent nerve terminals to modulate the chemosensory activity.

Fig. 6.3 Immunohistochemical demonstration of amino acid transmitters in the rat carotid body (CB). a, b Low- and higher-magnification images of CB showing details of GLU immunoreactivity in a subset of glomus cells (arrows) and in a few intraglomerular (arrowhead) and periglomerular nerve fibers. c Low-magnification overview of cell clusters demonstrating immunoreactivity for GABA in glomus cells and pericellular nerve fibers within the CB parenchyma. d The inset at higher magnification clearly indicates that immunostaining is found in a proportion of glomus cells (arrows) and a subset of sustentacular cells (arrowhead). GABA immunoreactive interglomerular and perivascular nerve fibers are observed as well. Scale bars = 100 μm in a, c, 50 μm in b, d

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Gamma-Aminobutyric Acid

The CB chemosensory discharge can be blunted by the concurrent action of inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) which diminishes the depolarizing action of CSN excitatory inputs (Nurse 2010; Leonard et al. 2018). Earlier immunohistochemical studies have revealed immunoreactivity for both GABA and its synthesizing enzyme glutamic acid decarboxylase (GAD) in almost all glomus cells of the mouse CB (Oomori et al. 1994, 1995). Further, weak GABA-immunopositive reaction has been shown in a subset of glomus cells of the cat CB (Pokorski and Ohtani 1999) and the presence of G-protein-coupled GABAB receptors is reported in these cells (Fearon et al. 2003). We have also observed a low number of GABA-immunoreactive glomus cells as well as numerous long pericellular nerve fibers that are weaker immunostained in the rat CB (Fig. 6.3c, d). Such findings suggest a presynaptic site of action for GABA in the CB. However, GABA-immunoreactivities are not found in the nerve fibers whose endings synapse on the chemoreceptor cells in mice and cats (Oomori et al. 1995; Pokorski and Ohtani 1999) and, moreover, no appreciable effect of GABA on the cat CSN discharges has been recorded (Nishi et al. 1979). Therefore, it seems unlikely that GABA is an essential presynaptic modulator of the chemoreceptor function. In addition to its presumed presynaptic role, there is evidence that GABA may act postsynaptically on ligand-gated GABAA receptors to inhibit via a shunting mechanism the sensory discharge in the cat and rat CB (Igarashi et al. 2009; Zhang et al. 2009). In line with this, several subunits of the ionotropic GABAA receptors have been identified in PG afferent terminals near glomus cells by immunohistochemistry and RT-PCR (Zhang et al. 2009). In addition, experimental evidence is provided for the expression of functional metabotropic GABAB receptor subunits (GABAB1 and GABAB2 ) on glomus cells (Fearon et al. 2003). Additional data for the transmitter role of GABA in the CB comprise the presence of several GABA transporters in both glomus cells and PG neurons, suggesting that both cell types are responsible for GABA clearance from the synapse (Zhang et al. 2009). It has also been shown that the general anesthetic propofol acting by a positive modulation of the inhibitory action of GABA through GABAA receptors has a depressant effect on peripheral CB chemosensitivity, possibly via interaction with cholinergic signaling (Jonsson et al. 2005). Thus, GABA appears to act as an inhibitory postsynaptic CB neurotransmitter.

6.2.2 Biogenic Amines Among the various neurotransmitters found in glomus cells from several different species, biogenic amines represent the largest group and are best characterized (reviewed in Alfes et al. 1977). They include acetylcholine, the catecholamines noradrenaline and dopamine, as well as serotonin and histamine. Notably, in the early studies acetylcholine and dopamine were considered leading transmitter candidates at synapses of glomus cells.

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6.2.2.1

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Acetylcholine: The Cholinergic Hypothesis

The so-called cholinergic hypothesis is based on several observations accumulated during the first half of the twentieth century (for references, see Fitzgerald 2000). It postulates a model of chemotransduction in which glomus cells release acetylcholine (ACh) as an essential transmitter capable of exciting the afferent CSN abutting on these cells. Thus, ACh has emerged as the early frontrunner for the major excitatory neurotransmitter in the CB (Hollinshead and Sawyer 1945) and, despite skepticism in the years to follow, it is still considered an attractive candidate (Fitzgerald 2000; Shirahata et al. 2007). Several lines of evidence show that ACh meets most of the criteria to be considered a classical excitatory transmitter in this organ: It is stored in the glomus cells, and its content remains unchanged after the section of the CSN or the removal of the superior cervical ganglion (SCG; Fitzgerald 2000); the enzymatic machinery for ACh biosynthesis and degradation has been demonstrated in glomus cells of several species, including rats (Nurse 1987; Nurse and Zhang 1999), cats, pigs and rabbits (Ballard and Jones 1972; Wang et al. 1989; Shirahata et al. 1996). However, our findings show that choline acetyltransferase (ChAT), the rate-limiting enzyme for ACh synthesis, is mostly localized in intraglomerular, periglomerular and perivascular nerve fibers, and in some intrinsic autonomic ganglion cells innervating the rat CB. In most cases, glomus cells are negative and only a small number of them express the enzyme (Fig. 6.4). On the other hand, a high affinity, sodium-dependent choline uptake mechanism, common to all cholinergic neurons, has been reported in the CB (Wang et al. 1989). Moreover, the cat CB in vitro releases ACh in response to electrical stimulation (Eyzaguirre and Zapata 1968). In the rat, the cholinergic marker vesicular acetylcholine transporter (VAChT) is detected in glomus cells in situ (Zhang and Nurse 2004). Furthermore, the exogenous application of ACh to the CB increases chemosensory discharge in a dose-dependent manner in most species (Iturriaga and Alcayaga 2004). Finally, immunocytochemical studies have shown the presence in glomus cells and PG perikarya and terminals of a4 and a7 subunits of the nicotinic ACh receptor (AChR) in cats (González et al. 1994; Ishizawa et al. 1996; Shirahata et al. 1998) as well as both M1 and M2 subtypes of muscarinic AChRs in the CB chemosensory system in cats (Shirahata et al. 2004) and rabbits (Shirahata et al. 2007). Taken together, these data strongly support the hypothesis that ACh is released in response to chemical stimuli from the glomus cells and increases the rate of chemoafferent discharge by acting on cholinergic receptors in the afferent nerve endings. However, activation of these receptors by ACh induces a wide variety of responses. Although an excitatory role for exogenous ACh seems to predominate in rat and cat CB, it is noteworthy that species differences may exist, since an inhibitory role for ACh has been proposed in the rabbit CB (Iturriaga and Alcayaga 2004; Shirahata et al. 2007). On the other hand, doubts about its importance have persisted because blockers of both nicotinic and muscarinic ACh receptors, which mediate its excitatory or inhibitory effects, respectively, cannot completely inhibit the CB response to natural stimulation (Douglas 1954; Fitzgerald 2000; Iturriaga and Alcayaga 2004; Nurse 2005). It is plausible, therefore, to assume

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Fig. 6.4 Immunohistochemical localization of ChAT in the rat carotid body (CB). a Low-power photomicrograph showing the expression of ChAT in the adult CB. b At a higher magnification note that numerous periglomerular (arrows) and intraglomerular (arrowheads) nerve fibers are ChATimmunoreactive. A group of intensely immunopositive small intrinsic autonomic neurons is also visible in close proximity to blood vessels in the CB. Scale bars = 100 μm in a, 50 μm in b

that ACh may have both pre- and postsynaptic roles during sensory transmission (Fitzgerald 2000; Nurse 2005), or that other excitatory neurotransmitter candidates such as ATP are involved as co-transmitters in hypoxic signaling (Zhang et al. 2000).

6.2.2.2

Catecholamines

The mammalian CB contains large amounts of catecholamines (CA), such as dopamine and noradrenaline, stored in the dense-cored vesicles of glomus cells (reviewed in Roumy et al. 1990; González et al. 1994). Accordingly, these cells express tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, and dopamine β-hydroxylase (DBH), a synaptic vesicular enzyme that converts dopamine into noradrenaline (Fidone and González 1986; Wang et al. 1991). No convincing evidence exists as to the adrenaline expression in the CB.

Dopamine: The Dopaminergic Hypothesis The dopaminergic hypothesis is based on the detection of high levels of dopamine (DA) within the CB of all mammalian species studied (see Fitzgerald et al. 2009a). Historically, DA is the predominant CA synthesized, taken-up and stored in densecored vesicles of glomus cells of several species and released in response to natural stimuli (González et al. 1994). In the rat CB, virtually all glomus cells (~ 10,000 cells per CB) contain DA and exhibit TH immunoreactivity (Fig. 6.5) (Laidler and Kay 1975; McDonald and Mitchell 1975; Bolme et al. 1977; Hess 1978; Vicario et al. 2000) and also express dopamine D2 receptor mRNA (Czyzyk-Krzeska et al. 1992). Similarly, immunoreactivity for TH is widely observed in the mouse glomus

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cells (Oomori et al. 1994) and the total number of TH-positive cells is estimated to be about 1500 cells per CB (Pardal et al. 2007). The cat CB is also a dopaminergic organ, and its TH content is similar to that found in the rat where the ratio DA/NA is about 5:1 (Vicario et al. 2000). Likewise, the rabbit glomus cells exhibit intense TH immunoreactivity (González et al. 1981) and express the dopamine D2 receptor gene (Verna et al. 1995). Our research demonstrates that this also stands true for the human glomus cells, which express not only the CA-synthesizing enzyme TH, but also the vesicular monoamine transporter 1 (VMAT1), a specific transporter for catecholamines and a synaptosomal-associated protein of 25 kDa (SNAP25), an important component of the neuroendocrine exocytotic apparatus. Moreover, the majority of them are richly endowed with dopamine D2 receptors (Lazarov et al. 2009, 2011). Recent evidence also suggests that the high level of TH in the glomus cells is a distinctive feature of the antenatal human CB (Otlyga et al. 2021). Thus, investigations on CBs in many different species have led to the conclusion that DA is a primary transmitter in the CB, because it meets most of the necessary criteria for such a role including its biosynthesis and storage by glomus cells, as well as Ca2+ -dependent release triggered by hypoxia. In addition, expression of functional DA receptors in afferent sensory neurons, effects of agonists or antagonists, genetic ablation studies and quantitative trait loci analysis are in line with this role (Huey et al. 2003; Tankersley 2003; Iturriaga and Alcayaga 2004). The initial suggestion of Fidone, González and coworkers that DA acts as an excitatory transmitter in the rabbit CB (Fidone et al. 1982; González et al. 1994) has since fallen into disfavor by demonstrating that depletion of DA with reserpine had little or no effect on the hypoxia-induced increase in CSN discharge (Donnelly 1996). Moreover, the absence of D1 receptors but the expression of inhibitory, hyperpolarizing D2 receptors by the glomus cells and PG neurons further indicate that DA may not serve as an excitatory transmitter (see Gauda 2002, and references therein). Subsequent physiological and pharmacological studies allow for more definitive characterization of dopaminergic profiles of glomus cells involved in hypoxic chemosensitivity and indicate that DA, acting via G-protein-coupled D2 receptors, appears to be, either presynaptically or postsynaptically, an inhibitory neuromodulator in almost all mammalian species except the rabbit (Iturriaga and Alcayaga 2004; Iturriaga et al. 2009). It is likely that dopaminergic inhibition represents a potential negative feedback pathway by which DA inhibits release of excitatory neurotransmitters (Nurse and Piskuric 2013). However, the effects of D2 receptor antagonists on CB chemoreception and PG neurons (Iturriaga and Alcayaga 2004) suggest that DA, at least within the cat CB, may also act as autocrine–paracrine modulator of the chemosensory activity (Nurse 2005), as proposed earlier (Zapata 1977). Thus, although the DA action in the CB remains controversial, the reported different roles in the generation of afferent chemosensory activity appear to reflect true species differences (Iturriaga et al. 2009).

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Fig. 6.5 Immunohistochemical expression of TH in the rat carotid body (CB). a Low-magnification overview of a glomic lobule demonstrating TH immunoreactive structures in the adult CB. b Higher magnification of the area inside the rectangle in a reveals that almost, if not all, glomus cells (arrows) are intensely TH-immunostained. c, d Hematoxylin counterstaining highlights the improved visualization and cellular localization of the precipitated DAB reaction product for TH-immunostaining in glomic lobules (arrows). Scale bars = 100 μm in a, c, 50 μm in b, d

Noradrenaline Noradrenaline (NA) is another catecholamine that exists in the glomus cells of the CB (González et al. 1994). An early immunofluorescence study has failed to find evidence for the presence of NA in the rat CB and claimed that virtually all glomus cells are dopaminergic (Hess 1978). The author assumes that most of NA found in the CB is in the autonomic nerve fibers. However, subsequent immunohistochemical studies have shown that some of the glomus cells in the rat and most of the glomus cells in the cat CB contain DBH and therefore may be sites of NA synthesis (Chen et al. 1985). The presence of NA-containing glomus cells is also observed in the rabbit CB (Schamel and Verna 1992; Verna et al. 1993). However, the NA content is 10 times larger in the cat than in the rabbit CB (Armengaud et al. 1988). In other species including dogs, guinea pigs and humans, certain chemoreceptor cells have been found to be immunopositive for either NA or DBH (Kobayashi et al. 1983; Varndell et al. 1982). Moreover, it has also been reported that SCG removal (sympathectomy) reduces the content of NA in the CB in several mammalian species about 50% and

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that decrease cannot be prevented by the transection of the CSN (Mir et al. 1982). Consistent with the immunohistochemical studies, electrophysiological studies have demonstrated that NA inhibits the Ca2+ current in glomus cells of rabbit CBs via α2 -adrenergic receptors (Almaraz et al. 1997; Overholt and Prabhakar 1999). These findings suggest that NA in glomus cells has an inhibitory role for the ongoing release of excitatory neurotransmitters.

6.2.2.3

Serotonin (5-Hydroxytryptamine)

Among the indolamines examined for a possible function as transmitters of chemoreceptor activity, serotonin (5-Hydroxytryptamine, 5-HT) fulfills some criteria, such as presence in the glomus cells in several species (Grönblad et al. 1983; Oomori et al. 1994; Zhang and Nurse 2000), in situ localization of its biosynthetic enzyme tryptophan hydroxylase and transporter in them (Yokoyama et al. 2013) and identification of 5-HT5a and 5-HT2a receptors on these cells (Wang et al. 2000; Zhang et al. 2003). Indeed, we have identified a subset of 5-HT-immunoreactive glomus cells specifically localized in cell clusters near blood vessels (Fig. 6.6). Nonetheless, a cardinal property, i.e., its release in response to hypoxia by the CB, has not been observed (Jacono et al. 2005), although it is released in vitro from CB cultures (Zhang and Nurse 2000). In addition, the exogenous application of 5-HT increases CSN activity. These data are consistent with a prominent presynaptic role for 5-HT involving a protein kinase C-mediated inhibition of K+ channels in glomus cells (Zhang et al. 2003; Nurse 2005). Blockade of G-protein-coupled 5-HT2a autoreceptors on them by ketanserin or ritanserin leads to inhibition of the hypoxia-induced postsynaptic PG response recorded in coculture (Zhang et al. 2003), and thus, the possibility cannot be excluded that 5-HT may have both presynaptic and postsynaptic roles during chemosensory transmission. Further studies are required to settle the inconsistencies between the two sets of data.

6.2.2.4

Histamine

Histamine (HIS), a biogenic diamine, has recently been implicated in hypoxic chemosensitivity. Indeed, we and others report that glomus cells contain all the biochemical machinery for the biosynthesis, storage and release of HIS, as well as its receptors. Histaminergic traits such as expression of the histamine molecule itself (Fig. 6.7a, b), its synthesizing enzyme histidine decarboxylase (HDC) and selective transporter VMAT2 (Fig. 6.7c, d), as well as components of the neuroendocrine exocytosis apparatus like SNAP25 and syntaxin 1 have been localized in glomus cells in rats (Koerner et al. 2004), cats (Del Rio et al. 2008) and humans (Lazarov et al. 2009). Moreover, H1 and H3 histamine receptors are detected not only in the glomus cells (as autoreceptors), but also in afferent PG neurons and in the structure they terminate, the solitary tract nucleus (Lazarov et al. 2006). In addition, the topic application of H1 and H3 receptor agonists to the CB causes an increased phrenic

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Fig. 6.6 Immunohistochemical localization of serotonin (5-Hydroxytryptamine, 5-HT) in the rat carotid body (CB). a Low-magnification overview of glomic lobules showing the expression of 5-HT in a subpopulation of glomus cells. b High-power view of the boxed area in a demonstrates a location of 5-HT-immunoreactive glomus cells close to blood vessels (arrows). Scale bars = 100 μm in a, c, 50 μm in b, d

nerve activity in a working heart–brainstem preparation indicating that HIS may also serve as a transmitter within the peripheral arterial oxygen sensors (Lazarov et al. 2006). Last but not least, radioenzymatic and immunohistochemical evidence points out that the storage of HIS in the glomus cells exceeds that of DA more than tenfold (Koerner et al. 2004). Nevertheless, its physiological role awaits further characterization. Although initially no effect of HIS on CB chemosensory discharges was observed (Landgren et al. 1954), an increase of the cAMP level in the rat CB has been reported after the application of exogenous HIS (Mir et al. 1983). Furthermore, it has been shown that the administration of HIS in an in vitro CB preparation produces a dosedependent increase in the chemosensory activity albeit its application to the isolated PG has no effect on CSN discharge (Del Rio et al. 2009). Although it has been proposed that HIS is an excitatory hypoxic transmitter between the glomus cells and the chemosensory nerve endings of PG neurons (Koerner et al. 2004), the current data indicate that rather HIS plays an autocrine and/or paracrine role in the generation of the chemosensory activity in the CB. Therefore, HIS is a modulator of the CB chemoreception, and the signal transmission at the pre- and postsynaptic levels may be differentially modulated through activation of its excitatory H1 and inhibitory H3 receptors (Del Rio et al. 2009).

6.2.3 Purines: The Purinergic Hypothesis As it now stands, the concept of purinergic signaling in the CB postulates that purine nucleotides and nucleosides such as adenosine-5' -triphosphate and adenosine are likely excitatory transmitters involved in CB hypoxic signaling (reviewed in Lahiri et al. 2007; Conde et al. 2017). The complex effects of both adenosine-5' -triphosphate

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Fig. 6.7 Immunohistochemical detection of HIS in the rat carotid body (CB). Representative photomicrographs at low and higher magnifications indicating that the clusters of glomus cells exhibit HIS (a, b) and its selective transporter VMAT2 (c, d). Insets show high-power profiles of CB glomeruli demonstrating that virtually all glomus cells (arrows) are immunopositive for HIS (b) and VMAT2 (d). Scale bars = 100 μm in a, c, 50 μm in b, d

and adenosine enable the hypoxic signal to be transduced from chemoreceptors to afferent neurons, with multiple receptors allowing for a nuanced transmission unavailable to single-action transmitters (see Lahiri et al. 2007). More recently, Murali and Nurse (2016) have suggested that the bidirectional crosstalk between glomus and sustentacular cells during chemotransduction is mediated by purinergic signaling and possibly, albeit to a lesser extent, by other CB neurotransmitters or neuromodulators (Leonard and Nurse 2023).

6.2.3.1

Adenosine 5I -Triphosphate

In addition to biogenic amines, a large quantity of adenine nucleotides, including adenosine triphosphate (ATP), has been found in the dense-core granules of glomus cells (Böck and Gorgas 1976; Böck 1980). ATP has been found to increase cytosolic calcium in glomus cells (Mokashi et al. 2003). The chemosensory excitatory effects of ATP observed upon its administration have initially been considered as part of its metabolic role, but the demonstration of membrane receptors for ATP in other tissues

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in the late 1990s made it necessary to consider its possible role as a transmitter during chemosensory transmission in the CB (see Fitzgerald et al. 2009a). Later it has also been shown that hypoxia and hypoglycemia stimulate Ca2+ -dependent ATP release from rat and cat CB chemoreceptor cells (Buttigieg and Nurse 2004; Fitzgerald et al. 2009b). ATP, now considered the main excitatory neurotransmitter in the processing of chemosensory stimuli in the CB of many species (Nurse 2010; Piskuric and Nurse 2013), appears to have excitatory afferent influence on the CSN via acting on P2X2/P2X3 receptors in PG neuronal perikarya and peripheral processes in the rodent CB (Spergel and Lahiri 1993; Zhang et al. 2000; Prasad et al. 2001; Nurse and Piskuric 2013). Apart from the presence of P2X ionotropic ligand-gated ion channel ATP receptors, functional P2Y metabotropic G-protein-coupled receptors have also been described in the rat CB, indeed localized not on glomus but sustentacular cells (Xu et al. 2003). These authors assume that the ATP-induced Ca2+ rise in sustentacular cells can mediate paracrine interactions within the CB. In addition, the presence of pannexin-1 channels, gap junction-like proteins, in the CB sustentacular cells raises the possibility that these cells participate in CB chemotransduction by acting as “ATP amplifiers” (Zhang et al. 2012; Leonard et al. 2018). Accordingly, the application of P2Y2 agonists to rat sustentacular cells opens nonselective cation channels with pharmacological properties of pannexin-1 channels (Zhang et al. 2012). Stimulating P2Y2 receptors mobilizes intracellular Ca2+ and opens pannexin1 channels on those cells, causing further (nonvesicular) release of ATP from them. Hence, ATP may also have additional presynaptic actions mediated by autocrine– paracrine stimulation of purinergic P2Y receptors. The application of P2X receptor blocker suramin reduces the CSN chemosensory discharge while the joint administration of suramin and hexamethonium, a nicotinic AChR antagonist, completely abolishes both the basal and the hypoxia-induced chemosensory excitation (Zhang et al. 2000; Nurse 2010), thus suggesting that ATP may participate in the generation of chemoafferent activity. However, the failure of ATP antagonists to eliminate CB chemosensory activity indicates that purinergic transmission is not a sufficient mechanism for such transfer of information (Zapata 2007). Such a possibility seems partly justified by Zhang et al. (2000) who have found that a co-release of ACh and ATP is necessary for the full expression of sensory excitation by hypoxia. Therefore, ATP might operate synergetically with ACh (or other transmitters) as effective excitatory co-transmitters between glomus cells and chemosensory nerve endings in different mammalian species (Zhang et al. 2000; Varas et al. 2003; Iturriaga and Alcayaga 2004; Nurse 2005; Alcayaga et al. 2006), which constitutes the so-called cholinergic-purinergic hypothesis for CB chemotransmission (reviewed in Fitzgerald et al. 2009a).

6.2.3.2

Adenosine

The purine nucleoside, adenosine, a breakdown product of ATP, is released in normoxic conditions by the CB and its release increases in response to hypoxia (for a review, see Conde et al. 2009). When applied exogenously, it can also stimulate

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the CSN chemosensory activity (McQueen and Ribeiro 1981) acting on G-proteincoupled adenosine receptors. Among the different adenosine receptors, A2A and A2B receptors are the predominant receptor subtypes localized in the CB chemoreceptor cells and, in addition, excitatory A2A receptors are also present postsynaptically on the CSN (Conde and Monteiro 2006). This effect is mimicked by adenosine analogs and inhibited by the A1/A2A selective antagonists theophylline and 8-phenyltheophylline, suggesting the presence and involvement of A2 receptors (McQueen and Ribeiro 1983). The mechanisms underlying the increase in chemoreceptor discharge from the CSN in response to hypoxia involve A2A receptor stimulation by adenosine, increased cAMP and protein kinase A, block of TASK-like (background) K+ channels, membrane depolarization, opening of voltage-gated Ca2+ channels, Ca2+ influx and transmitter release from glomus cells (González et al. 1994; López-López et al. 1997; Conde and Monteiro 2004). Levels of extracellular adenosine can also be altered by membrane nucleoside transporters (Lahiri et al. 2007). Even though current evidence suggests that the ATP extracellular catabolism and release of adenosine per se from glomus cells are the main sources of the total adenosine tool, the contribution of extracellular cAMP to adenosine production cannot be ruled out (Conde et al. 2017). Both exogenous and endogenous adenosine may increase cAMP content in glomus cells via A2A and A2B action on adenylyl cyclase, leading to the release of neurotransmitters (Conde et al. 2008). In particular, it has been shown that adenosine is involved in the release of catecholamines through an antagonist interaction between A2B and dopamine D2 receptors (Conde et al. 2008). Though the impact of hypoxia on adenosine receptors is unclear, it also provokes an increased release of ACh and decreased release of DA and NA from the cat CB (Fitzgerald et al. 2004). Thus, adenosine acts as a neuromodulator of the release of classical transmitters from CB chemoreceptor cells. Furthermore, its excitatory role in CB chemosensory activity is consensual and could be due to a direct action on postsynaptic sensory nerve endings as well as at presynaptic sites in chemoreceptor cells (Conde et al. 2008). It should be noted, however, that the presence of A1 receptors in the PG cell bodies suggests that adenosine might also have an inhibitory role in hypoxic chemotransmission (Gauda et al. 2000).

6.2.4 Neuropeptides and Peptide Hormones Besides classical transmitters, more recently described neuroactive peptides that serve as transmitters or modulators elsewhere in the nervous system are likely mediators in the CB. Pearse (1969) was the first to propose that a polypeptide which he provisionally named “glomin” is present in the CB.

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Substance P

Substance P (SP), the first member of the tachykinin family of peptides, has been shown to be stored in the CB in most species, being located in glomus cells (Smith et al. 1990) and also in fine afferent nerve fibers distributed in the parenchyma of the CB (Lundberg et al. 1979; Jacobowitz and Helke 1980; Kummer and Habeck 1991; Kusakabe et al. 1994) or associated with both structures (Cuello and McQueen 1980; Chen et al. 1986; Prabhakar et al. 1989; Kim et al. 2001; Atanasova and Lazarov 2012). Although SP-immunoreactivity is evident in some glomus cells, significant SP expression is visible in thick perivascular, periglomerular and intraglomerular nerve fibers as well as in varicosities that surround immunonegative glomus cells in a basket-like manner as our immunohistochemical data have demonstrated (Fig. 6.8). Evidence has since been accumulating to support the original hypothesis that SP is an important neurotransmitter/neuromodulator of hypoxic chemosensitivity (Helke et al. 1980). Indeed, exogenous SP administration increases the sensory response of the CB in several mammalian species, but not in goats, during normoxic and hypoxic conditions (Prabhakar et al. 1987, 1989). On the other hand, neuropharmacology studies have shown that SP, acting on NK-1 receptors, augments the spontaneous sensory discharge of the CB both in vivo and in vitro (McQueen 1980; Monti-Bloch and Eyzaguirre 1985). In accordance with this, selective peptide and nonpeptide SP (NK-1) receptor antagonists spantide and CP-96345, respectively, either attenuate or abolish the hypoxic response of the rat and cat CB while leaving the sensory response to hypercapnia unaffected (Prabhakar et al. 1993; Cragg et al. 1994; Kumar et al. 2000). More importantly, carboxypeptidase-E, a protease that is involved in its processing into biologically active form, and the enzyme neutral endopeptidase necessary for the degradation and inactivation of SP have been identified in the mammalian CB, and moreover, phosphoramidon, a potent inhibitor of this enzyme, potentiates the chemosensory response to hypoxia (Kumar 1997; Kumar et al. 2000). Finally, it has been shown that low oxygen enhances SP release from the glomus cells or afferent nerves in a Ca2+ -dependent manner (Kim et al. 2001). All these findings suggest that SP fulfills a number of the criteria required for conventional synaptic transmission. Possible interactions between SP and other signaling systems in the CB have not yet been persuasively elucidated.

6.2.4.2

Calcitonin Gene-Related Peptide

Calcitonin gene-related peptide (CGRP) is considered a specific marker for the identification of some, if not all, sensory nerve terminals in the PNS, and this peptide might subserve a sensory function. There is now convincing evidence that CGRPimmunoreactive nerve fibers are found around the blood vessels as well as around the clusters of glomus and sustentacular cells in the CB (Fig. 6.9) of a wide variety of species including human (Kummer and Habeck 1991), rat (Kondo and Yamamoto 1988; Atanasova et al. 2016b), cat (Torrealba and Correa 1995), guinea pig (Heym

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Fig. 6.8 SP-immunoreactivity in the rat carotid body (CB). a At a low magnification, immunoreactivity is observed in the nerve fibers distributed in the stroma around CB glomeruli and blood capillaries. b Higher magnification of the boxed area in a shows SP-immunostained fine pericellular arborizations (arrows) and thicker perivascular nerve fibers (arrowheads). Isolated immunopositive varicose nerve fibers are also visible in the interglomerular connective tissue in close association with blood vessels. Scale bars = 100 μm in a, 50 μm in b

and Kummer 1989; Kummer et al. 1989a) and chicken (Kameda 1989, 1998). Transection of the CSN in rats (Kondo and Yamamoto 1988) and the vagal nerve in chickens (Kameda 1998) leads to the complete absence of these fibers in the CB, thus suggesting that the vast majority of them originate from the PG or nodose ganglion, respectively. However, CGRP-immunoreactive sensory nerve fibers apposed to the glomus cells are shown to be few in number and, moreover, no synaptic membrane specializations are found at the site of apposition (Kondo and Yamamoto 1988). Therefore, CGRP is unlikely to be involved in chemoreception and it may subserve a different sensory function such as a regulation of the blood flow within this organ and/or the metabolic activity of glomus cells.

Fig. 6.9 Immunohistochemical distribution of CGRP in the rat carotid body (CB). a Low-power view of CB lobules showing immunoreactivity for CGRP in the nerve fibers distributed in the intervascular stroma around glomeruli and blood vessels. b At a higher magnification, the immunoreactive nerve fibers are clearly seen as enclosing small blood vessels (arrowheads) and cell clusters (arrows). Scale bars = 100 μm in a, 50 μm in b

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Fig. 6.10 Immunohistochemical demonstration of NPY in the rat carotid body (CB). Low-power photomicrograph a and higher-magnification view b of representative fields of the glomus organ displaying the distribution of abundant NPY-immunoreactive thick varicosities (arrows) associated with blood vessels (BV) and a few immunostained glomus cells scattered throughout the CB parenchyma. Scale bars = 100 μm in a, 50 μm in b

6.2.4.3

Neuropeptide Y

Neuropeptide Y (NPY) is a peptide co-localized with NA in many sympathetic nerves. Previous studies have reported the presence of a few NPY-immunoreactive glomus cells in the CB of young rats (Oomori et al. 1991). In addition, a dense network of NPY-immunoreactive nerve fibers innervating the glomus tissue is found around small blood vessels in the CB (Fig. 6.10) but not in synaptic contacts with the glomus cells in the rat (Kondo et al. 1986; Kummer et al. 1989b; Atanasova and Lazarov 2012), the guinea pig (Kummer 1990) and chickens (Yamamoto et al. 1989; Kameda 1999). Since all these fibers disappear after the removal of the adjacent SCG, they are considered to be postganglionic sympathetic noradrenergic fibers that influence the CB vasculature (Kondo et al. 1986; Kameda 1999).

6.2.4.4

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two important members of the glucagon/secretin superfamily, have been found in the CB of birds and various mammals as well as in its analogous structure in amphibians, the carotid labyrinth (for a recent review, see Gonkowski 2020). VIP, a peptide abundant in autonomic nerve fibers, is present in varicose nerve fibers around blood vessels and between glomus cell clusters in the CB (Fig. 6.11) of chickens (Kameda 1989, 1999), rats (Kummer et al. 1989b; Atanasova et al. 2016b), mice (Heym and Kummer 1989), cats (Lundberg et al. 1979; Wharton et al. 1980), guinea pigs (Heym and Kummer 1989; Kummer 1990), chipmunks (Ohtomo et al. 2002), monkeys (Heym and Triepel 1985) and man (Heath et al. 1988; Kummer and Habeck 1993) as well as in the carotid labyrinths of some amphibian species

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(Kusakabe et al. 1991, 1995). However, their number and distribution in the CB clearly depends on the animal species studied. Furthermore, immunohistochemical studies have also shown that contrary to other mammalian species, weak VIPimmunoreactivity is observed in glomus cells in humans (Smith et al. 1990) and rats (Atanasova et al. 2016b), although other experiments have failed to confirm these findings in chronically hypoxic or normoxic rat CB (Kusakabe et al. 1998b). It has been suggested that VIP-immunoreactive nerve fibers may have sensory (PG), sympathetic (SCG) and parasympathetic (CG) origins (Ichikawa 2002; Gonkowski 2020). Although the exact roles of VIP in the CB might be different in various animal species, taken together with previous immunohistochemical and physiological reports, it seems that this peptide regulates the intraorganic blood flow and may be associated with chemosensory mechanisms by controlling the local circulation in this organ under physiological conditions and during pathological states. The distribution of VPAC1 and VPAC2 receptors in the CB and the exact mechanisms of the VIP impact on the blood flow still await experimental confirmation in further studies. On the other hand, PACAP, which has 68% sequence homology with VIP, has been observed in the rat glomus cells, which are also endowed with its specific Gprotein-coupled receptor PAC1 (Lam et al. 2012). These authors have also found that the number of glomus cells containing PACAP protein and its receptor as well as PAC1 mRNA levels in them are significantly increased during chronic and intermittent hypoxia (Lam et al. 2012). In addition, some mechanisms observed during the stimulatory effect of PACAP on the CB are similar to those observed during hypoxia (Xu et al. 2008), which strongly suggests that this peptide has an important function in the regulation of CB activity under both normoxic and hypoxic conditions. Since PACAP has been shown to exert trophic effects during hypoxia, its possible role in the production of morphological hypoxic changes in the CB may also be hypothesized (Porzionato et al. 2008).

Fig. 6.11 Immunohistochemical localization of VIP in the rat carotid body (CB). a Representative photomicrograph at low magnification indicating the presence of VIP-immunoreactive structures in the glomus tissue. Boxed region in a is shown at higher magnification in b. Note the immunopositive perivascular varicose nerve fibers (arrowheads) and occasional immunostained glomus cells (arrows) in cell clusters. Scale bars = 100 μm in a, 50 μm in b

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Galanin

Galanin (GAL), a pleiotropic neuropeptide that is widely expressed in the nervous and endocrine systems, has been identified by immunohistochemistry in the nerve fibers innervating the rat (Ichikawa and Helke 1993; Finley et al. 1995) and chicken CB (Kameda 1989, 1999). Their origin and plasticity in the rat CB have been demonstrated after transection of the CSN and SCG removal. It has been shown that after a CSN transection with a combination of ganglionectomy all GAL-immunoreactive fibers disappear, whereas many immunostained glomus cells, which are not observed in the normal CB, appear in the operated CB within three days after axotomy (Ichikawa and Helke 1993). This suggests that SCG removal perhaps enhances the effect of the nerve cut on GAL content in glomus cells. Denervation (Ichikawa and Helke 1993) and retrograde tracing studies (Finley et al. 1995) have demonstrated that the majority of GAL-immunopositive fibers originate from the PG and have a sensory nature. In chicken CB, GAL-immunoreactive nerve fibers have been reported to derive from the 14th cervical ganglion of the sympathetic trunk (Kameda 1999). The presence of GAL in nerve fibers suggests the possibility of its local action on the CB. Identification of two receptor subtypes, GAL1 and GAL2, in the glomus cells in rats (Porzionato et al. 2010) and experimental studies on CB cell cultures may explain the trophic actions of this peptide on glomus cells. Lately, Di Giulio et al. (2015) reported on the selective expression of GAL in glomus cells in the human CB. Another study has provided further immunohistochemical evidence for the role of GAL as a modulator of neural stem cell function and its importance for CB plasticity and repair (Mazzatenta et al. 2014).

6.2.4.6

Enkephalins

Among endogenous opioid peptides, almost all glomus cells in the mammalian CB express enkephalins (ENK; Fidone and González 1986). In particular, met/ leu-ENK-like immunoreactivity has been found in the CB in cat (Lundberg et al. 1979; Wharton et al. 1980; Hansen et al. 1982; Varndell et al. 1982; Heym and Kummer 1989; Wang et al. 1992), rabbit (Hanson et al. 1986; Heym and Kummer 1989), piglet (Varndell et al. 1982), shrew and dog (Heym and Kummer 1989), human (Heath et al. 1988; Smith et al. 1990; Scraggs et al. 1992) and rat (Atanasova and Lazarov 2012) though in the rat CB only nerve fibers but not glomus cells have initially been shown to exhibit met-ENK-like immunoreactivity (Heym and Kummer 1989). Our further immunohistochemical experiments have additionally revealed that not only nerve fibers but also some glomus cells in the rat CB display intense mENK-immunoreactivity (Fig. 6.12). In addition, physiological chemoreceptor stimulation decreases met-ENK content in the rabbit CB indicating its release by hypoxia (Hanson et al. 1986). Experimental studies in cats with the application of selective opioid receptor agonists and antagonists have provided evidence that the depression of the chemosensory discharge and the sensory response to hypoxia

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Fig. 6.12 mENK-immunoreactivity in the rat carotid body (CB). a At a low magnification, mENK-immunoreactivity is mostly observed in the nerve fibers within and around cell clusters. A few intensely stained glomeruli are also seen. b Higher magnification of the boxed area in a shows numerous mENK-immunostained intraglomerular (arrow) and perivascular nerve fibers (arrowheads). Scale bars = 100 μm in a, 50 μm in b

caused by ENK involves delta-opioid receptors (Pokorski and Lahiri 1981; Kirby and McQueen 1986), and such receptors have been identified by immunohistochemistry in both rat CB glomus cells and nerve fibers (Ichikawa et al. 2005). Nonetheless, the functional significance of ENK in hypoxic sensory transmission of the CB has not been established yet.

6.2.4.7

Endothelin

Endothelin (ET), a potent endothelial cell-derived vasoactive peptide, is hardly detectable in mammalian glomus cells under normoxic conditions but its expression markedly increases during prolonged hypoxia (He et al. 1996). It excites CB chemoreceptors by acting on its G-protein-coupled receptor type A (ETA) and induces glomus cell mitosis both in vitro and in vivo in order to adapt the CB to chronic hypoxia (Paciga et al. 1999; Chen et al. 2002). Under such conditions, ET increases cAMP levels in the CB and the activated second messenger signaling pathways promote the phosphorylation of Ca2+ -channel protein, thereby enhancing hypoxia-evoked intracellular Ca2+ levels (Chen et al. 2000a, b). These studies suggest that ET is a potent modulator of the sensory response to hypoxia (for a review, see Rey and Iturriaga 2004).

6.2.4.8

Angiotensin

Angiotensin II (Ang II), a vasoactive peptide hormone, is a major component of the renin-angiotensin system (RAS) that exists under physiological conditions in the rat CB and plays a key role in its modulatory response to hypoxia (Lam and Leung 2002; Leung et al. 2003). RT-PCR has identified gene expression of both Ang II type

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Fig. 6.13 Immunohistochemical demonstration of AT1 receptors in the rat carotid body (CB). Low- (a) and high (b)-power photomicrographs indicating that a subset of CB glomeruli is AT1 receptor immunostained. Higher magnification of the rectangle in a reveals that the receptor protein is expressed perinuclearly in the glomus cells (arrows) while the surrounding blood vessels and nerve fibers in-between are immunonegative. Scale bars = 100 μm in a, 50 μm in b

1 (AT1) and type 2 (AT2) receptors in the rat CB (Fung et al. 2001), and AT1 receptors have also been identified in glomus cells (Fig. 6.13) by immunohistochemistry (Fung et al. 2001; Atanasova et al. 2018). Ang II is also known as a potent mediator of oxidative stress since it induces intracellular formation of ROS which could serve as important intermediates in several signal transduction pathways to increase the CB excitability in normoxia (Peng et al. 2011). Accordingly, it has been shown that multiple intermediate metabolites of RAS and their corresponding receptors can influence the glomus cell function and enhance afferent chemoreceptor activity (Fung 2015). Specifically, it has been established that Ang II itself increases rat CB efferent activity, presumably via the activation of the AT1 receptor (Allen 1998), and that chronic hypoxia intensifies AT1 receptor-mediated efferent activity as well (Leung et al. 2000; Lam et al. 2014; Fung 2015). In addition, it has been demonstrated that the blockade of AT1 receptors with losartan inhibits the intracellular signaling pathway, thus decreasing calcium levels in the glomus cells and, moreover, the losartan-mediated decrease of AT1 receptors in hypoxic rats significantly lessens the levels of oxidative stress in the CB (Lam et al. 2014).

6.2.4.9

Leptin

Leptin (LEP) is a peptide hormone predominantly released from adipose tissue that is now recognized as a central regulator of systemic energy homeostasis. In addition, circulating leptin has been implicated in the respiratory and cardiovascular control involving carotid chemoreceptors. Leptin and its receptor isoforms have been identified in rat and human glomus cells (Porzionato et al. 2011), suggesting that it may also play an important role in modulating CB chemosensitivity during hypoxic conditions. Indeed, it has recently been revealed that the hypoxia-induced increase in the

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CSN activity is enhanced by leptin administration and this effect is prevented by CB denervation (Caballero-Eraso et al. 2019). Accordingly, the expression of leptin in the CB is upregulated and the leptin receptors are downregulated by intermittent hypoxia (Messenger and Ciriello 2013). This study also shows that CB glomus cells, when activated by leptin during intermittent hypoxia, further increase their production of leptin so that it can either exert an effect on the cell itself or on neighboring cells to alter their ability to transduce changes in arterial PO2 . There is also evidence that leptin activates leptin receptors acting on TRPM7 channels, thus inducing an increase in sympathetic nerve activity and elevated blood pressure in mice (Shin et al. 2019). In addition, leptin increases the frequency of CSN discharge and the release of adenosine from the CB but does not modify intracellular Ca2+ in its chemoreceptor cells (Ribeiro et al. 2018). Altogether, these data suggest the possibility that leptin exerts modulatory effects at multiple levels throughout the chemoreceptor reflex pathway and support the concept that the CB activity may be a common link between hypertension and metabolic dysfunction. Nonetheless, the contribution of leptin within the CB to glucose homeostasis needs further investigation.

6.2.4.10

Insulin

Insulin (IN), a 51-amino acid peptide hormone, apart from its pivotal role in the regulation of glucose homeostasis, also serves as a vascular and sympathoexcitatory hormone in both the central nervous system and periphery. Accumulating evidence suggests that circulating insulin can activate the CB resulting in increased sympathetic tone and hyperventilation in rats and that CSN resection abolishes these effects (Bin-Jaliah et al. 2004; Ribeiro et al. 2013). A recent study has additionally revealed that the intracarotid insulin administration leads to augmented CSN activity and respiratory responses in dogs, suggesting a strong modulatory effect of IN on peripheral chemoreceptors that control the cardiorespiratory neuronal networks in the brainstem (Baby et al. 2023). In fact, IN acts on IN receptors present in the CB chemoreceptor cells (Ribeiro et al. 2013) eliciting an increase in intracellular Ca2+ and the release of neurotransmitters, such as dopamine and ATP (Conde et al. 2014). This IN-induced neurosecretory response in chemoreceptor cells is then transduced in an increase in ventilation and in an augmented sympathetic outflow (Conde et al. 2014). It has also been reported that IN triggers CB activation through the activation of Kv1.3 channels (Ribeiro et al. 2016). The CB overactivation is increasingly recognized as an important feature of metabolic diseases and since its denervation prevents metabolic and hemodynamic alterations in animal models, the CB may be a common denominator for metabolic dysfunction which could be implicated in IN resistance (Conde et al. 2018; Badoer 2020). As suggested by Conde et al. (2014), one way to modulate CB activity, and thus to treat metabolic diseases related with IN resistance, would be to directly target its effector, the sympathetic nervous system. Furthermore, controlling the overactive sympathetic outflow to the CB chemoreceptors by pharmacological agents and/or by bioelectronic modulation might be also beneficial for other pathophysiological

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conditions, such as hypertension, heart failure and sleep apnea (Baby et al. 2023). However, recent human studies in young, healthy individuals have demonstrated that CB desensitization does not attenuate the sympathoexcitatory response to hyperinsulinemia (Limberg et al. 2020). It has yet to be thoroughly tested whether this is also true in IN-resistant subjects. Nevertheless, it is tempting to suggest that targeting IN signaling in the CB could be another clinical approach to treat cardiorespiratory and metabolic disorders associated with CB hyperactivity (Holmes and Kumar 2023).

6.2.5 Calcium-Binding Proteins The presence of calcium-binding proteins (CaBPs), including calretinin (CR), calbindin D-28 k (CB) and parvalbumin (PV), has been shown in the rat CB. In particular, numerous CR- or CB-immunoreactive nerve fibers and very few PVimmunostained nerve fibers are found in the CSN and throughout the rat CB innervating mostly clusters of glomus cells but also associated with blood vessels (Ichikawa and Helke 1997; Kusakabe et al. 2000). Besides, CR-containing neurons are detected in the rostral portion of the PG and are scattered throughout the nodose and jugular ganglia (Ichikawa et al. 1991), while CB- and PV-containing neurons are found in the caudal portion of the PG and nodose ganglia and only rarely in the jugular ganglion (Ichikawa and Helke 1995, 1997). Moreover, a coexistence of CaBPs is found in neurons of the PG, nodose and jugular ganglia but not in the CB (Ichikawa and Helke 1997). Calbindin is also present in fine varicose nerve fibers in the chronically hypoxic rat CB though their density is decreased by 70% than in normoxic CB (Kusakabe et al. 2000). Similar changes in the distribution of NOScontaining nerve fibers in the chronically hypoxic rat CB have also been reported (Kusakabe et al. 1998a). These findings propose a possible coexistence of these two substances and suggest their common involvement in acclimatization to hypoxia. On the other hand, the altered innervation may indicate changes in transmitter release at synaptic regions, whose processes are involved in changes in calcium concentrations in neurons and, thus, an involvement of CaBPs in the neural pathway that modulates CB chemoreception.

6.2.6 Gaseous Messengers In addition to conventional neurotransmitters, it is being increasingly appreciated that all three major gaseous messenger molecules, i.e., nitric oxide, carbon monoxide and hydrogen sulfide, and the enzymes associated with their synthesis are broadly expressed in the CB and, moreover, they exert powerful influences on its ability to sense O2 via ion channel modulation in the glomus cells (reviewed in Prabhakar and Semenza 2012). Emerging evidence further suggests that nitric oxide and carbon monoxide are inhibitory, whereas hydrogen sulfide is an excitatory gasotransmitter in the CB.

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6.2.6.1

89

Nitric Oxide

Nitric oxide (NO), one of the most potent vasodilators and a neurotransmitter in the nervous system (Snyder 1992), is enzymatically produced from the amino acid l-arginine by a family of enzymes called NO synthases (NOS), which have three isoforms. The neuronal (nNOS) and the endothelial (eNOS) isoforms are constitutive and require Ca2+ for their activity, whereas the inducible one (iNOS) is expressed in response to environmental stimuli, including hypoxia. Expression of nNOS is not evident in glomus cells but is restricted to nerve fibers (Fig. 6.14) associated with lobules of chemosensory glomus cells as well as with the vascular endothelium of the CB in rats (Höhler et al. 1994), cats (Wang et al. 1993, 1994, 1995b; Grimes et al. 1995) and guinea pigs (Tanaka and Chiba 1994). In addition, it has been reported that the CB blood vessels are richly innervated by eNOS-immunoreactive varicosities (Wang et al. 1994). Denervation and retrograde tracing experiments have subsequently shown that the CB in rats is richly innervated by both afferent and efferent NOS-containing fibers from PG, SCG and autonomic microganglia neurons (Wang et al. 1993; Prabhakar 1999; Campanucci and Nurse 2007; Atanasova et al. 2016a). Also, electrical stimulation of the CSN, or CB exposure to hypoxia, causes an elevation in NO production that is prevented by specific nNOS antagonists (Prabhakar 1999). These effects of NO seem to be mediated by cyclic guanosine monophosphate (cGMP), consistent with the known ability of NO to activate a soluble form of guanylate cyclase (Wang et al. 1995a, b). It is known that the biological actions of NO are mediated by multiple pathways, which include activation of heme-containing guanylate cyclase and subsequent elevation of cGMP levels (Snyder 1992). The available evidence suggests that NO modulates the chemoreception process in the CB at different sites and modes, i.e., indirectly by modifying the vascular tone and oxygen delivery, and directly

Fig. 6.14 Expression of neuronal NOS in the rat carotid body (CB). a Low-power photomicrograph demonstrating the localization of nNOS-immunoreactivity in CB glomeruli. b Higher magnification of the area inside the rectangle in a shows that nNOS-immunostaining is present in thin varicosities (arrows) around the immunonegative glomus cells. Occasional nNOS-containing cells (arrowheads) are seen at the periphery of the CB. Scale bars = 100 μm in a, 50 μm in b

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through the modulation of the excitability of glomus cells and PG neurons. This idea is supported by the observations that physiological concentrations of NO produce tonic inhibition of the glomus cell activity in normoxia by increasing blood flow and oxygen delivery to them and reducing the augmented chemosensory discharges induced by hypoxia (reviewed in Moya et al. 2012). Given that molecular oxygen is a co-factor in the biosynthesis of NO, it seems that NO basal levels in normoxia act as amplifiers of molecular oxygen and keep the sensory discharge low, whereas the decreased synthesis of NO during hypoxia may contribute in part to the augmentation of sensory discharge (Prabhakar 1999). Summers et al. (1999) have demonstrated that NO selectively inhibits voltage-gated L-type (and not N-type) Ca2+ currents in rabbit glomus cells via a cGMP-independent way, providing a simple and direct mechanism to account for the inhibitory effects of NO on CB hypoxic sensitivity. Therefore, it is now believed that the inhibition of CB function is mediated by NO released from their efferent fibers (Prabhakar 1999; Campanucci et al. 2012). Besides, the release of the excitatory transmitter ATP under hypoxic conditions can excite afferent nerve fibers but can also activate Ca2+ influx into efferent neurons, thereby activating nNOS and generating NO in a negative feedback pathway. In line with this, neurons in the glossopharyngeal nerve that express nNOS also express P2X2, P2X3, P2X4 and P2X7 purinoceptors (Campanucci et al. 2006). In fact, the release of excitatory neurotransmitter ATP during hypoxic stress from chemoreceptors may trigger, via a variety of P2X receptors, additional NO synthesis, leading to CB inhibition (Campanucci et al. 2006). NO may inhibit glomus cells by activating Ca2+ -dependent K+ channels (Campanucci et al. 2006) or inhibiting L-type Ca2+ channels (Summers et al. 1999). Thus, the interaction of NO with multiple ion channels can account for its negative feedback control of CB output (Prabhakar and Peers 2014).

6.2.6.2

Carbon Monoxide

Carbon monoxide (CO) is endogenously generated in mammalian cells during degradation of heme by the enzyme heme oxygenase (HO). Its constitutively expressed isoform, HO-2 is found in the glomus cells and not in the nerve fibers or other cell types in the CB of cat, rat (Prabhakar et al. 1995) and mouse (Ortega-Sáenz et al. 2006), thus suggesting that glomus cells are the primary source of HO-2. It is also known that physiological levels of hypoxia inhibit HO activity and reduce CO generation (Prabhakar 2012). Studies with HO inhibitors have revealed that its blockade by zinc-protoporphyrin-9 stimulates the CB activity in a dose-dependent manner, whereas low concentrations of CO inhibit it (Prabhakar et al. 1995). Furthermore, the application of zinc-protoporphyrin-9 augments the hypoxic ventilatory response and bilateral sectioning of CSN abolishes this effect (Prabhakar 1998). Taken together, these pharmacological and denervation observations, along with amperometric recorded elevated CB baseline sensory activity in HO-2 knockout mice (Ortega-Sáenz et al. 2006), indicate that CO is an inhibitory gasotransmitter in the CB (Peng et al. 2010).

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Recent studies have provided interesting insights into the cellular mechanism(s) by which CO exerts its inhibitory influence on CB activity. The idea that CO might influence CB chemosensitivity via modulation of ion channels has yet to gain further credibility. It is likely that once formed by glomus cells, CO influences cGMP formation and/or K+ channel activity and cytosolic Ca2+ levels in adjacent glomus cells, a paracrine effect, or may act back on the same cell, an autocrine action (Prabhakar et al. 1995; Overholt et al. 1996; Prabhakar 1998). Although previous studies have reported that glomus cell secretory response often does not coincide with sensory excitation of the CB by hypoxia (Donnelly 1996), it is being increasingly recognized that under hypoxic conditions, tonic production of CO is inhibited, leading to channel closure, which could lead to depolarization, transmitter release, and CSN excitation (Prabhakar and Peers 2014).

6.2.6.3

Hydrogen Sulfide

Hydrogen sulfide (H2 S), together with NO and CO, is now considered to be the third member of the gaseous transmitter family. Emerging evidence suggests that H2 S, endogenously generated by the enzyme cystathionine-γ-lyase (CSE), is an excitatory gasotransmitter of the CB, enhancing its sensory response to hypoxia. It has been shown that glomus cells of rat, mouse and cat CB express CSE (Peng et al. 2010; Li et al. 2010; Fitzgerald et al. 2011). Unlike NO and CO, H2 S levels are low in normoxic CB but increase in isolated glomus cells challenged with hypoxia (Makarenko et al. 2012). Likewise, exogenous application of NaHS, an H2 S donor, stimulates CB activity in mice and rats in a concentration-dependent manner and, more importantly, the time course and magnitude of such responses resemble those evoked by hypoxia (Peng et al. 2010; Li et al. 2010). Moreover, genetic deletion or pharmacological inhibition of CSE significantly reduces basal H2 S levels in the CB and dramatically impairs hypoxic chemosensitivity (Peng et al. 2010). All these collective approaches strongly suggest that CSE-derived H2 S is a major physiological contributor to CB stimulation by hypoxia (Prabhakar and Semenza 2012). The excitatory effects of H2 S on CB activity seem to require Ca2+ influx (Peng et al. 2010; Li et al. 2010) and may involve inhibition of both known O2 -sensitive K channels in glomus cells: maxiK and TASK-like channels (Telezhkin et al. 2010; Buckler 2012). It has been hypothesized that the low levels of H2 S under normoxia are due to the inhibitory influence of CO on CSE and that hypoxia reduces HO-2 activity to reverse the inhibition and augment H2 S formation (Peng et al. 2010). Therefore, it is plausible that hypoxia-evoked H2 S generation in the CB requires the biochemical interaction of CSE with HO-2, and such interactions between them work in concert with ion channels (particularly K channels) in glomus cells. It is likely that the functional interaction between inhibitory (CO) and excitatory (H2 S) gaseous transmitters constitutes an important component of the chemosome hypothesis (Prabhakar 2006), as mentioned earlier. The precise molecular mechanisms by which CO inhibits H2 S generation from CSE in the CB, however, still await elucidation. These modulatory

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pathways probably play an essential role in mediating the plastic changes in chemoreceptor function which are important for physiological adaptations to chronic hypoxia in health and disease. A better understanding of their functional role may therefore be of clinical interest because the enzymes that generate gas messengers and redox regulation by hypoxia-inducible factors (HIFs) represent potential therapeutic targets for normalizing CB function and downstream autonomic output in the most prevalent cardiorespiratory diseases. In conclusion, neurotransmitters are critical for hypoxia-induced afferent nerve stimulation, e.g., some of them stimulate, whereas others inhibit it (see López-Barneo et al., 2016 and references therein). It appears that the CB chemosensory output reflects a fine balance of conventional excitatory and inhibitory signaling pathways, together with gliotransmission (Leonard and Nurse 2023). Among the various neurotransmitters found in glomus cells, ATP and ACh seem to activate the sensory afferent fibers, while other transmitters (e.g., 5-HT, GABA, and adenosine) and neuropeptides (e.g., opioids, ET, and SP) expressed in the CB or by efferent autonomic fibers modulate the glomus cell chemosensory response (for a review, see Nurse 2014). In addition, several gas compounds, such as NO, CO and H2 S are produced in the CB and alter the activity of ion channels in glomus cells. It is also clear that most of the aforementioned neuropeptides are often co-localized in the CB, and moreover, they are co-released during hypoxia in an autocrine or paracrine fashion. From the available evidence, it appears that the inhibitory messengers work in tandem with excitatory transmitters like a push–pull-like mechanism and their complex interactions might play a more important role in CB plasticity (Prabhakar 2006). Compliance with Ethical Standards This study was partially financed by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01 and by a scientific project No. 5/2022 funded by the Faculty of Medicine to the Trakia University, Stara Zagora, Bulgaria. The authors declare no conflict of interest. All applicable international, national, and/ or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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Wang ZZ, Dinger BG, Stensaas LJ, Fidone SJ (1995a) The role of nitric oxide in carotid chemoreception. Biol Signals 4:109–116 Wang ZZ, Stensaas LJ, Dinger BG, Fidone SJ (1995b) Nitric oxide mediates chemoreceptor inhibition in the cat carotid body. Neuroscience 65:217–229 Wang Z-Y, Keith IM, Beckman MJ, Brownfield MS, Vidruk EH, Bisgard GE (2000) 5-HT5a receptors in the carotid body chemoreception pathway of rat. Neurosci Lett 278:9–12 Wharton J, Polak JM, Pearse AGE, McGregor GP, Bryant MG, Bloom SR, Emson PC, Bisgard GE, Will JA (1980) Enkephalin-, VIP- and substance P-like immunoreactivity in the carotid body. Nature (London) 284:269–271 Woods RI (1967) Distribution of cytochrome oxidase, monoamine oxidase and carbonic anhydrase in the carotid body of the rabbit. Nature 213:1240 Woods RI (1975) Penetration of horseradish peroxidase between all elements of the carotid body. In: Purves MJ (ed) The peripheral arterial chemoreceptors. Cambridge Univ Press, London, pp 195–205 Xu J, Tse FW, Tse A (2003) ATP triggers intracellular Ca2+ release in type II cells of the rat carotid body. J Physiol 549:739–747 Xu F, Tse FW, Tse A (2008) Stimulatory actions of pituitary adenylate cyclase-activating polypeptide (PACAP) in rat carotid glomus cells. Adv Exp Med Biol 605:69–74 Yamamoto M, Kondo H, Nagatu I (1989) Immunohistochemical demonstration of tyrosine hydroxylase, serotonin and neuropeptide tyrosine in the epithelioid cells within arterial walls and carotid bodies of chicks. J Anat 167:137–146 Yokoyama T, Misuzu YY, Yamamoto Y (2013) Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat. Histochem Cell Biol 140:147–155 Yokoyama T, Nakamuta N, Kusakabe T, Yamamoto Y (2014) Vesicular glutamate transporter 2immunoreactive afferent nerve terminals in the carotid body of the rat. Cell Tissue Res 358:271– 275 Zapata P (1977) Modulatory role of dopamine on arterial chemoreceptors. Adv Biochem Psychopharmacol 16:291–298 Zapata P (2007) Is ATP a suitable co-transmitter in carotid body arterial chemoreceptors? Respir Physiol Neurobiol 157:106–115 Zhang M, Nurse CA (2000) Does endogenous 5-HT mediate spontaneous rhythmic activity in chemoreceptor clusters of rat carotid body? Brain Res 872:199–203 Zhang M, Nurse CA (2004) CO2 /pH chemosensory signaling in co-cultures of rat carotid body receptors and petrosal neurons: role of ATP and ACh. J Neurophysiol 92:3433–3445 Zhang M, Zhong H, Vollmer C, Nurse CA (2000) Co-release of ATP and ACh mediates hypoxic signalling at rat carotid body chemoreceptors. J Physiol 525:143–158 Zhang M, Fearon IM, Zhong H, Nurse CA (2003) Presynaptic modulation of rat arterial chemoreceptor function by 5-HT: role of K+ channel inhibition via protein kinase C. J Physiol (Lond) 551:825–842 Zhang M, Clarke K, Zhong H, Vollmer C, Nurse CA (2009) Postsynaptic action of GABA in modulating sensory transmission in co-cultures of rat carotid body via GABAA receptors. J Physiol 587:329–344 Zhang M, Piskuric NA, Vollmer C, Nurse CA (2012) P2Y2 receptor activation opens pannexin-1 channels in rat carotid body type II cells: potential role in amplifying the neurotransmitter ATP. J Physiol 590:4335–4350 Zhao C, Li C, Zhao B, Liu Y (2022) Expression of group II and III mGluRs in the carotid body and its role in the carotid chemoreceptor response to acute hypoxia. Front Physiol 13:1008073

Chapter 7

Neurochemical Plasticity of the Carotid Body

Abstract A striking feature of the carotid body (CB) is its remarkable degree of plasticity in a variety of neurotransmitter/modulator systems in response to environmental stimuli, particularly following hypoxic exposure of animals and during ascent to high altitude. Current evidence suggests that acetylcholine and adenosine triphosphate are two major excitatory neurotransmitter candidates in the hypoxic CB, and they may also be involved as co-transmitters in hypoxic signaling. Conversely, dopamine, histamine and nitric oxide have recently been considered inhibitory transmitters/modulators of hypoxic chemosensitivity. It has also been revealed that interactions between excitatory and inhibitory messenger molecules occur during hypoxia. On the other hand, alterations in purinergic neurotransmitter mechanisms have been implicated in ventilatory acclimatization to hypoxia. Chronic hypoxia also induces profound changes in other neurochemical systems within the CB such as the catecholaminergic, peptidergic and nitrergic, which in turn may contribute to increased ventilatory and chemoreceptor responsiveness to hypoxia at high altitude. Taken together, current data suggest that complex interactions among transmitters markedly influence hypoxia-induced transmitter release from the CB. In addition, the expression of a wide variety of growth factors, proinflammatory cytokines and their receptors have been identified in CB parenchymal cells in response to hypoxia and their upregulated expression could mediate the local inflammation and functional alteration of the CB under hypoxic conditions. Keywords Carotid body · Hyperoxia · Hypoxia · Neurochemical plasticity · Proinflammatory cytokines · Trophic factors · Ventilatory acclimatization to hypoxia

As stated in Chap. 4, the CB structure and function may undergo plastic modifications during development and aging. Another striking feature of the CB is its remarkable degree of plasticity in a variety of neurotransmitter/modulator systems in response to environmental stimuli, particularly during aging or following exposure of animals to both acute and chronic sustained hypoxia or perinatal hyperoxia, in order to maintain oxygen homeostasis (for recent reviews, see Kumar and Prabhakar 2012; De Caro © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_7

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et al. 2013; Prabhakar et al. 2015; Iturriaga 2018; Lazarov and Atanasova 2022; Di Giulio et al. 2023). Since the effects of chronic intermittent hypoxia on the CB are reversible, it has been proposed that it recruits additional neurotransmitters or modulators in the CB (Prabhakar 2001). Altered neurotransmitter profile of chemosensory cells in the CB also occurs during the course of ascent to high altitude.

7.1 Hypoxia-Induced Neurochemical Changes in the Carotid Body There is a growing consensus that in addition to the significant cellular rearrangement that culminates in structural plasticity, chronic hypoxia induces neurochemical changes in the chemosensory cells of the CB. It is well established that hypoxia causes glomus cells to depolarize and release (both excitatory and inhibitory) transmitters, which bind to autoreceptors or postsynaptic receptors on apposed chemoafferent nerve terminals (González et al. 1994). Multiple putative neurotransmitters are thought to mediate signals generated by hypoxia. The predominant excitatory transmitter synthesized and released by glomus cells in response to hypoxia is still a matter of debate. Current evidence suggests that acetylcholine (ACh) and adenosine triphosphate (ATP) are the two major excitatory transmitter candidates in the hypoxic CB and, accordingly, the cholinergic and purinergic hypotheses for hypoxic chemosensitivity, described in detail in the previous chapter, are introduced (reviewed by Iturriaga and Alcayaga 2004; Nurse 2005; Fitzgerald et al. 2009). Indeed, hypoxia induces an increased release of ACh in the rat (Nurse and Zhang 1999), rabbit (Kim et al. 2004), cat (Fitzgerald et al. 1997) and human CB (Kåhlin et al. 2014). Similarly, hypoxia-induced ATP release from glomus cells has been demonstrated in the whole CB and tissue slices in vitro (Buttigieg and Nurse 2004) and, moreover, this release and its consequent actions on the chemoreceptor discharge may increase in response to hypoxia (González et al. 1994; Conde and Monteiro 2004). However, ATP exerts strong negative feedback regulation on hypoxia signaling in the rat glomus cells via the closure of background conductance(s) other than the TASK-like K+ , maxiK or Na+ channels under normoxic conditions (Xu et al. 2005). Consequently, the co-release of ACh and ATP, which came up with the cholinergic-purinergic hypothesis, has been put forward to account for the main mechanism mediating hypoxic chemotransmission in the rat CB (Zhang et al. 2000). Another widely proposed, but still controversial, mechanism is represented by the inhibitory action of dopamine (DA) in CB chemoexcitation in humans (Lazarov et al. 2011). In particular, it has been reported that chronic hypoxia upregulates dopaminergic activity in glomus cells (Wang and Bisgard 2002) and that its effects may explain increased oxygen-sensitivity in chronically hypoxic CB (Bisgard 2000). Short-term hypoxia also induces an increase in tyrosine hydroxylase (TH) mRNA and protein expression in rat CB glomus cells (Czyzyk-Krzeska et al. 1992; Kato et al. 2010). Likewise, long-term hypoxia causes a dramatic increase in the number of glomus

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cells containing noradrenaline (NA) (Verna et al. 1993b; Schamel et al. 2016). These results suggest an alternative explanation based both on a dynamic balance between the inhibitory action of DA and the excitatory effect of NA (Schamel et al. 2016). However, it has been reported that hypoxia-induced sensory discharge in the CB is unrelated to catecholamine efflux, at least in rats and cats (Donnelly 1996; Iturriaga et al. 1996). On the other hand, there is convincing evidence that histamine (HIS) is released from the rat CB during hypoxia (Koerner et al. 2004). Our recent data have additionally proved the modulatory role of HIS as an essential excitatory modulator of the chemoreceptor activity upon hypoxia in man (Lazarov et al. 2011; Lazarov and Atanasova 2012). In line with this, there is recent experimental evidence for the presence of A2A -D2 and D2 -H3 heterodimers in both the glomus and sustentacular cells as well as in afferent terminals of the rat and human CB, and that these receptor– receptor interactions may play a role in the chemosensory modulation in response to hypoxic stimuli (Stocco et al. 2021, 2022). Recently, it has been reported that Ca2+ /calmodulin-dependent protein kinase II subunits activate the vesicular release of NA and ATP from the chemoreceptor cells and may also enhance glutamatergic transmission from nerve endings in the rat CB, thus modulating reciprocal synaptic transmission between glomus cells and nerve terminals of petrosal neurons (Saito et al. 2023). In addition, it has lately been shown that the activation of group II or III mGluR subunits presynaptically attenuates the CB response to acute hypoxia by inhibiting glutamate (GLU) release and/or facilitating gamma-aminobutyric acid (GABA) release from glomus cells (Zhao et al. 2022). In fact, these authors report that the application of their agonists (LY379268 or L-SOP, respectively) lower hypoxia-induced enhancement of carotid sinus nerve activity but the exact mechanisms by which these receptor subunits reduce the CB chemoreflex response to hypoxia still need to be clarified. There is also limited evidence about the role of inhibitory GABAergic signaling on CB responses to acute hypoxia. Neuropeptide release from the rabbit CB in response to hypoxia has also been reported (Hanson et al. 1986; Kim et al. 2001). Indeed, some credible evidence claims that altered peptidergic innervation of the chronically hypoxic CB is another feature of hypoxic adaptation. It has been demonstrated that the absolute number of vasoactive intestinal peptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers in the chronically hypoxic CB is significantly increased, while that of substance P (SP)and calcitonin gene-related peptide (CGRP)-containing fibers remains unchanged (Kusakabe et al. 1998b). Moreover, these authors have postulated that since both VIP and NPY are vasoactive neuropeptides in nature, altered CB circulation may contribute to modulation of the chemosensory mechanisms by chronic hypoxia. It is also revealed that chronic intermittent hypoxia increases the expression of another vasoactive peptide, endothelin (ET) in the CB and that bosentan, a pan ET receptor antagonist prevents the induced sensitization of the hypoxic sensory response (Rey et al. 2007, 2008). On the other hand, met/leu-enkephalin levels are significantly reduced in the hypoxic rabbit CB (Hanson et al. 1986). Likewise, studies by Prabhakar et al. (1993) have demonstrated that nitric oxide synthase (NOS) activity is noticeably inhibited in CB extracts exposed to hypoxia. In addition, Kusakabe and coworkers have reported that the density of the nitrergic and calbindin D-28k fibers in

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the chronically hypoxic rat CB is markedly decreased (Kusakabe et al. 1998a, 2000). Since NO is an inhibitory neuronal messenger in the normoxic CB, the authors conclude that the sensory mechanisms in the hypoxic CB may be involved in ‘disinhibition’ resulting from reduced NO synthesis. However, Iturriaga et al. (2000) have not been able to find alterations in NO levels during hypoxia in an ex vivo cat CB preparation. Therefore, the possibility of coexistence of these two substances, mentioned above, suggests their interaction during acclimatization to hypoxia and may indicate an involvement of a calcium-binding protein for NOS activation. In a series of experiments, Lahiri and his coworkers have found that heme proteins are also involved in sensory excitation by hypoxia (see Lahiri et al. 2006, and references therein). It is known that chronic hypoxia is also associated with elevated sympathetic activity and hypertension. Considering the involvement of the CB and the putative role of certain neuroactive substances in the pathogenesis of hypertension, discussed in Chap. 8, we have recently established the altered neurotransmitter profile of CB chemosensory cells in the hypertensive state (Atanasova et al. 2020, 2023). Specifically, in the hypertensive CB, the catecholamine and serotonin content is markedly higher while GLU and GABA levels are significantly reduced compared to normotensive controls (Figs. 7.1 and 7.2). Our results also reveal a statistically significant decrease in the apparent density of SP- and VIP-immunoreactive nerve fibers, whereas that of NPY- and CGRP-immunostained fibers remain almost unchanged in the hypertensive CB (Figs. 7.3 and 7.4). We have further demonstrated that under hypertensive conditions, the production of nitric oxide is impaired (Figs. 7.5 and 7.6) and that the components of the neurotrophin signaling system (neurotrophins and their receptors) display an abnormally enhanced expression (Figs. 7.7, 7.8 and 7.9) which possibly modulates the chemosensory processing. Furthermore, hypoxia seems to share a similar neurochemical phenotype with the aging process in both humans and animals. Indeed, there is recent evidence that the morphological changes during aging are accompanied by age-dependent reductions in the release of essential signaling molecules like hypoxia-inducible factor one-alpha (HIF-1α), vascular endothelial growth factor (VEGF) and NOS which are engaged in the process of aging (Di Giulio et al. 2023).

7.2 Transmitter Mechanisms of Acclimatization The CB plasticity mechanisms underlie the so-called ventilatory acclimatization to hypoxia (VAH), an adaptive process to high altitudes. It has been reported that VAH occurs in humans and several animal models exposed to chronic hypoxia (Powell et al. 1998) and is characterized by enhanced CB chemosensory responses (Vizek et al. 1987). It is largely determined as functional changes occurring in the expression of ion channels, resulting in increased glomus cell excitability, and by neurochemical interactions between a variety of neurotransmitter systems and their receptors in the glomus cells. Moreover, the structural changes and complex interrelationships among

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Fig. 7.1 Immunohistochemical localization of classical neurotransmitters in the hypertensive carotid body (CB) of the rat. Representative photomicrographs showing the expression of TH(a, b), 5-HT- (c, d), GLU- (e, f) and GABA-immunoreactivity (g, h) in the CB of normotensive Wistar rats (NWR; left column) and their age-matched spontaneously hypertensive rats (SHR; right column). Please note the stronger and more distinct TH (b) and 5-HT (d) immunostaining in the cytoplasm of glomus cells in hypertensive than in control animals. Also note that larger number of GLU- (e) and GABA-immunoreactive (g) glomus cells is found in the CB parenchyma of the normotensive rats in contrast to their number and staining intensity in hypertensive rats, where it is markedly weaker (f and h, respectively). Scale bars = 50 μm

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Fig. 7.2 Statistical comparison of the staining intensity of tyrosine hydroxylase (TH), serotonin (5HT), glutamate (GLU) and gamma-aminobutyric acid (GABA) in control NWR rats (green boxes, n = 5) and SHRs (orange boxes, n = 5). The data are compared using the Mann–Whitney’s U test, where p < 0.001 in comparison with the NWR control group and presented as box plots diagram, where the line within the box represents the median

transmitters markedly influence hypoxia-induced responses in the human CB, thus implying its essential role in VAT (Lazarov and Atanasova 2022). The glomus cells are the chemoreceptive elements of the CB critical in defining the ventilatory response to hypoxia and hypercapnia. Given the central role of ATP and adenosine in CB chemoexcitation, it is not surprising that alterations in purinergic neurotransmitter mechanisms, and particularly in adenosine signaling, have been implicated in VAH (Conde et al. 2012; Leonard et al. 2018). Hypoxia does not seem to significantly alter the expression of P2X receptors in afferent chemosensory fibers, but it has been shown that exposure of rats to sustained hypoxia for 4–5 days leads to the upregulation of surface-located ectonucleotidases, which catalyze the conversion of ATP to adenosine (Salman et al. 2017). Chronic hypoxia also induces profound changes in other neurochemical systems within the CB such as catecholaminergic, peptidergic and nitrergic systems (Wang and Bisgard 2002). Specifically, TH, DA and D2 receptor expression is downregulated in glomus cells of mouse (López-Barneo et al. 2008), rat (Czyzyk-Krzeska et al. 1992; Joseph et al. 2002) and cat (Tatsumi et al. 1995; Wang et al. 1998) while other studies fail to support this finding in goats (Janssen et al. 1998) or human subjects (Pedersen et al. 1999). Besides, experimental data have demonstrated that NA content does not increase and no change in noradrenergic receptor sensitivity occurs during hypoxic acclimatization in the goat CB (Ryan et al. 1993), but it gradually increases upon hypoxia by inducing its synthesis in glomus cells in the rat and rabbit CB (Pequignot et al. 1987; Verna et al. 1993a, b). It seems that DA, whose utilization increases in the early stage of hypoxia, might exert neurochemical effects on the chemoreceptors throughout the whole hypoxic

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Fig. 7.3 Immunohistochemical distribution of neuropeptides in the hypertensive carotid body (CB) of the rat. Representative light microscopic images indicating the immunohistochemical expression of SP- (a, b), CGRP- (c, d), VIP- (e, f) and NPY-immunoreactivity (g, h) in the CB of control normotensive Wistar rats (NWR; left column) and their age-matched spontaneously hypertensive rats (SHR; right column). Note that the density of SP- (b) and VIP-immunoreactive nerve fibers (f) is diminished, whereas that of NPY- (h) and CGRP-containing fibers (d) remains almost unchanged in the hypertensive CB compared to normotensive CB (a, c, e and g). Scale bars = 50 μm

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Fig. 7.4 Box plot diagrams depicting a statistical comparison of the density of SP-, CGRP-, VIPand NPY-immunoreactive nerve fibers in the control NWR (green boxes, n = 5) and SHR (orange boxes, n = 5). Note that the density of SP- and VIP-immunostained fibers is significantly reduced in the CB of hypertensive compared to control rats. No statistically significant difference in the density of CGRP- and NPY-immunoreactive nerve fibers in the two breeds of animals is observed. The data are compared using the Mann–Whitney’s U test. The level of significance is p < 0.001 in comparison with the control NWR group

exposure, whereas NA, whose utilization is stimulated later, might play a significant role only after a delay (Olson et al. 1983; Pequignot et al. 1987). Thus, it is likely that a down regulation of catecholaminergic inhibition may contribute to increased ventilatory and chemoreceptor responsiveness to hypoxia at high altitude. On the other hand, nicotinic cholinergic receptors are induced in CB postsynaptic afferent fibers during chronic hypoxia, thereby enhancing the excitatory effect of acetylcholine (Bisgard 2000). Furthermore, hypoxia overall inhibits ACh release via activation of cholinergic and dopaminergic autoinhibitory receptors in the CB (Kim et al. 2004). It has recently been shown that glomus cells have the ability to synthesize, package and release HIS in response to hypoxia, thereby contributing to the modulation of respiration within the rat (Koerner et al. 2004; Burlon et al. 2009). In particular, it has been demonstrated that hypoxia significantly increases HIS release from isolated neonatal rat CB (Koerner et al. 2004), but the subsequent activation of HIS receptors on glomus cells does not augment Ca2+ entry into them and, therefore, HIS is unlikely to provide a presynaptic positive feedback mechanism during chemotransduction (Burlon et al. 2009). It is more probable that HIS acts in synergy with other transmitters such as ATP or ACh to amplify their excitatory properties in response to hypoxia.

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Fig. 7.5 Visualization of nitrergic structures in the hypertensive carotid body (CB) of the rat. a, b Histochemical demonstration of NADPH-d activity in the CB of control NWR and SHR. NADPHd positive nerve fibers are located near the glomus cells and surround the blood vessels. Note that the perivascular and periglomerular NADPH-d-reactive varicose fibers are less numerous in SHR than in NRW. c, d Immunohistochemical localization of nNOS enzyme in the CB of NWR and SHR. A weaker staining intensity of immunoreactive nerve fibers is seen in the SHR compared to control NWR. Scale bars = 50 μm

Further, there is evidence that long-term hypoxia also induces neurochemical changes in the nitrergic and peptidergic activities. In particular, it increases the protein expression of all three NOS isoforms, ET and ET-1 receptors (Mosqueira and Iturriaga 2019), which participate in the increase of CB responsiveness induced by chronic hypoxia via upregulation of the Ca2+ current amplitude in glomus cells (Chen et al. 2002). It is believed that these two signaling pathways interfere with the protein expression of each other in response to hypoxia. In addition, a marked reduction in SP levels is observed in the cat CB glomus cells following long-term hypoxia (Wang et al. 1998). These changes suggest that some neuropeptides play a significant role in the adaptive mechanisms of chemoreceptor function which occur in response to chronic physiological stimulation. There is now compelling evidence that under physiological conditions a locally generated renin-angiotensin system exists in the rat CB (Lam and Leung 2002; Leung et al. 2003), and that this system is not only involved in the modulation of CB function under conditions of normoxia but also plays a key role in its modulatory response to

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Fig. 7.6 Densitometrical analysis of NADPH-d and nNOS-stained nerve fibers in the CB of normotensive NWR (green boxes, n = 5) and SHR (orange boxes, n = 5). The data are shown as box plots, where the boxes comprise the second and third quartiles (25–75%) and the line within the box represents the median. The whiskers extending parallel from the boxes indicate variability outside the upper and lower quartiles. Outliers are plotted as dots. The data are compared by Mann–Whitney’s U test where p < 0.001 in comparison with the NWR group

hypoxia. However, the possibility that certain less well-studied neuropeptides such as Ang II and ET might contribute to the mechanisms underlying VAT requires future investigation. Taken together, current data suggest that complex interactions among transmitters markedly influence hypoxia-induced transmitter release from the CB. Whether similar neurochemical interactions occur during pathophysiological conditions that lead to increased CB chemosensitivity is further discussed.

7.3 Trophic Responses to Hypoxia and Hyperoxia As already discussed, the CB shows higher density and hypoxic sensitivity of K+ channels and an increased hypoxic Ca2+ response during maturation. Functional maturation of the CB may also be explained with reference to the developmentally regulated expression of trophic factors and their receptors (Porzionato et al. 2008) which play a major role in its neurochemical aspects (De Caro et al. 2013).

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Fig. 7.7 Immunohistochemical demonstration of neurotrophic factors in the hypertensive carotid body (CB) of the rat. High-power magnifications of light photomicrographs demonstrate that the cell clusters of control NWR (left column) and hypertensive SHR (right column) exhibit immunoreactivity for NGF (a, b), BDNF (c, d), NT-3 (e, f) and GDNF (g, h). Note the enhanced immunostaining in the cytoplasm of a large number of neuron-like glomus cells and in a subset of glial-like sustentacular cells in the hypertensive compared to that in normotensive CB. Scale bars = 50 μm

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Fig. 7.8 Immunohistochemical visualization of neurotrophin receptors in the hypertensive carotid body (CB) of the rat. The majority of glomus cells are immunoreactive for p75 (a, b), TrkA (c, d), TrkB (e, f), TrkC (g, h) and GFRα1 receptors (i, j) in NWR (left column) and SHR (right column). Please note that both breeds of animals have almost similar expression of the low-affinity pan neurotrophic receptor p75, whereas the expression of TrkA, TrkB, TrkC and GFRα1 receptors in glomus cells is stronger in hypertensive than that in normotensive rats. Scale bars = 50 μm

In turn, neurotrophic mechanisms may influence neurotransmitter metabolism in chemosensory glomus cells. A wide variety of growth factors and their specific receptors have been identified in CB parenchymal cells in response to environmental stimuli such as hypoxia or hyperoxia (for a recent review, see Stocco et al. 2020) and under hypertensive conditions (Atanasova and Lazarov 2014). Specifically, chronic hypoxia is found to upregulate more than twice the expression of the proinflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα), and their corresponding receptors IL-1R1,

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Fig. 7.9 Box plot charts comparing the staining intensity for neurotrophic factors (a) and their corresponding receptors (b) in NWR (green boxes, n = 5) and SHR (orange boxes, n = 5). The data are presented as box and whisker plots, where the boxes represent the second and third quartiles (25–75%) and the line within the box represents the median. The lines extending parallel from the boxes are whiskers, which indicate variability outside the upper and lower quartiles. Outliers are plotted as individual dots that are in line with the whiskers. The data are compared using the Mann–Whitney’s U test, where p < 0.001 in comparison with the NWR control group. Note that only in the pan neurotrophic receptor p75, no difference in the staining intensity between the two breeds of animals is registered

glycoprotein (gp)130 and TNFR1 into the rat glomus cells during the early phases of exposure (Porzionato et al. 2013). However, long-term hypoxic exposure causes an elevation more than fivefold for IL-6, indeed not only in glomus cells but also in sustentacular cells, whereas expression of the other cytokines recovers to normal levels (Liu et al. 2009). Moreover, these authors have also provided experimental evidence in support of the hypothesis that chronic hypoxia-induced inflammation responses in the rat CB could be mediated by ETA receptors expressed by both chemosensory glomus cells and immune cells (Liu et al. 2009). An increased release of IL-1β, IL-4, IL-6, IL-8 and IL-10, as well as an enhanced expression of their receptors IL-1R1 and IL-6R, has been reported in the human glomus cells, too (Kåhlin et al. 2014). Such upregulation of IL-1β, IL-6, TNFα and VEGF is also observed under chronic intermittent hypoxia conditions (Lam et al. 2012; Del Rio et al. 2011a, b; Porzionato et al. 2013). Exogenous cytokines are found to enhance the intracellular calcium response to acute hypoxia in dissociated glomus cells (Lam et al. 2012), and these play critical roles in mediating phenotypic adjustments in glomus cells during sustained hypoxia as well. In fact, exposure to cytokines plus hypoxia significantly increases CB cell excitability and is further correlated to the upregulation of the transcription factor HIF-1α and to consequent overexpression of specific hypoxiasensitive genes such as adrenomedullin in glomus cells (Liu et al. 2013). Therefore, the upregulated expression of proinflammatory cytokines could mediate the local inflammation and functional alteration of the CB under chronic hypoxic conditions (Lam et al. 2012) but does not seem to be linked to the mechanisms underlying the potentiation of the CB chemosensory response to acute hypoxia (Del Rio et al. 2012). This suggestion still needs to be validated in human subjects.

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On the other hand, it has been revealed that long-lasting normobaric hyperoxia affects the oxygen-sensitive mechanism in the glomus cells in rats by an agedependent decreased chemosensory response to hypoxia (Di Giulio et al. 1998). Hyperoxia-treated rats tend to have lower resting ventilation and more variable breathing patterns as neonates (Bavis et al. 2010) and adult rats exhibit a diminished hypoxic ventilatory response (Bavis et al. 2003). In addition, short exposures to mild-to-moderate hyperoxia elicit developmental plasticity in this response (Bavis et al. 2023). It has been hypothesized that these developmental changes of the respiratory control system are partially related to the altered expression of neurotrophic factors in the CB following chronic exposure to perinatal hyperoxia (Dmitrieff et al. 2011). Indeed, chronic exposure to hyperoxia decreases message and protein levels of brain-derived neurotrophic factor (BDNF) which do not correspond to the increased mRNA and protein expression of its receptor TrkB (Dmitrieff et al. 2011; ChavezValdez et al. 2012). Conversely, hyperoxia does not change the expression levels for the glial cell line-derived neurotrophic factor (GDNF) mRNA or protein, but the transcript levels for its receptor RET are downregulated in the hyperoxic rat CB (Dmitrieff et al. 2011). It is possible that decreased BDNF signaling through TrkB receptors, and probably the inadequate GDNF signaling, on the glomus cell membrane may contribute to CB hypoplasia seen in hyperoxia-treated rats (Erickson et al. 1998; Wang and Bisgard 2005) and would lead to the degeneration and/or abnormal maturation of chemoafferent neurons (Hertzberg et al. 1994). Compliance with Ethical Standards This study was partially funded by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01. The authors declare no conflict of interest. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

References Atanasova DY, Lazarov NE (2014) Expression of neurotrophic factors and their receptors in the carotid body of spontaneously hypertensive rats. Respir Physiol Neurobiol 202:6–15 Atanasova DY, Dandov AD, Dimitrov ND, Lazarov NE (2020) Histochemical and immunohistochemical localization nitrergic structures in the carotid body of spontaneously hypertensive rats. Acta Histochem 122:151500 Atanasova DY, Dandov AD, Lazarov NE (2023) Neurochemical plasticity of the carotid body in hypertension. Anat Rec 306:2366–2377 Bavis RW, Olson EB Jr, Vidruk EH, Bisgard GE, Mitchell GS (2003) Level and duration of developmental hyperoxia influence impairment of hypoxic phrenic responses in rats. J Appl Physiol 95:1550–1559

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Hanson G, Jones L, Fidone S (1986) Physiological chemoreceptor stimulation decreases enkephalin and substance P in the carotid body. Peptides 7:767–769 Hertzberg T, Fan G, Finley JCW, Erickson JT, Katz DM (1994) BDNF supports mammalian chemoafferent neurons in vitro and following peripheral target removal in vivo. Dev Biol 166:801–811 Iturriaga R (2018) Translating carotid body function into clinical medicine. J Physiol 596:3067–3077 Iturriaga R, Alcayaga J (2004) Neurotransmission in the carotid body: transmitters and modulators between glomus cells and petrosal ganglion nerve terminals. Brain Res Rev 47:46–53 Iturriaga R, Alcayaga J, Zapata P (1996) Dissociation of hypoxia-induced chemosensory responses and catecholamine efflux in cat carotid body superfused in vitro. J Physiol 497(Pt 2):551–564 Iturriaga R, Villanova S, Mosqueivar M (2000) Dual effects of nitric oxide on cat carotid body chemoreception. J Appl Physiol 89:1005–1012 Janssen PL, O’Halloran KD, Pizarro J, Dwinell MR, Bisgard GE (1998) Carotid body dopaminergic mechanisms are functional after acclimatization to hypoxia in goats. Resp Physiol 111:25–32 Joseph V, Soliz J, Soria R, Pequignot J, Favier R, Spielvogel H, Pequignot JM (2002) Dopaminergic metabolism in carotid bodies and high-altitude acclimatization in female rats. Am J Physiol Regul Integr Comp Physiol 282:R765–R773 Kåhlin J, Mkrtchian S, Ebberyd A, Hammarstedt-Nordenvall L, Nordlander B, Yoshitake T, Kehr J, Prabhakar P, Poellinger L, Fagerlund MJ, Eriksson LI (2014) The human carotid body releases acetylcholine, ATP and cytokines during hypoxia. Exp Physiol 99:1089–1098 Kato K, Yamaguchi-Yamada M, Yamamoto Y (2010) Short-term hypoxia increases tyrosine hydroxylase immunoreactivity in rat carotid body. J Histochem Cytochem 58:839–846 Kim D-K, Oh EK, Summers BA, Prabhakar NR, Kumar GK (2001) Release of substance P by low oxygen in the rabbit carotid body: evidence for the involvement of calcium channels. Brain Res 892:359–369 Kim D-K, Prabhakar NR, Kumar GK (2004) Acetylcholine release from the carotid body by hypoxia: evidence for the involvement of autoinhibitory receptors. J Appl Physiol 96:376–383 Koerner P, Hesslinger C, Schaefermeyer A, Prinz C, Gratzl M (2004) Evidence for histamine as a transmitter in rat carotid body sensor cells. J Neurochem 91:493–500 Kumar P, Prabhakar NR (2012) Peripheral chemoreceptors: function and plasticity of the carotid body. Compr Physiol 2:141–219 Kusakabe T, Matsuda H, Harada H, Hayashida Y, Gono Y, Kawakami T, Takenaka T (1998a) Changes in the distribution of nitric oxide synthase immunoreactive nerve fibers in the chronically hypoxic rat carotid body. Brain Res 795:292–296 Kusakabe T, Hayashida Y, Matsuda H, Gono Y, Powell FL, Ellisman MH, Kawakami T, Takenaka T (1998b) Hypoxic adaptation of the peptidergic innervation in the rat carotid body. Brain Res 806:165–174 Kusakabe T, Matsuda H, Hirakawa H, Hayashida Y, Ichikawa T, Kawakami T, Takenaka T (2000) Calbindin D-28k immunoreactive nerve fibers in the carotid body of normoxic and chronically hypoxic rats. Histol Histopathol 15:1019–1025 Lahiri S, Roy A, Baby SM, Hoshi T, Semenza GL, Prabhakar NR (2006) Oxygen sensing in the body. Prog Biophys Mol Biol 91:249–286 Lam SY, Leung PS (2002) A locally generated angiotensin system in rat carotid body. Regul Pept 107:97–103 Lam SY, Liu Y, Ng KM, Lau CF, Liong EC, Tipoe GL, Fung ML (2012) Chronic intermittent hypoxia induces local inflammation of the rat carotid body via functional upregulation of proinflammatory cytokine pathways. Histochem Cell Biol 137:303–317 Lazarov N, Atanasova D (2012) The human carotid body in health and disease. Acta Morphol Anthropol 19:135–140 Lazarov N, Atanasova D (2022) The human carotid body and its role in ventilatory acclimatization to hypoxia. Acta Morphol Anthropol 29:63–68

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Lazarov N, Atanasova D, Reindl S, Gratzl M (2011) Dopamine and histamine: two major transmitters in hypoxic chemosensitivity in the human carotid body. Clujul Med 2(Suppl 2):84–88 Leonard EM, Salman S, Nurse CA (2018) Sensory processing and integration at the carotid body tripartite synapse: neurotransmitter functions and effects of chronic hypoxia. Front Physiol 9:225 Leung PS, Fung ML, Tam MS (2003) Renin-angiotensin system in the carotid body. Int J Biochem Cell Biol 35:847–854 Liu X, He L, Stensaas L, Dinger B, Fidone S (2009) Adaptation to chronic hypoxia involves immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. Am J Physiol Lung Cell Mol Physiol 296:L158–L166 Liu X, He L, Dinger B, Stensaas L, Fidone S (2013) Sustained exposure to cytokines and hypoxia enhances excitability of oxygen-sensitive type I cells in rat carotid body: correlation with the expression of HIF-1α protein and adrenomedullin. High Alt Med Biol 14:53–60 López-Barneo J, Ortega-Sáenz P, Pardal R, Pascual A, Piruat JI (2008) Carotid body oxygen sensing. Eur Respir J 32:1386–1398 Mosqueira M, Iturriaga R (2019) Chronic hypoxia changes gene expression profile of primary rat carotid body cells: consequences on the expression of NOS isoforms and ET-1 receptors. Physiol Genomics 51:109–124 Nurse CA (2005) Neurotransmission and neuromodulation in the chemosensory carotid body. Auton Neurosci 120:1–9 Nurse CA, Zhang M (1999) Acetylcholine contributes to hypoxic chemotransmission in co-cultures of rat type 1 cells and petrosal neurons. Respir Physiol 115:189–199 Olson EB Jr, Vidruk EH, McCrimmon DR, Dempsey JA (1983) Monoamine neurotransmitter metabolism during acclimatization to hypoxia in rats. Resp Physiol 54:79–96 Pedersen MEF, Dorrington KL, Robbins PA (1999) Effects of dopamine and domperidone on ventilatory sensitivity to hypoxia after 8 h of isocapnic hypoxia. J Appl Physiol 86:222–229 Pequignot JM, Cottet-Emard JM, Dalmaz Y, Peyrin L (1987) Dopamine and norepinephrine dynamics in rat carotid body during long-term hypoxia. J Auton Nerv Syst 21:9–14 Porzionato A, Macchi V, Parenti A, De Caro R (2008) Trophic factors in the carotid body. Int Rev Cell Mol Biol 269:1–58 Porzionato A, Macchi V, De Caro R, Di Giulio C (2013) Inflammatory and immunomodulatory mechanisms in the carotid body. Respir Physiol Neurobiol 187:31–40 Powell FL, Milsom WK, Mitchell GS (1998) Time domains of the hypoxic ventilatory response. Respir Physiol 112:123–134 Prabhakar NR (2001) Oxygen sensing during intermittent hypoxia: cellular and molecular mechanisms. J Appl Physiol 90:1986–1994 Prabhakar NR, Kumar GK, Chang CH, Agani FH, Haxhiu MA (1993) Nitric oxide in the sensory function of the carotid body. Brain Res 625:16–22 Prabhakar NR, Peng YJ, Kumar GK, Nanduri J (2015) Peripheral chemoreception and arterial pressure responses to intermittent hypoxia. Compr Physiol 5:561–577 Rey S, Corthorn J, Chacon C, Iturriaga R (2007) Expression and immunolocalization of endothelin peptides and its receptors, ETA and ETB, in the carotid body exposed to chronic intermittent hypoxia. J Histochem Cytochem 55:167–174 Rey S, Del Rio R, Iturriaga R (2008) Contribution of endothelin-1 and endothelin A and B receptors to the enhanced carotid body chemosensory responses induced by chronic intermittent hypoxia. Adv Exp Med Biol 605:228–232 Ryan ML, Hedrick MS, Pizarro J, Bisgard GE (1993) Carotid body noradrenergic sensitivity in ventilatory acclimatization to hypoxia. Resp Physiol 92:77–90 Saito H, Yokoyama T, Nakamuta N, Yamamoto Y (2023) Immunohistochemical distribution of Ca2+ /calmodulin-dependent protein kinase II subunits in the rat carotid body. Acta Histochem 125:152043

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Salman S, Vollmer C, McClelland GB, Nurse CA (2017) Characterization of ectonucleotidase expression in the rat carotid body: regulation by chronic hypoxia. Am J Physiol Cell Physiol 313:C274–C284 Schamel A, Chaouti A, Douma M, Sabour B (2016) Morphological and neurochemical plasticity of the carotid body after long-term hypoxia: vascular and cellular involvement, morphometric study in Meriones shawi rats. Der Pharma Chem 8:82–98 Stocco E, Barbon S, Tortorella C, Macchi V, De Caro R, Porzionato A (2020) Growth factors in the carotid body—an update. Int J Mol Sci 21:E7267 Stocco E, Sfriso MM, Borile G, Contran M, Barbon S, Romanato F, Macchi V, Guidolin D, De Caro R, Porzionato A (2021) Experimental evidence of A2A –D2 receptor–receptor interactions in the rat and human carotid body. Front Physiol 12:645723 Stocco E, Sfriso MM, Barbon S, Emmi A, Guidolin D, Di Giulio C, Macchi V, De Caro R, Porzionato A (2022) D2–H3 receptor-receptor interactions in the carotid body: a descriptive multispecies study. Ital J Anat Embryol 126(Suppl 1):52 Tatsumi K, Pickett CK, Weil JV (1995) Possible role of dopamine in ventilatory acclimatization to high altitude. Respir Physiol 99:63–73 Verna A, Schamel A, Pequignot JM (1993a) Noradrenergic glomus cells in the carotid body: an autoradiographic and immunocytochemical study in the rabbit and rat. Adv Exp Med Biol 337:93–100 Verna A, Schamel A, Pequignot JM (1993b) Long-term hypoxia increases the number of norepinephrine-containing glomus cells in the rat carotid body: a correlative immunocytochemical and biochemical study. J Auton Nerv Syst 44:171–177 Vizek M, Pickett CK, Weil JV (1987) Increased carotid body hypoxic sensitivity during acclimatization to hypobaric hypoxia. J Appl Physiol 63:2403–2410 Wang Z-Y, Bisgard GE (2002) Chronic hypoxia-induced morphological and neurochemical changes in the carotid body. Microsc Res Tech 59:168–177 Wang Z-Y, Bisgard GE (2005) Postnatal growth of carotid body. Respir Physiol Neurobiol 149:181– 190 Wang ZZ, Dinger B, Fidone SJ, Stensaas LJ (1998) Changes in tyrosine hydroxylase and substance P immunoreactivity in the cat carotid body following chronic hypoxia and denervation. Neuroscience 83:1273–1281 Xu J, Xu F, Tse FW, Tse A (2005) ATP inhibits the hypoxia response in type I cells of rat carotid bodies. J Neurochem 92:1419–1430 Zhang M, Zhong H, Vollmer C, Nurse CA (2000) Co-release of ATP and ACh mediates hypoxic signalling at rat carotid body chemoreceptors. J Physiol 525:143–158 Zhao C, Li C, Zhao B, Liu Y (2022) Expression of group II and III mGluRs in the carotid body and its role in the carotid chemoreceptor response to acute hypoxia. Front Physiol 13:1008073

Chapter 8

Carotid Body Dysfunction and Mechanisms of Disease

Abstract Emerging evidence shows that the carotid body (CB) dysfunction is implicated in various physiological and pathophysiological conditions. It has been revealed that the CB structure and neurochemical profile alter in certain human sympatheticrelated and cardiometabolic diseases. Specifically, a tiny CB with a decrease of glomus cells and their dense-cored vesicles has been seen in subjects with sleep disordered breathing such as sudden infant death syndrome and obstructive sleep apnea patients and people with congenital central hypoventilation syndrome. Moreover, the CB degranulation is accompanied by significantly elevated levels of catecholamines and proinflammatory cytokines in such patients. The intermittent hypoxia stimulates the CB, eliciting augmented chemoreflex drive and enhanced cardiorespiratory and sympathetic responses. High CB excitability due to blood flow restrictions, oxidative stress, alterations in neurotransmitter gases and disruptions of local mediators is also observed in congestive heart failure conditions. On the other hand, the morpho-chemical changes in hypertension include an increase in the CB volume due to vasodilation, altered transmitter phenotype of chemoreceptor cells and elevated production of neurotrophic factors. Accordingly, in both humans and animal models CB denervation prevents the breathing instability and lowers blood pressure. Knowledge of the morphofunctional aspects of the CB, a better understanding of its role in disease and recent advances in human CB translational research would contribute to the development of new therapeutic strategies. Keywords Congestive heart failure · COVID-19 · Inflammation · Hypertension · Metabolic syndrome · Obstructive sleep apnea · Schizophrenia · Sleep disordered breathing · Sudden infant death syndrome

The CB has lately gained medical attention, particularly in respiratory medicine, due to its implication in the pathophysiology of various conditions, some of them highly prevalent in the human population (reviewed in Prabhakar and Peng 2004; Lazarov and Atanasova 2012; Lopez-Barneo et al. 2016; Iturriaga 2018; OrtegaSáenz and López-Barneo 2020; Iturriaga et al. 2021; López-Barneo 2022). Recent advances in human CB translational research and in understanding morphological © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_8

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and physiological mechanisms that operate in it reveal that its histological structure and neurochemical profile alter in certain cardiovascular, respiratory and metabolic disorders, thus implying the CB as a common denominator for cardiovascular and metabolic dysfunction (Badoer 2020). As important sensors of key metabolic and endocrine signals associated with stress-induced disorders, the CB could be the missing link between sleep disordered breathing and dysmetabolism (Conde et al. 2023). Emerging evidence also supports a causal relationship between CB dysfunction and augmented sympathetic outflow which is a common hallmark of human sympathetic-related and cardiometabolic diseases leading to target organ damage in cardiac and renal failure, increased chemical loop gain in apnea, total peripheral resistance in hypertension and glucose intolerance in diabetes. Given the clinical implications of the abnormal enhanced CB chemosensory discharge in autonomic dysfunction, a better understanding of the CB role in these diseases and conditions may help reveal their pathological mechanisms and would contribute to the development of new therapeutic strategies for them.

8.1 The Carotid Body and Sudden Infant Death Syndrome In the healthy fetus, the CB does not significantly contribute to breathing, though its activity is necessary for establishing rhythmic breathing at birth (Jansen et al. 1981). Moreover, during the early postnatal life, human infants seem to be particularly vulnerable to hypoxic and hypercapnic episodes during sleep. The main reason is that peripheral chemoreceptors are not fully developed at birth and their plasticityinduced changes, mediated by environmental exposures such as the extremes of oxygen tension, result in altered chemosensitivity which may be one of the factors contributing to a higher incidence of sudden infant death syndrome (SIDS), a disease responsible for unexpected deaths in newborns (Gauda et al. 2007). CB morphological findings in SIDS victims include a marked reduction of the glomic tissue volume, the number and size of neurosecretory granules in glomus cells and proliferation of progenitor cells (Naeye et al. 1976; Perrin et al. 1984a; Pávai et al. 2005; Porzionato et al. 2013b). Similarly, a tiny CB with a considerable decrease in the number of glomus cells and their dense-cored vesicles has been seen in subjects with congenital central hypoventilation syndrome (Cutz et al. 1997). However, the CB degranulation in SIDS patients is accompanied by abnormalities in their catecholamine content, in particular significantly elevated levels of dopamine (tenfold) and noradrenaline (threefold) (Perrin et al. 1984b; Porzionato et al. 2013b). Another possible mechanism underlying SIDS pathogenesis could be the exceedingly high cytokine concentrations, mainly for interleukin (IL)-6, causing the so-called cytokine storm that can explain some SIDS deaths (Vennemann et al. 2012). Prematurity and other environmental factors such as nicotine exposure due to maternal tobacco smoking, commonly abused substances or hyperoxia that alters the chemoreceptive function of the CB have also been implicated in SIDS pathogenesis (Holgert et al. 1995; reviewed in Gauda et al. 2007; Porzionato et al. 2013b). For example, evidence is

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presented that the CB and PG of newborn rats perinatally exposed to nicotine display alterations in the gene expression of catecholamine biosynthetic enzymes and peptide neuromodulators including as substance P (Porzionato et al. 2013b).

8.2 The Carotid Body and Obstructive Sleep Apnea Obstructive sleep apnea (OSA) is a common clinical syndrome and a well-established significant risk factor for cardiovascular disease and mortality (Gao et al. 2014). It is characterized by intermittent hypoxia and sleep disordered breathing. During sleep, OSA patients suffer complete or partial episodes of airflow obstruction produced by the collapse of the upper airways, leading to hypoxia and hypercapnia, which stimulates the CB and causes sympathetic hyperactivity, eliciting ventilatory, autonomic and vasopressor reflexes (Gozal and Kheirandish-Gozal 2008; Garvey et al. 2009). The enhanced chemoreflex drive arising from the CB chemoreceptors and their maladaptive responses of the paracrine signaling to hypoxia represents a common feature of this condition (Mansukhani et al. 2015; Prabhakar et al. 2023) and, moreover, its severity is associated with high CB chemosensitivity (Li et al. 2021). Indeed, both OSA patients and rodents exposed to chronic intermittent hypoxia, the goldstandard animal model of OSA, develop enhanced cardiorespiratory and sympathetic responses to acute hypoxia and hypertension (Peng and Prabhakar 2004; Huang et al. 2009; Iturriaga et al. 2009; Del Rio et al. 2010; Prabhakar et al. 2023). Since CB activation is not associated with changes in the morphology of the chemoreceptor tissue (Peng et al. 2003; Del Rio et al. 2011), it seems that the altered function is due to direct effects of hypoxia on glomus cells sensing. In addition, chemoreceptor cells of the CB in experimental animals and humans are sensitive to inflammatory cytokines and immunogenic molecules in the circulation (Fung 2014), and a significantly increased local expression of proinflammatory cytokines, cytokine receptors and inflammatory mediators in glomus cells is reported in the CB under hypoxic conditions (Fung 2015). Activation of the cytokine pathways leads to local inflammation of the CB which plays a pathogenic role in OSA. Besides, the upregulated expression of angiotensin II (Ang II) receptors and other locally produced components of the renin–angiotensin–aldosterone system in the CB also play a role in the augmented activity of the carotid chemoreceptor in intermittent hypoxia relevant to sleep apnea (Fung 2015). Finally, recent data show that interactions between reactive oxygen species (ROS) and gaseous transmitters cause CB hyperactivity by intermittent hypoxia (Prabhakar et al. 2018). It seems that ROS generation by nontranscriptional and transcriptional mechanisms involving dysregulated hypoxiainducible factor (HIF)-1 and HIF-2 as well as epigenetic mechanisms is critical for CB activation by intermittent hypoxia, thus contributing to cardiovascular morbidities which are associated with OSA in a murine model and human subjects (Prabhakar et al. 2023). Whether glutamate (GLU) and GABA are also involved in the CB chemoreflex modulation during this disease remain to be validated but recent evidence has shown that NMDA receptor activation enhances carotid sinus nerve

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(CSN) activity in a rat model of chronic intermittent hypoxia (Gold et al. 2022). Taken together, the findings suggest that the elevation of these mediators could alter the response of glomus cells to hypoxia which may be one of the major cellular mechanisms underlying the altered functions of the CB relevant to breathing instability in sleep apnea. Accordingly, bilateral CB ablation or denervation decreases the sympathetic overflow (Fletcher et al. 1992; Calverley 1999), and in both humans and sleeping animal models, this procedure prevents the apnea and periodic breathing that normally follow transient ventilatory overshoots (Smith et al. 2003). Furthermore, morphine-induced respiratory depression in conscious rats is greatly enhanced after bilateral CB denervation, suggesting a protective rather than causative role of the CB in this common and potentially life-threatening complication associated with opioid treatment or abuse (Baby et al. 2018). It has recently been reported that in rats chronic intermittent hypoxia induces a slight increase in the CB volume with a marked rearrangement of cellular elements of its neuronal lineage and rapid differentiation of CB progenitors, which then mature into glomus cells (Caballero-Eraso et al. 2023). In fact, this rearrangement of the cellular composition of the CB germinal niche along with aforementioned functional and chemical changes provides a novel perspective to the pathogenesis of CB-mediated sympathetic hyperactivation that may lead to the development of new treatment strategies for OSA (discussed in Chap. 10).

8.3 The Carotid Body and Congestive Heart Failure Available data from both congestive heart failure (CHF) patients and animal models indicate enhanced chemoreceptor reflexes in its early stages which in turn contribute to morbidity due to the reflex activation of sympathetic nerve activity and destabilization of breathing (for reviews, see Prabhakar and Peng 2004; Schultz et al. 2013, 2015; Toledo et al., 2017). Furthermore, high chemosensitivity is strongly correlated with a high risk of mortality and poor prognosis in patients with CHF. The main factors that enhance CB excitability in CHF include blood flow restrictions to the CB region, oxidative stress mediated by Ang II and disruptions of local mediators. Indeed, during the progression of CHF there is a marked reduction in the expression of the blood flow-sensitive transcription factor, Kruppel-like factor 2 (KLF-2), and its viral transduction in the CB from rabbits with CHF normalizes CB function and reduces sympathetic output despite the chronic reduction of blood flow to the tissue (Schultz et al. 2015). Conversely, both circulating and local tissue levels of the pro-oxidant Ang II peptide are elevated in CHF (Li et al. 2006). Other pro-oxidant mediators such as endothelin and inflammatory cytokines are elevated in CHF as well and they can stimulate production of ROS (Schultz et al. 2015). Besides, it has recently been revealed that the aberrant ATP transmission in the CB triggers episodic discharges that via P2X3 receptors play a crucial role in disease progression (Lataro et al. 2023). In addition, alterations in neurotransmitter gases, and in particular downregulation of both nitric oxide (NO) and carbon monoxide (CO) production in the CB

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during heart failure, contribute to its exaggerated function observed in CHF (Schultz et al. 2012). For example, the decreased endogenous nNOS activity in the CB plays an important role in the enhanced activity of the CB chemoreceptors and peripheral chemoreflex function in both CHF patients and experimental animal models (Li et al. 2005; Moya et al. 2012). Further, the reduced blood flow to the CB may play an important role in regulating Ang II metabolism and NO effects on CB chemoreceptor function in CHF. These findings suggest that multiple factors come together in the CB in CHF to alter its afferent activity. Therefore, a better understanding of the physiological and molecular mechanisms responsible for CB hyperactivity will contribute to the development of effective therapeutic approaches such as CB ablation to improve survival and life quality and to decrease mortality of CHF patients (Del Rio et al. 2013). However, the translation of these procedures to clinical settings must be done with caution because CB ablation may potentially trigger cardiovascular events (Pijacka et al. 2018; Lataro et al. 2023). For instance, bilateral CB ablation has shown to improve the autonomic imbalance in CHF patients, but it has also increased the occurrence of nocturnal hypoxia, particularly in patients with concomitant sleep apnea (Niewinski et al. 2017). An alternative to CB resection could be the systemic administration of HIF-2α antagonists to decrease CB overactivation, as recently suggested (see López-Barneo 2022 and references therein).

8.4 The Carotid Body and Hypertension It is generally accepted that essential hypertension is characterized by hypersensitivity to hypoxia and is associated with endothelial dysfunction, increased oxidative stress in blood vessels and activation of the inflammatory mediators (Schulz et al. 2011). It has lately been revealed that the CB input plays a fundamental role in both the genesis and maintenance of hypertension (Abdala et al. 2012). There is increasing evidence that the CB chemoreflex-evoked sympathoexcitatory responses are enhanced as hypertension develops and that hyperoxic inactivation of the CB produces a rapid and pronounced reduction in arterial pressure in both patients with primary hypertension (Sinski et al. 2012) and spontaneously hypertensive rat (SHR), a well-known experimental model of hypertension (McBryde et al. 2013; Paton et al. 2013). Additionally, it has been established that hypertension is abolished by CB removal (Abdala et al. 2012) and particularly that the bilateral CB ablation in some hypertensive patients causes an immediate and sustained fall in blood pressure (Paton et al. 2013) and lowered blood pressure in SHRs (Pijacka et al. 2018), thus suggesting that selective carotid glomectomy may be an effective procedure for the treatment of resistant hypertension (McBryde et al. 2013). Recent evidence also suggests that ablation of the CB reduces chronic intermittent hypoxia-induced hypertension (Del Rio et al. 2016). The morpho-chemical alterations of the CB in essential hypertensive patients and in SHRs involve an increase in its total volume due to a dilation of blood vessels and an elevated catecholamine content of its cell population (Heath et al. 1985; Przybylski

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et al. 1990; Habeck 1991; Kumar and Prabhakar 2012; Kato et al. 2012). Our ongoing experiments provide immunohistochemical evidence that the altered transmitter phenotype of CB chemoreceptor cells and elevated production of neurotrophic factors modulate the chemosensory processing in hypertensive animals which contributes to autonomic dysfunction and elicits sympathetic hyperactivity, consequently leading to elevated blood pressure (Atanasova et al. 2022). We have also suggested that GABA possibly exerts a depressant effect on CB chemosensitivity under hypertensive conditions (Atanasova et al. 2023), but future work will be required to establish, whether GLU signaling may also play a role in maladaptive plastic alterations associated with enhanced chemoreflex activity in hypertension. Further, prior immunohistochemical data have indicated that the neuropeptide profile of the CB cell population in the SHR is altered and this may activate its chemosensitivity (Takahashi et al. 2011). Recent research has also revealed a crucial role of purinergic signaling via P2X3 receptors within the CB in the regulation of blood pressure, thus highlighting the therapeutic potential for modulating purinergic transmission in hypertension (Bardsley et al. 2021; Li et al. 2023). Furthermore, these purinergic receptors are upregulated by ATP which in turn could elicit the release of GLU, raising excitation in the hypertensive CB. In addition, compelling evidence suggests that NO increases the CB chemosensory activity and this enhanced peripheral chemoreflex sensitivity contributes to sympathoexcitation and consequent pathology in hypertension (Prabhakar 1999; Iturriaga 2001; Campanucci and Nurse 2007; Prabhakar and Semenza 2012). Our subsequent study shows that in the hypertensive CB nNOS and eNOS protein expression is significantly downregulated, whereas iNOS expression is upregulated in the glomic tissue compared to normotensive controls (Atanasova et al. 2020). Experimental evidence suggests that the endothelial dysfunction in hypertension may be associated not only with an impaired NO metabolism but also with an increased production of Ang II and Ang II-induced superoxide anion generation (reviewed in Consolim-Colombo and Bortolotto 2018). Accordingly, our follow-up research has demonstrated that the glomus cells are richly endowed with Ang II type 1 (AT1) receptors (Atanasova et al. 2018). A more recent study has additionally indicated that the CB-evoked increase in blood pressure in renovascular hypertension is partly mediated by the AT1 receptor (Sohn et al. 2022). Simultaneous activation of this system and the interaction of NO with Ang II may potentiate even more the direct effects of NO on blood vessels. We further report that in glomus cells of hypertensive animals not only the production of NO is impaired but also components of the neurotrophin signaling system display enhanced expression (Atanasova and Lazarov 2014; Lazarov and Atanasova 2019). Taken together, these results suggest that the altered expression of NOS isoenzymes elicits chemosensory potentiation via an increase in oxidative stress and Ang II levels and a decrease in NO production in the CB, and the heightened chemosensory discharge might elicit vasoconstriction, enhanced vascular dysfunction and consequent blood pressure elevation. In addition, it has lately been shown that CB hyperexcitability is boosted by excessive activity of its sympathetic innervation from the superior cervical ganglion (SCG) and that sensitization of CB-evoked reflex sympathoexcitation appears to be mediated by α1adrenoceptors located either on the vasculature and/or glomus cells (Felippe et al.

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2023). Given the distinct connectivity between subsets of glomus cells and reflex pathways linked recently to a ribbon cable or “private line” of communication (Zera et al. 2019), it is tempting to speculate that the CB and SCG may also represent novel therapeutic targets for the treatment of hypertensive patients. Moreover, it is likely that the interconnection between them could be a potential translational approach for normalizing CB sensitivity and the selective denervation of the ganglioglomerular nerve in humans would be of a great value in the hypertension treatment (Felippe et al. 2023).

8.5 The Carotid Body, Obesity and Metabolic Syndrome Metabolic syndrome, a set of metabolic abnormalities, is newly attributed to the CB, a single peripheral sensor for multiple metabolic parameters that is involved in the normal control of metabolism (Conde et al. 2018). Growing evidence indicates that the exaggerated sympathetic outflow evoked by CB overactivation may play a major role in the progression and maintenance of cardiometabolic morbidity associated with systemic hypertension, glucose intolerance, insulin resistance and dyslipidemia (for recent reviews, see Iturriaga et al. 2016, 2021; Kim and Polotsky 2020; Conde et al. 2023). In addition, patients with metabolic disorders and obesity also have increased levels of leptin, ROS and proinflammatory cytokines (Iturriaga et al. 2016). In fact, the overactivation of the CB creates a metabolic vicious cycle that involves increased sympathetic activity leading to insulin resistance and to the disruption of glucose homeostasis. Proposed molecular mechanisms implicated in sympathetic hyperactivity induced by an enhanced CB chemosensory discharge include the transcriptional modifications of the oxygen-labile HIF alpha subunits, HIF-1α/2α, during hypoxia, the balance of gasotransmitters in the CB and the activation of purinergic receptors P2X2/3 and the leptin-mediating transient receptor potential cation channel subfamily M member 7 (TRPM7) channels (Kim and Polotsky 2020; Shin et al. 2021). Accordingly, the CB may represent a potential pharmacological target for the treatment of metabolic diseases. Indeed, bilateral surgical resection of the CSN or CB ablation have been shown to cause remission of pathological manifestations in chronic conditions associated with metabolic disturbances (Del Rio et al. 2013; Paton et al. 2013; Ribeiro et al. 2013) and improve insulin sensitivity in mice (Shin et al. 2014). Nonetheless, evidence for the efficacy of pharmacological interventions targeting the CB in diabetes, obesity and dyslipidemia is still lacking.

8.6 The Carotid Body and Inflammatory Responses Recent evidence has shown that the CBs can recognize the presence of immunogens and cytokines in the blood, and thus, they provide an alternative route to vagal afferences for signaling between the immune system and central nervous system

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(CNS) structures involved in the organization of the systemic inflammatory responses (Zapata et al. 2011). Inflammation that commonly occurs in premature infants may adversely affect the structure and function of the carotid chemoreceptors, thereby contributing to intractable apnea that occurs in some of them (Gauda et al. 2013). Consequently, inflammatory/immunological factors and their receptors have been demonstrated to play a role in the physiology and plasticity of the CB (Stocco et al. 2020). Indeed, several proinflammatory cytokines such as IL-1β, IL-6 and TNF-α are considered autocrine/paracrine modulators of CB chemosensory function, rather than transmitters released by hypoxia between glomus cells and CSN terminals (Zapata et al. 2011; Porzionato et al. 2013a; Fung 2015). For instance, it has been shown that IL-1β injections depolarize glomus cells, increase their intracellular calcium concentration (Shu et al. 2007) and enhance the expression of tyrosine hydroxylase in the rat CB (Zhang et al. 2007). Such an enhanced intracellular free calcium in cultured glomus cells and catecholamine release have also been reported in response to IL-6 application (Fan et al. 2009). On the other hand, the physiological effects of Il-8 and IL-10 on CB chemoreception are largely unknown. A histopathological condition in humans affecting the CB is the so-called chronic carotid glomitis which may be interpreted as its inflammatory response to the known degenerative changes with age (Khan et al. 1989) or opiate addiction (Porzionato et al. 2005). It is characterized by the presence of lymphocyte aggregates throughout its structure, mainly sub or intralobularly. As stated already, inflammation and proinflammatory cytokines have also potential role in the alterations of the carotid chemoreceptor function and clinical implications for other cardiorespiratory conditions such as SIDS (Porzionato et al., 2013b). A potential contributive role of inflammatory cytokines in CB adaptation to chronic intermittent hypoxia, the main characteristic of OSA, has also been proposed (Iturriaga et al. 2009). Specifically, it has been revealed that chronic hypoxia induces upregulation of cytokines and causes CB hypersensitivity (reviewed in Porzionato et al. 2013a). The contribution of oxidative stress and proinflammatory cytokines to the enhanced CB chemosensory responsiveness to systemic hypertension has been described as well (Iturriaga et al. 2015). In particular, chronic intermittent hypoxia for 3 and 7 days upregulates mRNA and protein expression of interleukins IL-1β, IL-6 and TNF-α and corresponding receptors IL1-RI, gp130 and TNF-RI in the rat CB (Lam et al. 2012). It is believed that the main factor playing a role in inflammatory and immune responses in the CB is probably promoted nuclear factor-kappa B (NF-κB), which is activated by oxidative stress and hypoxia (Belaiba et al. 2007). In the rat CB, chronic intermittent hypoxia for 21 days causes a significant increase in the levels of the p65 subunit of NF-kB which activates the expression of many genes involved in inflammatory and immune responses, particularly that of cytokines IL-6, TNF-α and IL-1β (Del Rio et al. 2012). Proinflammatory cytokines IL-1β, IL-4, IL-8 and IL-10 are found in human CB tissue as well (Kåhlin et al. 2014). As noted previously, a higher cytokine concentration has been proposed to explain some SIDS deaths (Vennemann et al. 2012). The effects of proinflammatory cytokines on the neurogenesis of CB have lately been investigated, and the treatment with IL-1β is suggested to increase the plasticity of CB, while the extracellular signal-regulated kinase (ERK) ½, which determines the neuronal

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progenitor cell fate, seems to play a role in neurogenic signaling in the CB (Xue et al. 2015). In summary, the available evidence indicates that besides their capacity to express proinflammatory cytokines and respective receptors, CB chemoreceptors may also serve as an additional afferent sensory pathway for signaling systemic immune signals to the CNS, thus contributing to the anti-inflammatory reflex response (Iturriaga et al. 2022). Therefore, it is likely that the CB functions as a sensor of systemic inflammation. Further, research is needed to better understand how inflammation and systemic inflammatory molecules alter ion channel function, intracellular calcium concentration, transmitter release in glomus cells and their potential implications in inflammatory diseases.

8.7 The Carotid Body and COVID-19 Notably, the CB could be an alternative entry pathway of SARS-CoV-2 invasion. Indeed, both immunohistochemistry and RT-PCR have detected the presence of the virus in the human CB (Lambermont et al. 2021; Porzionato et al. 2021). In consequence, viral infection of glomus cells could disrupt the mitochondrial-based O2 sensor and thus cause hypoxemia even before producing any structural damage to the cells (Villadiego et al. 2021). Besides, the viral infection of endothelial cells may induce local inflammatory reactions which could further intrinsically contribute to chemoreception derangement through local or systemic ACE1/ACE2 imbalance (Porzionato et al. 2020). Indeed, histopathological findings such as inflammatory aggregates, microthromboses, blood congestion and microhemorrhages have been described in the CB of patients with COVID-19 (Porzionato et al. 2021). The authors claim that these pathological changes could be ascribed to local and/or systemic actions of SARS-CoV-2 and could potentially affect chemoreception. As the CB also recognizes blood immunogens and cytokines, potential effects of SARS-CoV2 cytokine storm on carotid chemoreception cannot be excluded either, even in the absence of local invasion of the CB (Porzionato et al. 2013a, 2020). The CBinduced increase in sympathetic activity could obviously have negative effects also on pulmonary, cardiovascular, renal and metabolic homeostasis which may explain the so-called silent hypoxemia (strong hemoglobin desaturation without dyspnea and tachypnea) in some COVID-19 patients (Ottestad et al. 2020; Machado and Paton 2021; Villadiego et al. 2021). At more advanced stages, SARS-CoV-2 infection could lead to inflammation and glomus cell death, thereby reducing the amount of chemosensitive elements in the CB and, henceforth, the ability to respond to hypoxemia (Villadiego et al. 2021). Thus, increased CB chemosensitivity and reflex sympathetic outflow could contribute to the detrimental effects in COVID-19, which in turn would further increase hypoxic chemosensitivity and sympathoactivation in a vicious circle (Porzionato et al. 2020). Nevertheless, it is still unknown what is the CB sensitivity in COVID-19 patients with hyperventilatory versus silent responses to

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hypoxia. In this regard, it has been proposed that the level of sensitivity of the peripheral chemoreceptors may contribute to the symptoms and outcomes of COVID-19 and, thus, the CB may be central to survival in patients with COVID-19 (Machado and Paton 2021).

8.8 The Carotid Body and Prion Diseases Prion diseases are a family of transmissible, progressive neurodegenerative disorders that affect both humans and animals. Yet there is considerable uncertainty about the exact portal of entry of pathogenic agents called prions into the CNS following exposure to infected blood. Given the highly vascular nature of the CB, its strategic location and target innervation, it is believed that the CB could serve as a possible direct route for prion neuroinvasion. Indeed, it has recently been described that the mast cells in the human CB express the cellular prion protein (PrPC ), a cell surface glycoprotein normally expressed in the CNS (Sweetland et al. 2023). As these cells are in close proximity to blood vessels, afferent nerves that are synaptically connected to the brainstem and sympathetic postganglionic fibers which are linked to the intermediolateral column of the thoracic spinal cord, it is likely that they may be a source of PrPC which is spread through these nerves to the CNS where prions accumulate and replicate in neurons, causing their destruction. Therefore, the CB may be a new and more efficient route of prion neuroinvasion than the oral and nasal mucosal pathways of prion infection.

8.9 The Carotid Body and Schizophrenia A possible relationship between the CB and schizophrenia has also been investigated. In the course of routine forensic necropsies, it has been observed that the CB in schizophrenics is smaller than average and its reduced size is due to contracted, thickened arterioles and empty vessels (Geertinger 1978). With respect to vascularization and efferent innervation, the CB is structurally identical to the cutaneous glomera (Adams 1958) which are considered the local anatomical substrate for the vascular response leading to acrocyanosis, a pathognomonic feature of schizophrenia. Accordingly, the increased sympathetic tone in the schizophrenic, which is a prerequisite for cyanosis of the hands, must also affect the CB, producing vasoconstriction, reduced blood flow, and oxygen depletion within it, thus resulting in chemoreceptor malfunction (Geertinger 1978). Recently, it has been suggested that inflammation and neurovascular endothelial dysfunction seem to be an interrelated etiological mechanism of schizophrenia and a possible explanation of the increased cardiovascular risk of such patients (Najjar et al. 2017). In line with this, a more recent study has reported the predictive value of circulating vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1), which are well-known biomarkers

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of endothelial inflammation and dysfunction, in the onset of schizophrenia (Radu et al. 2020). Therefore, increasing understanding of the role of inflammation will enable new therapeutic approaches and will provide new methods to reduce the cardiovascular risk in schizophrenia patients. Compliance with Ethical Standards This study was partially funded by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01. The authors declare no conflict of interest. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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Sohn CS, Chang JW, George B, Chen S, Ramchandra R (2022) Role of the angiotensin type 1 receptor in modulating the carotid chemoreflex in an ovine model of renovascular hypertension. J Hypertens 40:1421–1430 Stocco E, Barbon S, Tortorella C, Macchi V, De Caro R, Porzionato A (2020) Growth factors in the carotid body—an update. Int J Mol Sci 21:E7267 Sweetland GD, Eggleston C, Bartz JC, Mathiason CK, Kincaid AE (2023) Expression of the cellular prion protein by mast cells in the human carotid body. Prion 17:67–74 Takahashi M, Matsuda H, Hayashida Y, Yamamoto Y, Tsukuda M, Kusakabe T (2011) Morphological characteristics and peptidergic innervation in the carotid body of spontaneously hypertensive rats. Histol Histopathol 26:369–375 Toledo C, Andrade DC, Lucero C, Schultz HD, Marcus N, Retamal M, Madrid C, Del Rio R (2017) Contribution of peripheral and central chemoreceptors to sympatho-excitation in heart failure. J Physiol 595:43–51 Vennemann MM, Loddenkötter B, Fracasso T, Mitchell EA, Debertin AS, Larsch KP, Sperhake JP, Brinkmann B, Sauerland C, Lindemann M, Bajanowski T (2012) Cytokines and sudden infant death. Int J Leg Med 126:279–284 Villadiego J, Ramírez-Lorca R, Cala F, Labandeira-García JL, Esteban M, Toledo-Aral JJ, LópezBarneo J (2021) Is carotid body infection responsible for silent hypoxemia in COVID-19 patients? Function (Oxf) 2:zqaa032 Xue F, Liu L, Fan J, He S, Li R, Peng ZW, Wang BR (2015) Interleukin-1β promotes the neurogenesis of carotid bodies by stimulating the activation of ERK1/2. Respir Physiol Neurobiol 219:78–84 Zapata P, Larraín C, Reyes P, Fernández R (2011) Immunosensory signalling by carotid body chemoreceptors. Respir Physiol Neurobiol 178:370–374 Zera T, Moraes DJA, Silva MD, Fisher JP, Paton JFR (2019) The logic of carotid body connectivity to the brain. Physiology 34:264–282 Zhang XJ, Wang X, Xiong LZ, Fan J, Duan XL, Wang BR (2007) Up-regulation of IL-1 receptor type I and tyrosine hydroxylase in the rat carotid body following intraperitoneal injection of IL-1β. Histochem Cell Biol 128:533–540

Chapter 9

Stem Cell Niche in the Mammalian Carotid Body

Abstract Accumulating evidence suggests that the mammalian carotid body (CB) constitutes a neurogenic center that contains a functionally active germinal niche. A variety of transcription factors is required for the generation of a precursor cell pool in the developing CB. Most of them are later silenced in their progeny, thus allowing for the maturation of the differentiated neurons. In the adult CB, neurotransmitters and vascular cytokines released by glomus cells upon exposure to chronic hypoxia act as paracrine signals that induce proliferation and differentiation of pluripotent stem cells, neuronal and vascular progenitors. Key proliferation markers such as Ki-67 and BrdU are widely used to evaluate the proliferative status of the CB parenchymal cells in the initial phase of this neurogenesis. During hypoxia sustentacular cells which are dormant cells in normoxic conditions can proliferate and differentiate into new glomus cells. However, more recent data have revealed that the majority of the newly formed glomus cells is derived from the glomus cell lineage itself. The mature glomus cells express numerous trophic and growth factors, and their corresponding receptors, which act on CB cell populations in autocrine or paracrine ways. Some of them initially serve as target-derived survival factors and then as signaling molecules in developing vascular targets. Morphofunctional insights into the cellular interactions in the CB stem cell microenvironment can be helpful in further understanding the therapeutic potential of the CB cell niche. Keywords Carotid body · Cell proliferation and differentiation · Germinal niche · Growth and trophic factors · Neural crest-derived stem cells · Peripheral neurogenesis · Plasticity · Progenitor cells · Signaling molecules · Transcription factors

Over the last decade, a large body of literature has evolved on the subject that the adult mammalian CB constitutes a neurogenic center that contains a functionally active germinal niche of quiescent multipotent stem cells and immature committed progenitors with the capacity to differentiate into both neuronal and vascular cell types (for reviews, see Pardal et al. 2010; Pardal and López-Barneo 2012; Sobrino et al. 2019a, b,; Pardal 2023). Remarkably, the neurogenic niche is present even at advance © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_9

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age in the human CB (Ortega-Sáenz et al. 2013). However, the CB germinal center does not seem to be permanently active and is only turned on in the context of the homeostatic adaptive response to chronic hypoxia (Pardal et al. 2007). As mentioned previously in Chap. 4, the adult CB has an extraordinary structural plasticity, and its growth in response to hypoxia is mediated by a population of neural crest-derived stem cells, which can proliferate and differentiate into new oxygen-sensitive glomus cells (Pardal et al. 2007; Sobrino et al. 2018). Upon exposure to hypoxia, neurotransmitters and neuromodulators released by glomus cells act as paracrine signals that induce proliferation and differentiation of multipotent stem cells and progenitors, thus causing CB hypertrophy and an increased sensory output to the respiratory center (Sobrino et al. 2020). However, the regenerative potential of cultured CB stem cells, although qualitatively similar in several species, is less potent in mouse and human samples than in the rat (Ortega-Sáenz et al. 2013).

9.1 Markers of Cell Proliferation Cell proliferation can be assessed by quantifying protein levels of key proliferation markers such as the nuclear protein Ki-67 in proliferating cells or by measuring the level of DNA synthesis through incorporation of the thymidine analog bromodeoxyuridine into newly synthesized DNA of replicating cells.

9.1.1 Ki-67 Protein The cell proliferation antigen Ki-67 also known as marker of proliferation Ki-67 (MKI67) or briefly Ki-67 is a 359-kD nuclear protein that is widely used as a sensitive and specific marker of proliferating cells. Its expression has been used to evaluate cell proliferation and CB growth in hypoxic conditions (Platero-Luengo et al. 2014) and to assess neoplastic proliferation and cancer prognosis in CB carcinoma (Teh et al. 2017). To address the proliferative status of the parenchymal cells in the hypertensive CB, we performed Ki-67 immunostaining and found that it is mainly expressed in the glial-like sustentacular cells but also in some peripherally located glomus cells in several glomic lobules (Fig. 9.1) (Atanasova and Lazarov 2014). It seems that the hypertensic adaptation of the rat CB involves proliferation of sustentacular cells.

9.1.2 Bromodeoxyuridine Bromodeoxyuridine (BrdU), a synthetic thymidine analog that is stably incorporated into cells undergoing DNA synthesis, is commonly used in the detection of proliferating cells in living tissues. Previous research has revealed that sustentacular type II

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Fig. 9.1 Immunohistochemical visualization of Ki-67 protein in the hypertensive carotid body (CB) of the rat. a Low-magnification of the glomic islets in a spontaneously hypertensive rat showing the expression of Ki-67 in CB parenchyma. b The higher magnification of the outlined area in a reveals that the spindle-shaped sustentacular cells (arrowheads), distinguished by their brownstained nuclei, are highly Ki-67 immunoreactive. Note that some glomus cells (arrow) situated at the periphery of the cell clusters are immunostained as well. Scale bars = 100 μm in a, 50 μm in b

cells are dormant (or slowly dividing) cells in normoxic-resting conditions but during exposure to hypoxia they are responsible for the progenitor activity both in vivo and in vitro and could facilitate acclimatization to hypoxia (Pardal et al. 2007; Sobrino et al. 2018). The cell fate mapping experiments using BrdU have shown that the newly formed glomus cells are derived from sustentacular cells (Pardal et al. 2007). The former are highly catecholaminergic, synthesize GDNF and have chemoreceptive and electrophysiological properties of mature in situ glomus cells (Pardal et al. 2007). On the other hand, using cell lineage-labeling technology of glomus cells and sustentacular cells, respectively, some authors claim that the vast majority of the increased in number glomus cells during hypoxia is derived from the glomus cell lineage itself and not via transdifferentiation from sustentacular cells (Fielding et al. 2018). The existence of predifferentiated, immature glomus cells, which express TH and are capable of rapid division in response to hypoxia, has also been suggested in the adult CB (Sobrino et al. 2018). Unlike mature glomus cells, which are postmitotic, predifferentiated CB neuroblasts, that are smaller in size and located at the periphery of the glomerulus, can undergo mitotic divisions before final maturation. By combining Ki-67 with BrdU immunostaining, the time course of cell divisions was studied, and a transient period of fast and intense neurogenesis has been observed in the hypoxic CB (Sobrino et al. 2018). This unprecedented “fast neurogenesis” is stimulated by ATP and acetylcholine released from mature glomus cells. In addition to mature glomus cells and sustentacular cells, the resting CB parenchyma also contains restricted progenitors belonging to either neuronal or vascular lineages. The vascular progenitors are resting in normoxia but rapidly proliferate and migrate toward blood vessels to contribute to angiogenesis in response to hypoxia (Navarro-Guerrero et al. 2016; Annese et al. 2017; Sobrino et al. 2019a). A common anatomical feature shared by all progenitor cells is their localization in the proximity of glomus cells. This structural disposition supports the hypothesis that

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glomus cells function as master regulators that, by means of the release of neurotransmitters and vascular cytokines, can modulate the activity of the various progenitors in the niche (reviewed in Pardal and López-Barneo 2016; Sobrino et al. 2020).

9.2 Signaling Molecules and Transcription Factors Involved in the Carotid Body Organogenesis Ongoing work in the CB organogenesis has been devoted to the embryonic origin of CB stem cells which remains controversial. Based on embryonic development of the CB, Kameda (2020, 2021) recently proposed that the glomus cell progenitors in mammals and birds arise from the neural crest precursors of the sympathoadrenal lineage in the neighboring ganglion, i.e., the superior cervical ganglion (SCG) and the nodose ganglion, respectively, which then migrate into the CB primordium, constituting a solid cell cluster. Accordingly, CB glomus cells share some characteristics with sympathetic neurons. On the other hand, the sustentacular cells are derivatives of mesenchymal neural crest cells which colonize the third pharyngeal arch and form the wall of the third arch artery. Hence, the transdifferentiation of mesenchymal lineages into neuronal lineages appears to be an unlikely event. It has been demonstrated by gene-targeting experiments and expression studies of each gene that a variety of transcription factors and signaling molecules are required for the development of the elements necessary for the CB organogenesis (reviewed in Kameda 2020, 2021).

9.2.1 Mash1 and Hand2 The basic helix–loop–helix (bHLH) transcription factors control the generation of committed neural precursors in the central and peripheral nervous systems in early embryos (Johnson et al. 1990). Evidence shows that Mash1, a mammalian homolog of the Drosophila achaete-scute complex (asc), is transiently expressed in the developing sympathetic nervous system and is turned off before the precursors differentiate into neurons (Guillemot et al. 1993). In Mash1 null mutant mice, the CB primordium forms normally in the wall of the third arch artery, but it does not contain neuronal precursors, and no glomus cells are generated in the absence of Mash1 (Kameda 2005). Further, newborn Mash1 null mutants have exhibited severe hypoplasia of their CBs, associated with a marked decrease in cell proliferation (Kameda 2005). The mutant CB at birth consists of sustentacular cells or their precursors and many S100-expressing cells appear during its fetal development (Kameda 2005). Therefore, Mash1 is required for the genesis of glomus cells but not for sustentacular cells (Kameda 2005, 2020).

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The DNA-binding protein Hand2, another member of the twist family of bHLH transcription factors, has also been shown to play a role for the differentiation and proliferation of the neural precursor pool of sympathetic neurons and is important in determination of their noradrenergic phenotypes as well (Hendershot et al. 2008). Hand2 is required for early embryonic survival and is expressed in the CB of wild-type mice at E16.5 and in a subset of neural crest-derived cells in the carotid bifurcation region of conditional knockout mice (Hockman et al. 2018).

9.2.2 Hes1 The transcription factor Hes1, a member of the family of Hes genes, plays a critical negative role in regulating the development of neural crest derivatives by repressing the activities of proneural bHLH factors which as mentioned above are essential for the differentiation of CB glomus cells. The lack of Hes1 causes severe defects of the CB, including its hypoplasia. Indeed, the volume of CB in the null mutants is only half its value in wild-type embryos, and in case of missing third arch artery, the CB is absent in the mutants (Kameda et al. 2012).

9.2.3 Phox2b The homeodomain transcription factor Phox2b is expressed in all central and peripheral noradrenergic neurons and is a master regulator of their differentiation (Pattyn et al. 1999). It is also essential for the initial specification of sympathetic neurons from neural crest progenitors. In particular, the development of the CB is prevented in Phox2b null mutants, and only a small CB made up of sustentacular cells can be detected in the carotid bifurcation region at E 13.5 in the null mutant mice (Pattyn et al. 1999; Dauger et al. 2003). In Phox2b null mutants, differentiation of chromaffin cells is arrested at an earlier neuroblast stage, and they fail to give rise to the glomus cell progenitors due to the absence of SCG in the mutants (Kameda 2014).

9.2.4 Hoxa3 The homeobox gene Hoxa3 plays a crucial role for the formation of the third pharyngeal arch and pouch (Chisaka and Capecchi 1991). Kameda and coworkers (2002) have shown that in Hoxa3 homozygous null mutant mice, the CB rudiment does not form, whereas the adjacent SCG is enlarged in volume compared with that in wild types. The authors have postulated that Hoxa3 is essential for the formation of the CB in mouse embryos and its lack is due to the defect of development of the third

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arch artery, resulting in malformation of the carotid artery system (Kameda et al. 2002).

9.2.5 Sox4 and Sox11 The SoxC group transcription factors, Sox4 and Sox11, play critical role in the development of sympathetic neurons. Specifically, Sox 11 is needed for their proliferation at early embryonic stages, while Sox4 is important for their survival at later stages (Potzner et al. 2010). Both Sox4 and Sox11 are expressed in the CB of wild-type mouse embryos at E16.5 and their loss in the knockout mice results in the presence of only a few glomus cells in the small CB and numerous sustentacular cells which are diffusely distributed in the carotid bifurcation region of the mutants (Hockman et al. 2018).

9.2.6 Pax6 Pax6, a member of the paired-box (Pax) gene family, is a transcription factor essential for neural stem cell proliferation, multipotency and neurogenesis in the central nervous system. It has initially been found to be a master regulator of the eye development and later a master control gene in maintaining the normal function of certain cells in adulthood (Haubst et al. 2005). Recently, analyses of wholemount preparations of Pax6 mutant mice embryos have revealed that Pax6 specifically facilitates neuronal progenitor domain formation within the vertebrate hindbrain (Huettl et al. 2016). More recently, its role in regulating the specification of brainstem respiratory neurons has been described (Xia et al., 2022), but so far, no evidence has been provided for the involvement of this transcription factor in the development of CB parenchymal and/or SCG cells. Our preliminary data show that Pax6 is widely expressed in the developing CB and SCG (Fig. 9.2) of the wild-type mouse embryos, and its expression is abolished in the Pax6 mutant mouse line (unpublished). These findings suggest that CB and SCG neuronal precursors require the transcription factor Pax6 for their survival, proliferation and differentiation during the embryonic development.

9.2.7 HIF-1α and HIF-2α The hypoxia-inducible factors (HIF) are transcriptional activators that function as a master regulator of oxygen homeostasis. HIF-1α and HIF-2α, which are activated by oxygen deprivation, are expressed in the CB during the perinatal transition (Kameda 2020). For example, downregulation of HIF-1α expression and no

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Fig. 9.2 Immunohistochemical expression of Pax6 in the carotid body (CB) and superior cervical ganglion (SCG) of wild-type (+/+) and Pax6sey/sey mutant mice at E17.5. Frontal sections of the CB (a) and SCG (b) immunostained with Pax6 antibody showing that virtually all glomus cells (arrows) and sympathetic neurons of wild-type mouse embryos are immunopositive. Frontal sections stained with H&E (c) demonstrate that the size of CB (rectangle) and the adjacent SCG primordium are considerably smaller while the immunostaining with the TH antibody (d) reveals that the glomus cell number is notably reduced in E17.5 Pax6sey/sey mutants. Scale bars = 100 μm in a, 200 μm in c, 50 μm in b, d

significant changes in HIF-2α expression in glomus cells have been reported postnatally (Roux et al. 2005). However, HIF-1α increases the expression of VEGF and is essential for vascularization of the CB during embryonic development (Roux et al. 2005). On the other hand, transgenic overexpression of HIF-2α elicits CB hypertrophy and a significantly increased number of glomus cells (Macias et al. 2014), while its absence during embryogenesis results in CB atrophy (Macías et al. 2018). Therefore, HIF-2α is required for the growth, survival and function of the glomus cells during development (Kameda 2021).

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9.3 Trophic and Growth Factors The glomus cells in the adult mammalian CB express numerous trophic and growth factors, and their corresponding receptors, which act on its cell populations in autocrine or paracrine ways (for reviews, see Porzionato et al. 2008; Ortega-Sáenz et al. 2015; Stocco et al. 2020). We have recently found that glomus cells and a subset of sustentacular cells in the adult rat CB highly express certain neurotrophic factors of the nerve growth factor family such as nerve growth factor (NGF), brainderived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) as well as a member of the glial cell line-derived neurotrophic factor (GDNF) superfamily of ligands, GDNF (Atanasova and Lazarov 2013, 2014). In particular, we have observed that the majority of glomus cells and fewer sustentacular cells exhibit NGF- (Fig. 9.3a, b), BDNF- (Fig. 9.4a, b) and NT-3-immunoreactivity (Fig. 9.5a, b) and the immunostaining is stronger in the glomus cells compared to that of the sustentacular cells. Besides, both glomus and sustentacular cells in the rat CB are richly endowed with their high-affinity neurotrophin membrane receptors TrkA (Fig. 9.3c, d), TrkB (Fig. 9.4c, d) and TrkC (Fig. 9.5c, d) and the low-affinity pan-neurotrophin p75 receptor (Fig. 9.6). We have also identified a large number of glomus cells which are immunopositive for GDNF (Fig. 9.7a, b) and its cognate receptor GFRα1 (Fig. 9.7c, d). Current evidence suggests that some of these neurotrophins initially serve as target-derived survival factors and, subsequently, as signaling molecules in developing vascular targets. Their expression in the CB may thus be developmentally regulated (De Caro et al. 2013). Early studies have demonstrated that anti-NGF antibodies exposure in rat fetal and early postnatal life cause CB hypoplasia (Aloe and Levi Montalcini 1980) and that NGF can determine the conversion of human CB cells to neuron-like cells with extensive dendritic processes (Lawson 1980). However, late fetal and neonatal glomus cells are reported to survive in vitro in the absence of NGF (Fishman and Schaffner 1984). Further, BDNF and its receptor TrkB have been suggested to have a role in the CB prenatal and postnatal maturation, survival, plasticity and innervation of glomus cells, and additionally, BDNF produced by glomus cell may support local vascular morphogenesis (Brady et al. 1999; Bavis et al. 2015). On the other hand, no data regarding the expression of the other neurotrophins of this family, NT-3 and NT-4/5, and/or their corresponding receptors in the CB during embryonic and fetal life are available to date. Since the production of BDNF by the CB falls in the late fetal period, it has been hypothesized that chemoafferent neurons change their trophic requirements from BDNF to other factors during late fetal development (Brady et al. 1999). Indeed, GDNF has been considered to play a critical role in the prenatal/postnatal development and in vivo innervation of the CB. In situ hybridization, RT-PCR and immunohistochemical experiments have revealed strong labeling for GDNF mRNA and protein in the CB of rats at E17, E19, E21, and onward until birth (Nosrat et al. 1996; Erickson et al. 2001; Leitner et al. 2005). In addition, the RET receptor tyrosine kinase has also been identified in glomus cells in the fetal and early postnatal life both in vivo and in vitro (Leitner et al. 2005;

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Fig. 9.3 Immunohistochemical demonstration of NGF and its receptor TrkA in the rat carotid body (CB). Light photomicrographs at lower (a, c) and higher magnifications (b, d) showing the strong immunoreactivity for NGF and its specific receptor TrkA in the glomic lobules. Note that the immunostaining is observed in the cytoplasm of a large number of glomus cells and in a subset of sustentacular cells in the CB. BV, blood vessels. Scale bars = 100 μm in a, c, 50 μm in b, d

Izal-Azcárate et al. 2008). Furthermore, the human CB also expresses high levels of the dopaminotrophic GDNF and its production and the number of progenitor and glomus cells are preserved in individuals of advanced age (Ortega-Sáenz et al. 2015). Among the different forms of fibroblast growth factors (FGFs), only basic FGF (bFGF) is reported to be expressed in glomus cells. Nurse and Vollmer (1997) have demonstrated that in cell cultures from CB of E17 to E19 rat pups bFGF promotes survival and BrdU incorporation, while in postnatal P1-P3 cultures it still stimulates DNA synthesis without affecting cell survival. The authors have also shown that in fetal rat glomus cells, bFGF stimulates neuronal differentiation, producing neurite outgrowth and inducing neurofilament immunoreactivity, but the changes can no longer be detected in postnatal cultures (Nurse and Vollmer 1997). They conclude that such local concentrations of bFGF may be particularly high during expansion of the vascular bed and the development of innervation. Consequently, the possible effects of chronic exposure to hypoxia on the differentiation of CB neural stem cells into mature glomus cells have been investigated. Specifically, it has been shown that hypoxic conditions induce an increase in expression/secretion of a potent proliferative agent, endothelin (ET) by mature glomus

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Fig. 9.4 Immunohistochemical detection of BDNF and its receptor TrkB in the rat carotid body (CB). Light photomicrographs at lower (a, c) and higher magnifications (b, d) showing the strong immunoreactivity for BDNF and its cognate receptor TrkB in the glomic lobules. Note that the immunostaining is observed in many glomus cells and a fewer sustentacular cells in the CB. BV, blood vessels. Scale bars = 100 μm in a, c, 50 μm in b, d

cells, which establish synaptic-like contacts with stem cells expressing endothelin receptors, and so ET evokes proliferation of CB progenitors to induce organ growth in response to low blood oxygen levels (Platero-Luengo et al. 2014). In summary, the discovery of diverse stem and intermediate progenitor cells present within the CB parenchyma has revealed a much higher structural and functional plasticity in the adult CB than previously anticipated. The identification of a physiologically relevant germinal niche in the adult CB has biomedical interest for understanding the molecular and cellular mechanisms underlying its functioning and therapeutic potential. Furthermore, the characterization of CB stem cells has provided new target sites for the development of cell replacement therapies that could be used to treat a variety of neurologic and mental illnesses (discussed in Chap. 10). Compliance with Ethical Standards This study was financed in part by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01 and by the Bulgarian Ministry of Education and Science within the framework of the National Recovery and Resilience Plan of Bulgaria,

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Fig. 9.5 Immunohistochemical localization of NT-3 and its receptor TrkC in the rat carotid body (CB). Light photomicrographs at lower (a, c) and higher magnifications (b, d) showing the strong immunoreactivity for NT-3 and its major receptor TrkC in the glomic lobules. Note that a large number of glomus cells express both NT-3 and its cognate receptor TrkC. Scale bars = 100 μm in a, c, 50 μm in b, d

Fig. 9.6 Immunohistochemical visualization of the low-affinity pan-neurotrophin p75 receptor in the rat carotid body (CB). Low-power photomicrograph (a) and enlarged image (b) of the boxed region in a illustrating the immunoreactivity for p75 in most of the glomus and sustentacular cells, and in some nerve fibers within and around the cell clusters in the CB. Scale bars = 100 μm in a, 50 μm in b

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Fig. 9.7 Immunohistochemical demonstration of GDNF and its specific receptor GFRα1 in the rat carotid body (CB). a, b Photomicrographs indicating at low and higher magnifications the presence of GDNF-immunoreactive cell groups in the CB glomeruli. Note that only the glomus cells are immunostained. c, d Expression of GFRα1 receptor protein in virtually all glomus cells in the CB parenchyma. Scale bars = 100 μm in a, c, 50 μm in b, d

Component “Innovative Bulgaria”, project No. BG-RRP-2.004-0006-C02 “Development of research and innovation at Trakia University in service of health and sustainable well-being”. The authors declare no conflict of interest. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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Kameda Y (2020) Molecular and cellular mechanisms of the organogenesis and development of the mammalian carotid body. Dev Dyn 249:592–609 Kameda Y (2021) Comparative morphological and molecular studies on the oxygen-chemoreceptive cells in the carotid body and fish gills. Cell Tissue Res 384:255–273 Kameda Y, Nishimaki T, Takeichi M, Chisaka O (2002) Homeobox gene Hoxa3 is essential for the formation of the carotid body in the mouse embryos. Dev Biol 247:197–209 Kameda Y, Saitoh T, Nemoto N, Katoh T, Iseki S (2012) Hes1 is required for the development of the superior cervical ganglion of sympathetic trunk and the carotid body. Dev Dyn 241:1289–1300 Lawson W (1980) The neuroendocrine nature of the glomus cells: an experimental, ultrastructural, and histochemical tissue culture study. Laryngoscope 90:120–144 Leitner ML, Wanga LH, Osborne PA, Golden JP, Milbrandt J, Johnson EM (2005) Expression and function of GDNF family ligands and receptors in the carotid body. Exp Neurol 191:S68–S79 Macías D, Fernández-Agüera MC, Bonilla-Henao V, López-Barneo J (2014) Deletion of the von Hippel-Lindau gene causes sympathoadrenal cell death and impairs chemoreceptor-mediated adaptation to hypoxia. EMBO Mol Med 6:1577–1592 Macías D, Cowburn AS, Torres-Torrelo H, Ortega-Sáenz P, López-Barneo J, Johnson RS (2018) HIF-2α is essential for carotid body development and function. eLife 7:e34681 Navarro-Guerrero E, Platero-Luengo A, Linares-Clemente P, Cases I, López-Barneo J, Pardal R (2016) Gene expression profiling supports the neural crest origin of adult rodent carotid body stem cells and identifies CD10 as a marker for mesectoderm-committed progenitors. Stem Cells 34:1637–1650 Nosrat CA, Tomac A, Lindqvist E, Lindskog S, Humpel C, Stromberg I, Ebendal T, Hoffer BJ, Olson L (1996) Cellular expression of GDNF mRNA suggests multiple functions inside and outside the nervous system. Cell Tissue Res 286:191–207 Nurse CA, Vollmer C (1997) Role of basic FGF and oxygen in control of proliferation, survival, and neuronal differentiation in carotid body chromaffin cells. Dev Biol 184:197–206 Ortega-Sáenz P, Pardal R, Levitsky K, Villadiego J, Muñoz-Manchado AB, Durán R, BonillaHenao V, Arias-Mayenco I, Sobrino V, Ordóñez A, Oliver M, Toledo-Aral JJ, López-Barneo J (2013) Cellular properties and chemosensory responses of the human carotid body. J Physiol 591:6157–6173 Ortega-Sáenz P, Villadiego J, Pardal R, Toledo-Aral JJ, López-Barneo J (2015) Neurotrophic properties, chemosensory responses and neurogenic niche of the human carotid body. In: Peers C, Kumar P, Wyatt C, Gauda E, Nurse CA, Prabhakar N (eds) Arterial chemoreceptors in physiology and pathophysiology, vol 860. Springer, Cham, pp 139–152 Pardal R (2023) The adult carotid body: a germinal niche at the service of physiology. In: Conde SV, Iturriaga R, del Rio R, Gauda E, Monteiro EC (eds) Arterial chemoreceptors, ISAC XXI 2022, vol 1427. Springer, Cham, pp. 13–22 Pardal R, López-Barneo J (2012) Neural stem cells and transplantation studies in Parkinson’s disease. In: López-Larrea C, López-Vázquez A, Suárez-Álvarez B (eds) Stem cell transplantation, vol 741. Springer, New York, pp 206–216 Pardal R, López-Barneo J (2016) Mature neurons modulate neurogenesis through chemical signals acting on neural stem cells. Dev Growth Differ 58:456–462 Pardal R, Ortega-Sáenz P, Durán R, López-Barneo J (2007) Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body. Cell 131:364–377 Pardal R, Ortega-Sáenz P, Durán R, Platero-Luengo A, López-Barneo J (2010) The carotid body, a neurogenic niche in the adult peripheral nervous system. Arch Ital Biol 148:95–105 Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF (1999) The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399:366–370 Platero-Luengo A, González-Granero S, Durán R, Díaz-Castro B, Piruat JI, García-Verdugo JM, Pardal R, López-Barneo J (2014) An O2 -sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia. Cell 156:291–303 Porzionato A, Macchi V, Parenti A, De Caro R (2008) Trophic factors in the carotid body. Int Rev Cell Mol Biol 269:1–58

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Chapter 10

Carotid Body and Cell Therapy

Abstract During the past decade, the carotid body (CB) has been considered an innovative therapeutic target for the treatment of certain cardiorespiratory and metabolic diseases most of which are sympathetically mediated. It has recently been revealed that CB stem cells provide new target sites for the development of promising cell-based therapies. Specifically, generation of CB progenitors in vitro which can differentiate into functionally active glomus cells may be a useful procedure to produce the cell mass required for replacement cell therapy. Due to their dopaminergic nature, adult glomus cells can be used for an intrastriatal grafting in neurodegenerative brain disorders including Parkinson’s disease. The beneficial effect of throphic factors such as glial cell-derived neurotrophic factor synergistically released by the transplanted cells then enables the transplant to survive. Likewise, intracerebral administration of CB cell aggregates or dispersed cells has been tested for the treatment of an experimental model of stroke. The systematic clinical applicability of CB autotransplants following glomectomy in humans is under investigation. In such autotransplantation studies, cell aggregates from unilaterally resected CB might be used as autografts. In addition, stem cells could offer an opportunity for tissue expansion and might settle the issue of small number of glomus cells available for transplantation. Keywords Carotid body autotransplantation · Glomectomy · Neurodegenerative disorders · Paraganglioma · Parkinson’s disease · Replacement cell therapy · Stem and progenitor cells · Stroke · Sympathetically mediated diseases

Proliferation and differentiation of CB progenitors in vitro which can differentiate into functionally normal glomus cells may be a useful procedure to produce a cell mass, thus permitting the successful development of neurological cell replacement therapy (for a review, see Yu et al. 2005). Moreover, the CB offers potential clinical advantages because its unilateral surgical resection has no significant side effects and therefore can be used for autografts (Arjona et al. 2003). The CB is also a therapeutic target for the treatment of sympathetically mediated cardiovascular, respiratory and metabolic diseases (reviewed by Paton et al. 2013). © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_10

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10.1 Carotid Body-Based Cell Therapy in Parkinson’s Disease Parkinson’s disease, one of the most common neurodegenerative brain disorders, is characterized by a profound loss of dopaminergic neurons in the substantia nigra, leading to dopamine (DA) deficiency in the striatum. In addition to the current pharmacological treatment with the DA precursor levodopa (L-DOPA), an alternative therapeutic strategy for Parkinson’s disease has been assayed based on the replacement of damaged neurons by new cells that could help to restore the dopaminergic nigrostriatal pathway (Pardal and López-Barneo 2012). So far, the intraputamenal grafting of fetal mesencephalic DA-producing neurons has provided the best clinical results. Because of their dopaminergic nature, glomus cells have lately been tested in intrastriatal CB autotransplantation studies in animal models of Parkinson’s disease (Espejo et al. 1998; Luquin et al. 1999; Toledo-Aral et al. 2002) and in clinical trials in Parkinson’s patients (Arjona et al. 2003; Mínguez-Castellanos et al. 2007). Moreover, the CB resistance to hypoxic environments may have enabled the transplant to survive. However, it has been suggested that the beneficial effect of CB transplants is due to a trophic action exerted on nigrostriatal neurons which is mediated by the abundant expression of the glial cell line-derived neurotrophic factor (GDNF) in the transplanted cells rather than because of exogenous DA delivery (Villadiego et al. 2005; Muñoz-Manchado et al. 2013). As mentioned in the previous chapter, adult glomus cells selectively produce GDNF and, thus, they appear to be prototypical devices for the synergistic endogenous delivery of DA and GDNF and can be used for a promising antiparkinsonian cell therapy (López-Barneo et al. 2009). Nevertheless, one of the main disadvantages of the procedure is the limited amount of CB tissue and the small number of glomus cells available for transplantation (Pardal and López-Barneo 2012). For this purpose, CB stem cells could offer an opportunity for tissue expansion prior to transplantation by providing an efficacious graft source of DA cells and consequently might settle the issue. Indeed, it has recently been demonstrated that in vitro-generated and expanded CB dopaminergic glomus cells could be used as a promising source to increase the amount of tissue available for grafting in Parkinson’s patients (Villadiego et al. 2023). Moreover, the authors have also shown that these cells could induce both neurotrophic protection and repair of damaged dopaminergic nigral neurons and thus represent a new therapeutic option for the treatment of Parkinson’s disease.

10.2 Carotid Body Grafting in Stroke Additional potential application of the CB tissue for cell therapy is based on its survival in hypoxic environments, similar to those existing in the brain parenchyma after a tissue graft (López-Barneo et al. 2009). Indeed, intracerebral administration

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of CB cell aggregates or dispersed cells has been tested for the treatment of an experimental model of stroke as the CB autotransplantation significantly reduces strokeinduced behavioral deficits and cerebral infarction accompanied by increased levels of neurotrophic factors in CB-grafted stroke animals (Yu et al. 2004). The authors further suggest that the early intracerebral transplantation of CB cells produces neuroprotection, possibly through the synergistic release of neurotrophic factors (Yu et al. 2004). Because of the overlapping pathophysiologic mechanisms and some common precipitating factors in degenerative disorders and ischemic brain diseases (Mattson et al. 2001), it is likely that CB cell transplantation may exert similar beneficial effects on other neurodegenerative diseases.

10.3 Implications of Carotid Body Autotransplants in Tumorigenesis The discovery of CB stem cells raises the question of their possible role in the pathophysiology of a relatively frequent CB tumor, formerly called chemodectoma and now paraganglioma. It is speculated that disruption of CB stem cell homeostasis leads to tumor transformation. Specifically, it has been hypothesized that conversion of glial fibrillary acidic protein (GFAP)-positive latent stem cells to aberrant GFAPnegative proliferating progenitors could give rise to cancer stem cells leading to CB tumorigenesis (Pardal et al. 2007). In this regard, it remains an open question, whether CB tumorigenesis is related to the proliferative potential of the CB neurogenic niche. The systematic clinical applicability of CB autotransplants following glomectomy due to tumor surgery in humans is under investigation. Given the role of hypoxiainducible factor (HIF)-2α in glomus cell proliferation and CB growth discussed in Chap. 8 and López-Barneo (2022), it would be interesting to ascertain in the future studies if human paraganglioma adequately responds to HIF-2 antagonists as well.

10.4 Carotid Body and Treatment of Sympathetically Mediated Diseases The data from preclinical and clinical studies demonstrate the importance of the CB in the progression of sympathetic-related diseases, including neurogenic hypertension, sleep apnea, and chronic heart failure. As discussed in Chap. 8, the CB has been highlighted as a potential therapeutic target for their treatment (McBryde et al. 2013; Paton et al. 2013; Bardsley et al. 2021; Lataro et al. 2023). Surgical bilateral CB resection in humans could be a useful though not always entirely safe method to reverse enhanced CB chemosensory discharges and chemoreflexes and thus to improve autonomic dysfunction in sympathetic-related diseases. Yet the pharmacological modulation of CB chemosensory activity by targeting

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specific receptors or ion channels is an optimal therapeutic alternative to surgery that could favor the translation of CB research to a clinical reality. Bioelectric modulation of the carotid sinus nerve activity is another relatively noninvasive intervention that could be explored (Iturriaga et al. 2016). On the other hand, comprehensive understanding of the cellular interactions in the CB stem cell microenvironment (cell niche) and the molecular events responsible for the maintenance of multipotency of CB stem cells would improve the current cell therapies (for a recent comprehensive review, see Pardal 2023). Nonetheless, the primary interest in restorative and regenerative approaches including stem cell therapy has lately shifted from cell replacement to neuroprotection because efficient generation of neural subtypes with correct phenotype from pluripotent cells remains a challenge. Compliance with Ethical Standards The authors declare no conflict of interest. This chapter is a review of previously published research, and as such, no animal or human studies were performed.

References Arjona V, Mínguez-Castellanos A, Montoro RJ, Ortega A, Escamilla F, Toledo-Aral JJ, Pardal R, Méndez-Ferrer S, Martín JM, Pérez M, Katati MJ, Valencia E, García T, López-Barneo J (2003) Autotransplantation of human carotid body cell aggregates for treatment of Parkinson’s disease. Neurosurgery 53:321–328 Bardsley EN, Pen DK, McBryde FD, Ford AP, Paton JFR (2021) The inevitability of ATP as a transmitter in the carotid body. Auton Neurosci 234:102815 Espejo EF, Montoro RJ, Armengol JA, López-Barneo J (1998) Cellular and functional recovery of Parkinsonian rats after intrastriatal transplantation of carotid body cell aggregates. Neuron 20:197–206 Iturriaga R, Del Rio R, Idiaquez J, Somers VK (2016) Carotid body chemoreceptors, sympathetic neural activation, and cardiometabolic disease. Biol Res 49:13 Lataro RM, Moraes DJA, Gava FN, Omoto ACM, Silva CAA, Brognara F, Alflen L, Brazão V, Colato RP, do Prado JC Jr, Ford AP, Salgado HC, Paton JFR (2023) P2X3 receptor antagonism attenuates the progression of heart failure. Nat Commun 14:1725 López-Barneo J (2022) Neurobiology of the carotid body. Handb Clin Neurol 188:73–102 López-Barneo J, Pardal R, Ortega-Sáenz P, Durán R, Villadiego J, Toledo-Aral JJ (2009) The neurogenic niche in the carotid body and its applicability to antiparkinsonian cell therapy. J Neural Transm 116:975–982 Luquin MR, Montoro RJ, Guillen J, Saldise L, Insausti R, Del Río J, López-Barneo J (1999) Recovery of chronic parkinsonian monkeys by autotransplants of carotid body cell aggregates into putamen. Neuron 22:743–750 Mattson MP, Duan W, Pedersen WA, Culmsee C (2001) Neurodegenerative disorders and ischemic brain diseases. Apoptosis 6:69–81 McBryde FD, Abdala AP, Hendy EB, Pijacka W, Marvar P, Moraes DJ, Sobotka PA, Paton JF (2013) The carotid body as a putative therapeutic target for the treatment of neurogenic hypertension. Nat Commun 4:2395 Mínguez-Castellanos A, Escamilla-Sevilla F, Hotton GR, Toledo-Aral JJ, Ortega-Moreno A, Méndez-Ferrer S, Martín-Linares JM, Katati MJ, Mir P, Villadiego J, Meersmans M, PérezGarcía M, Brooks DJ, Arjona V, López-Barneo J (2007) Carotid body autotransplantation in

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Parkinson disease: a clinical and positron emission tomography study. J Neurol Neurosurg Psychiatry 78:825–831 Muñoz-Manchado AB, Villadiego J, Suárez-Luna N, Bermejo-Navas A, Garrido-Gil P, LabandeiraGarcía JL, Echevarría M, López-Barneo J, Toledo-Aral JJ (2013) Neuroprotective and reparative effects of carotid body grafts in a chronic MPTP model of Parkinson’s disease. Neurobiol Aging 34:902–915 Pardal R (2023) The adult carotid body: a germinal niche at the service of physiology. In: Conde SV, Iturriaga R, del Rio R, Gauda E, Monteiro EC (eds) Arterial chemoreceptors, ISAC XXI 2022, vol 1427. Springer, Cham, pp 13–22 Pardal R, López-Barneo J (2012) Neural stem cells and transplantation studies in Parkinson’s disease. In: López-Larrea C, López-Vázquez A, Suárez-Álvarez B (eds) Stem cell transplantation, vol 741. Springer, New York, pp 206–216 Pardal R, Ortega-Sáenz P, Durán R, López-Barneo J (2007) Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body. Cell 131:364–377 Paton JFR, Sobotka PA, Fudim M, Engelman ZJ, Hart EC, McBryde FD, Abdala AP, Marina N, Gourine AV, Lobo M, Patel N, Burchell A, Ratcliffe L, Nightingale A (2013) The carotid body as a therapeutic target for the treatment of sympathetically mediated diseases. Hypertension 61:5–13 Toledo-Aral JJ, Méndez-Ferrer S, Pardal R, López-Barneo J (2002) Dopaminergic cells of the carotid body: physiological significance and possible therapeutic applications in Parkinson’s disease. Brain Res Bull 57:847–853 Villadiego J, Méndez-Ferrer S, Valdés-Sánchez T, Silos-Santiago I, Fariñas I, López-Barneo J, Toledo-Aral JJ (2005) Selective glial cell line-derived neurotrophic factor production in adult dopaminergic carotid body cells in situ and after intrastriatal transplantation. J Neurosci 25:4091–4098 Villadiego J, Muñoz-Manchado AB, Sobrino V, Bonilla-Henao V, Suárez-Luna N, Ortega-Sáenz P, Pardal R, López-Barneo J, Toledo-Aral JJ (2023) Protection and repair of the nigrostriatal pathway with stem-cell-derived carotid body glomus cell transplants in chronic MPTP parkinsonian model. Int J Mol Sci 24:5575 Yu G, Xu L, Hadman M, Hess DC, Borlongan CV (2004) Intracerebral transplantation of carotid body in rats with transient middle artery occlusion. Brain Res 1015:50–56 Yu G, Fournier C, Hess DC, Borlongan CV (2005) Transplantation of carotid body cells in the treatment of neurological disorders. Neurosci Biobehav Rev 28:803–810

Chapter 11

The Carotid Body: A Tiny Structure with Many Roles

Abstract Over the last century, the structure of the mammalian carotid body (CB) has repeatedly been studied, and our present understanding of its normal morphology is comprehensive. It has been demonstrated that the CB has an intricate internal structure and a remarkable ability to release a wide variety of neurotransmitters and neuromodulators in response to different chemical stimuli. The advances in modern cellular/molecular biological methods and newly developed single-cell electrophysiological techniques have provided an additional insight into the precise working mechanisms and roles of the CB in health and disease. Emerging experimental evidence has also shown that the CB exhibits an extraordinary structural and functional plasticity as a consequence of various environmental stimuli. Lately, the CB has attracted much clinical interest because its dysfunction relates to a number of cardiovascular and respiratory disorders. Expanding knowledge about the pathophysiological mechanisms that alter the CB cell function would certainly help to facilitate the translational research. Recent progress in cell fate experiments has further revealed that the CB is a neurogenic center with a functionally active germinal niche. This may lead to the development of promising new candidate therapies to combat these diseases and improve the quality of human life. Thus, the CB has entered the twenty-first century with its actual designation. Keywords Carotid body · Chemoreception · Functional and neurochemical anatomy · Hypoxia · Neurogenic niche · Neurotransmitters and modulators · Stem cells

In retrospective, since its discovery in the middle of the eighteenth century, the carotid body (CB) has attracted increasing attention as a beautiful yet enigmatic organ. Looking back on a large number of previous studies, its morphofunctional organization has been consistently demonstrated. Based on them, it has been widely believed that hypoxia is transduced by glomus cells organized in cell clusters where they make both reciprocal chemical and electrical synapses with each other. Glomus cells receive sensory innervation from petrosal ganglion chemoafferents and are intimately associated with sustentacular cells and the blood supply. Recent advances © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0_11

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in CB research and in understanding morphological and physiological mechanisms that operate in it have revealed that chemoreception involves the interaction between glomus cells themselves, between glomus and sustentacular cells, and, most importantly, between glomus cells and chemosensory nerve terminals. Such a morphological arrangement constituting a “tripartite synapse” is ideally suited for both the autocrine and paracrine regulation of the glomus cell function. It can be inferred that the CB is a much more complex structure than hitherto thought. It has an intricate internal organization and a remarkable ability to release in response to different chemostimuli a broad variety of both conventional and unconventional transmitter agents that act in concert at the CB tripartite synapse and provide clues on its important role in the integrative sensory response and the homeostatic maintenance of the whole organism during physiological and pathological stress. Thus, as recently suggested, at an organ-level, the CB is comparable to a miniature brain with compartmentalized discrete regions of clustered glomus cells defined by their neurotransmitter expression and receptor profiles and with connectivity to mediate coordinated outputs to a given stimulus (Gold et al. 2022). Knowledge of the CB physiology and pathophysiology has progressed immensely in the recent years and its functional anatomy appears to be well established. Newly developed technologies are applied to study single-cell physiology, i.e., techniques for producing and maintaining CB cell cultures used for studying their behavior under in vitro conditions, laser-capture microdissection to obtain homogeneous cell populations, microarray analyses performed on mRNA extracted from these cell populations and studies of proteomics to provide complete and accurate profiles of protein expression in isolated or cultured CB cells in response to exposure to various external stimuli. Still, the role of glomus cells as multimodal sensors and, particularly, the molecular mechanisms underlying their responsiveness to changes in plasma levels of metabolites such as glucose and lactate, hormones like insulin and leptin, or alterations in blood flow, temperature and osmolality remain to be clarified. Research in the field of CB neurogenic niche has lately revealed the contribution of multipotent adult stem cells to CB plasticity. Future work should determine whether the interaction between mature neurons and stem cells is a natural phenomenon. In parallel with the cellular studies, detailed pharmacological and functional systemic analyses in recent years have defined. In turn, expanding knowledge about the pathophysiological mechanisms that alter its function would certainly help facilitate the CB translational research and will enable new therapies to be proposed to moderate the severity of human cardiovascular and pulmonary pathologies. Hence, understanding these mechanisms can have benefits to human health. However, the theory that aims at CB regulation and deactivation as a potential novel therapy for diseases mediated by the sympathetic nervous system requires further investigation in human trials to determine whether the CB is a safe and effective target. We have now come to appreciate that this remarkable organ, though tiny in size and seemingly vestigial, is much more complex than originally thought, and its ability to transduce and integrate chemosensory information involves the coordinated action of multiple endogenous neurotransmitters and neuromodulators. Henceforth, more

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than two and half centuries of study and almost 100 years after its identification as an arterial chemoreceptor, the CB no more remains an organ of mystery.

Reference Gold OMS, Bardsley EN, Ponnampalam AP, Pauza AG, Paton JFR (2022) Cellular basis of learning and memory in the carotid body. Front Synaptic Neurosci 14:90231

Summary

The current paradigm considers the carotid body (CB) a complex polymodal chemosensory organ that plays an essential role in initiating respiratory and cardiovascular adjustments to maintain blood gas homeostasis. It is strategically located in the bifurcation region of each common carotid artery. The organ consists of small clusters called glomeruli composed of two juxtaposed cell types, glomus and sustentacular cells, interspersed with blood vessels and nerve bundles and separated by connective tissue. The neuron-like glomus or type I cells contain numerous cytoplasmic organelles and dense-cored vesicles that store and release neurotransmitters. They form both conventional chemical and electrical synapses between each other and are contacted by peripheral nerve endings of petrosal ganglion afferent neurons. Such a morphological arrangement and synaptic organization are favorable for an autocrine and paracrine regulation of the glomus cell function. The glial-like sustentacular cells, in addition to their supporting role, maintain physiologic neurogenesis in the adult CB and are thus supposed to be progenitor cells. This new source of adult stem cells may be potentially useful for tissue repair after injury or for cell replacement therapy against neurodegenerative diseases that could favor the translation of CB research to a clinical reality. The CB is a highly vascularized organ and its intraorgan hemodynamics possibly plays a role in the process of chemoreception. There is also evidence that chronic hypoxia induces marked morphological and neurochemical changes within the CB, but the detailed molecular mechanisms by which these affect the hypoxic chemosensitivity still remain to be elucidated. Dysregulation of the CB function is implicated in various physiological and pathophysiological conditions, including ventilatory altitude acclimatization and sleep-disordered breathing. This concise and highly informative book will provide an update to the current knowledge of the structural organization and new insights into neurochemical anatomy of the mammalian CB, its function and dysfunction, and will contribute to our better understanding of respiratory homeostasis and cardiovascular responses in health and disease.

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. E. Lazarov and D. Y. Atanasova, Morphofunctional and Neurochemical Aspects of the Mammalian Carotid Body, Advances in Anatomy, Embryology and Cell Biology 237, https://doi.org/10.1007/978-3-031-44757-0

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