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NEUROSCIENCE RESEARCH PROGRESS

LONG-TERM MEMORY

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MECHANISMS, TYPES AND DISORDERS

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PERSPECTIVES ON COGNITIVE PSYCHOLOGY

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LONG-TERM MEMORY

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MECHANISMS, TYPES AND DISORDERS

ARSENI K. ALEXANDROV AND

LAZAR M. FEDOSEEV EDITORS

Nova Science Publishers, Inc. New York

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Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Long-term memory : mechanisms, types and disorders / editors, Arseni K. Alexandrov and Lazar M. Fedoseev. p. cm. Includes index. ISBN HERRN 1. Long-term memory. 2. Memory disorders. I. Alexandrov, Arseni K. II. Fedoseev, Lazar M. BF378.L65L66 2011 153.1'3--dc23 2011051315

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Long-Term Memory : Mechanisms, Types and Disorders, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook

CONTENTS Preface Chapter 1

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Chapter 2

Chapter 3

Chapter 4

Chapter 5

vii Molecular Mechanisms Integrating Adenylyl Cyclase Responsiveness to Metabolic Control on Long-Term Emotional Memory and Associated Disorders A. Bennun Long-Term Memory and Topographical Disorientation in Healthy Elderly People: Preliminary Results of a New Diagnostic Tool Maria Luisa Rusconi, Claudia Zamin, and Laura Carelli Magnetic Bubbles in Brain Neocortex: A Bio-Magnetite Based Model for Long-Term Memory Marcos Arturo Martínez Banaclocha PARP1 Activation Is Required for Long-Term Memory Malka Cohen-Armon Effects of Afobazole on the Searching Activity and Food-Procuring Skill of the Offspring of Rats Exposed to Hypoxia during Fetal Development O. V. Shreder, E. D. Shreder, V. V. Zabrodina and A. D. Durnev

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1

45

75

103

117

vi Chapter 6

Chapter 7

Contents The Benefits of Expressive Writing on Long-Term Memory Performance Tracy Linderholm and Lise Abrams

131

The Effect of Visuo-Spatial Attention on Long-Term Memory Encoding Stephan Josef Stegt and Cristina Massen

147

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Index

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171

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PREFACE This book presents current research in the study of the mechanisms, types and disorders of long-term memory. Topics included in this compilation include the role of long-term memory in aging and its correlation with topographic disorientation; the diversity and complexity of neurocortical connections, circuits, maps and their relationships with superior cognitive function; PARP1 activation for long-term memory; the effects of afobazole on cognitive activity; the benefits of expressive writing on long-term memory performance; and visuo-spatial attention on long-term memory encoding. Chapter 1 - The individual and separate effects of Mg2+, Mn2+, Ca2+, ATP4- and their complexes on the kinetics of rat’s brain adenylyl cyclase (AC), were jointly investigated with AC of fat cells and the insulin receptor tyrosine kinase (IRTK) of liver. The purpose was to evidence the integration of neurotransmitter responsiveness of AC, with the regulatory feedbacks of the metabolic network, supporting neuronal aerobic glycolysis. MgCl2 saturation curves of AC show cooperative interactions at three sites: i) An obligatory free Mg2+ requirement for the enzyme responsiveness to noradrenaline (NA), with a cooperative value n=4 and 300% increase of Vmax. The NA effect may mediate an emotionally activated long-term memory, which requires a sudden increase of glucose and oxygen consumption. ii) Memory affirmation may require a restless basal AC-activity: Km(MgATP)=1.05 mM. The active site requires an excess of free Mg2+ over substrate, to prevent dead end inhibition by ATP4- and/or CaATP. Basal AC-activity shows that the substrate MnATP increases affinity Km(MnATP)=0.06 mM and 200% the Vmax. AC-dependent memory may have Mn2+-activated switches, for differentiable gating of nerve impulses, along specific synapses networks. iii) Basal activity shows a cooperativity value, n=2.6, surging from the interaction between active and

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viii

Arseni K. Alexandrov and Lazar M. Fedoseev

regulatory sites. The latter, affinity values: K0.5(E-Mg2+)=4.7 mM, K0.5(EMn2+)=3.8 mM and K0.5(Ca2+)=0.02 mM. The AC kinetics could be integrated in a random rapid equilibrium (RARE BiBi), with dead end inhibition Ki(ATP4-)=0.27 mM and Ki(CaATP)=0.01 mM. An increase in chelating metabolites increases both dead end inhibitors. Alternatively, a decrease in chelating metabolites decreases CaATP, strongly activating AC-mediated and cAMP-dependent activation of pathways for memory affirmation. The insulinindependent uptake of glucose, supports anaerobic glycolysis in the erythrocyte, allowing maximal activity of 2,3-DPG mutase, at the cerebral spinal fluid (CSF) pH=7.4. The control of the erythrocyte 2,3-DPG level fulfills, the role of a glucose level sensorial capability of the Hb-red cell system. The molecular dynamics proposed allows that the sharing of some of the Hb R-groups allow binding of O2 and the activatory ligands Mg2+ or Zn2+, with exclusion of 2,3-DPG or viceversa. Hence, allowing matching of the rates of glucose and O2 consumption during brain aerobic glycolysis. The overall metabolic feedback by the “in common” dependence on free Mg2+ or Mn2+ of AC of brain, fat cells and the insulin receptor tyrosine kinase (IRTK) in liver, allows homoestatic network integration. The Na+/K+-pump by breaking ATP4into ADP2- and phosphate (Pi2-) increases the concentration of ionic Mg2+. The latter, by activating AC links the nerve impulse with the cAMP activation of protein kinases and the cAMP response element binding proteins. Hence, the dominant role of ATP within brain energy consuming processes, allows metabolic feedbacks which operate as a unificatory backbone of the processes leading to long-term memory consolidation. Chapter 2 - In the physiological aging, a reduction in processing speed, episodic memory and working memory has been established. However, little is known about navigational abilities in elderly people and few ecological tools are available. Recently, some evidences have suggested topographical disorientation (TD) as a possible indicator of conversion from amnesic Mild Cognitive Impairment (a-MCI) to Alzheimer’s disease (AD). Our purpose was to create and validate a new ecological instrument in healthy elderly subjects to be used subsequently in MCI and AD patients. Sample: we enrolled 30 healthy volunteer participants, 15 male and 15 female, mean age 68.13 (SD = 8.56). Instruments: we administered a neuropsychological assessment and experimental tasks that consist of bidimensional stimuli and a plastic city with several subtests. Results: we found significant correlations among the experimental test and spatial memory tasks and executive functions. In navigation tasks, no differences were found according to gender while age resulted to play an important role. Younger

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Preface

ix

elderly subjects showed better performances in execution times, learning of different paths and creating and manipulating a mental map. Conclusion: Remembering the correct place of landmarks seems to be a useful but not a sufficient ability in orientation into surrounding environments: binding together landmarks in a correct framework seems to be the key’s feature of orientation. These interesting results deserve to be experimented also in MCI and early AD patients. Chapter 3 - It is thought that there is a very close structure-function relationship in brain neocortex. However, the diversity and complexity of neurocortical connections, circuits, maps and their relationships with superior cognitive functions are not fully understood, requiring a re-evaluation of the classical approach. I propose that human cerebral neocortex is organized fundamentally in two complementary and closely interrelated threedimensional physical structures formed by the neuronal and the astroglial networks, which in addition to chemical and electrical signals may communicate with each other through magnetic signals. This magnetic interplay may result in complex but specific and self-organized patterns of magnetic bubbles bound to the astroglial matrix through magnetite nanoparticles bio-mineralization, supporting memory and other cognitive representations. The present model opens new perspectives for solving some important problems in neurobiological investigation, in which classical and modern physical approaches are fundamental. Chapter 4 - PolyADP-ribose-polymerase -1 (PARP1) is activated in neurons that mediate several forms of long-term memory in the marine slug Aplysia as well as in mammals. Because PARP1 activation is a response to DNA damage in virtually all eukaryotic cells, it was surprising that this protein is activated during learning and is required for long-term memory. Recently disclosed features of PARP1 may underlie this activity: (1) Its fast and transient activation in response to membrane depolariziation and stimulation of G-protein coupled receptors and receptor tyrosine kinases in the cell memebrane, (2) relaxation of the chromatin structure by PARP1 activation renders the DNA more accessible to transcription (3) PARP-1 activation in the absence of damaged DNA up-regulates ERK-triggered expression of immediate early genes, some of which are involved in long-term memory formation. Based on both in-vivo and in-vitro experimental findings, this review integrates currently known data on the activity of PARP1 in mechanisms underlying long-term memory formation during learning, and provides new insight on molecular mechanisms in the chromatin in the context of memory formation.

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Chapter 5 - In the model of the formation of spatial and food-getting skills in Ж-maze showed that hemic hypoxia leads to the disruption of spatial memory and cognitive behaviour in offspring of rats. More pronounced impairment of the orientation-exploration reactions and food-procuring behavior observed in females. Afobazole (1 or 10 mg / kg, per os), administered to pregnant rats on the background of hemic hypoxia and / or during breast feeding (200 mg / kg) increased the possibility of orientation and restored learning offspring. In animals treated with afobazole was similar to the control of the formation of short-and long-term memory in learning. Thus, afobazole corrects the disturbances of food-getting movements of learning and memory in rats exposed to hypoxia hemic during fetal development. Chapter 6 - Previous research has shown that expressive writing can be beneficial to cognition by increasing working memory performance. The present study examined whether expressive writing about negative life events could facilitate long-term memory performance. Participants either wrote about a negative life event or completed a neutral, non-writing task involving math calculations. Participants then completed a fan effect task involving long-term memory, where they learnedsubject-location sentences (subjects were paired with multiple locations) and later attempted to recognize a particular subject-location as quickly and as accurately as possible. The results showed that following expressive writing, subject-location pairs were learned faster relative to the neutral task. There was also some evidence that participants who wrote expressively recognized subject-location pairingsmore quickly and more accurately than participants in the neutral condition. We conclude that expressive writing has the potential to facilitate cognition beyond immediate recall, which has important educational implications. Chapter 7 - Existing studies about the effects of attention on long-term memory encoding focused on the effects of ‘frontal’, executive attentional mechanisms and made use of paradigms of divided attention. We investigated the impact of visuo-spatial (‘posterior’) attention on long-term memory encoding. In four experiments, we used a standard spatial cuing paradigm and presented words either under valid or invalid cuing conditions. Performance in yes-no-recognition, cued recall and free recall was enhanced in the valid cuing-condition, whereas no effect was found for source-monitoring. The results show not only divided, but visuo-spatial attention considerably influences memory encoding, and the impact of visuo-spatial attention is different from that of divided attention.

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In: Long-Term Memory ISBN: 978-1-61942-699-3 Editors: A. Alexandrov and L. Fedoseev © 2012 Nova Science Publishers, Inc.

Chapter 1

MOLECULAR MECHANISMS INTEGRATING ADENYLYL CYCLASE RESPONSIVENESS TO METABOLIC CONTROL ON LONG-TERM EMOTIONAL MEMORY AND ASSOCIATED DISORDERS

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A. Bennun Graduate School, Rutgers University (R) CONICET, Argentina

ABSTRACT The individual and separate effects of Mg2+, Mn2+, Ca2+, ATP4- and their complexes on the kinetics of rat’s brain adenylyl cyclase (AC), were jointly investigated with AC of fat cells and the insulin receptor tyrosine kinase (IRTK) of liver. The purpose was to evidence the integration of neurotransmitter responsiveness of AC, with the regulatory feedbacks of the metabolic network, supporting neuronal aerobic glycolysis. MgCl2 saturation curves of AC show cooperative interactions at three sites: i) An obligatory free Mg2+ requirement for the enzyme responsiveness to noradrenaline (NA), with a cooperative value n=4 and 300% increase of Vmax. The NA effect may mediate an emotionally activated long-term memory, which requires a sudden increase of glucose and oxygen consumption. ii) Memory affirmation may require a restless basal ACactivity: Km(MgATP)=1.05 mM. The active site requires an excess of

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2

A. Bennun free Mg2+ over substrate, to prevent dead end inhibition by ATP4- and/or CaATP. Basal AC-activity shows that the substrate MnATP increases affinity Km(MnATP)=0.06 mM and 200% the Vmax. AC-dependent memory may have Mn2+-activated switches, for differentiable gating of nerve impulses, along specific synapses networks. iii) Basal activity shows a cooperativity value, n=2.6, surging from the interaction between active and regulatory sites. The latter, affinity values: K0.5(E-Mg2+)=4.7 mM, K0.5(E-Mn2+)=3.8 mM and K0.5(Ca2+)=0.02 mM. The AC kinetics could be integrated in a random rapid equilibrium (RARE BiBi), with dead end inhibition Ki(ATP4-)=0.27 mM and Ki(CaATP)=0.01 mM. An increase in chelating metabolites increases both dead end inhibitors. Alternatively, a decrease in chelating metabolites decreases CaATP, strongly activating AC-mediated and cAMP-dependent activation of pathways for memory affirmation. The insulin-independent uptake of glucose, supports anaerobic glycolysis in the erythrocyte, allowing maximal activity of 2,3-DPG mutase, at the cerebral spinal fluid (CSF) pH=7.4. The control of the erythrocyte 2,3-DPG level fulfills, the role of a glucose level sensorial capability of the Hb-red cell system. The molecular dynamics proposed allows that the sharing of some of the Hb R-groups allow binding of O2 and the activatory ligands Mg2+ or Zn2+, with exclusion of 2,3-DPG or viceversa. Hence, allowing matching of the rates of glucose and O2 consumption during brain aerobic glycolysis. The overall metabolic feedback by the “in common” dependence on free Mg2+ or Mn2+ of AC of brain, fat cells and the insulin receptor tyrosine kinase (IRTK) in liver, allows homoestatic network integration. The Na+/K+pump by breaking ATP4- into ADP2- and phosphate (Pi2-) increases the concentration of ionic Mg2+. The latter, by activating AC links the nerve impulse with the cAMP activation of protein kinases and the cAMP response element binding proteins. Hence, the dominant role of ATP within brain energy consuming processes, allows metabolic feedbacks which operate as a unificatory backbone of the processes leading to longterm memory consolidation.

INTRODUCTION Long term memory requires the construction of new synapses [1] [2] Changes in synaptic strength participate in learning and memory. The process involves neuro-transmitters, receptors and new synapses generating pathways which reinforce the strength of connectivity between neurons. Noradrenalin (NA) binding activates various subtypes of adrenergic receptors, for a differential role of signaling pathways, which functionally operate by inducing synthesis of second messengers.

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The striatum and the hippocampus have been shown by Functional Magnetic Resonance Imaging (FMRI), to interact cooperatively to support episodic memory formation [3]. New long-lasting details of specific events could be lost to people that show amnesia, a dysfunction that can be related to hippocampus damage [4]. The cerebral cortex is capable of semantic memory, the ability to learn facts and relationships mediated by changes in the connections between cells, related by their conducting of specific types of information [5]. The ventromedial hypothalamus (VMH) has been shown to respond to catecholamine released into the glucose-sensing region, through a counterregulatory response, to hypoglycemia by stimulation of the beta-2-adrenergicreceptor (B2AR) [6]. Emotional charged events have been related to activation of the locus coeruleus release of noradrenalin [7], activating a second messenger system [8]. Noradrenalin activates AC in membrane preparations obtained from rat’s cerebral cortex, corpus striatum and hypothalamus [8]. However, overexposure to noradrenalin of AC during pre-incubation, an assay previous to the one measuring the activatory effects of the neurotransmitter [9], results in inactivation of the enzyme. Hence, it could be inferred that noradrenalin-dependent psychosomatic damage, lead in the course of an evolutionary selection process to a costly protection mechanism. This one, turns-off the enzyme rather than allowing a too perdurable permanence of the organism into the fight or flight state. The results of the experimentally obtained prolonged association of NA with its receptor may correspond to a stressing of the quaternary structure, during the activatory state [9]. This catecholamine-dependent inactivatory event was prevented by a pre-incubation condition, in which the presence of MgATP protects from NA, by the formation of an enzyme substrate complex. Hence, the NA-AC complex manifests thermic instability, whereas the NA-ACMgATP allows to protect the enzyme activity, but to the cost of decreasing its responsiveness to a catecholamine activatory effect [8] [9] [10]. Stress mediated by increasing adrenalin secretion, usually shows a pattern of hyperfunction follow by hypofunction, which when the stressed organ is brain it could be associated with a symptomatology of persistent anxiety followed by depression [9]. Under prolonged stress, the synthesis of new AC, may not match the rate of noradrenalin-dependent inactivation. The viability of functional synapses may be controlled by the speed of synthesis of the proteins, which make-up the hormonal receptor sites and/or AC-itself, versus the rate of inactivation or

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destruction of AC. This generalized molecular modeling for the evolution of psychosomatic diseases [9] could be tested, with regard to more specific physiological conditions and permit prognosis of dysfunction, allowing preventing treatments. Already medical applications have been developed, based in the use of Beta blockers to interfere with a stressful condition, attenuating the memory of a traumatic event. Characterization of the NA activation of brain AC indicated that could participate in the formation of molecular memory pathways [8] [11]. This proposition was extended to show its role in cAMP-dependent pathways, involved in the development of behavior patterns [12] [13] [14] [15] [16]. In absence of noradrenalin AC was shown to have a basal activity subject to upregulation by Mg2+ and Mn2+ ions and their ATP complexes MgATP and MnATP as substrates [11]. Na+-pump activity changes ionic equilibrium by the neuronal transfer out of Na+ coupled to K+ intake. Additional changes, relate to ATPase activity splitting ATP4- into ADP3- and Pi2-, with an ionic equilibriums allowing the endogenous release of free Mg2+ ions. The breakdown of ATP also decreases the concentration ATP4- and CaATP, which were shown to act as strong inhibitors of AC activity [11]. More recent studies obtained from the molecular, behavioral cognitive, signal transduction, physiologic and pathological research, has shown the relevance of cAMPmediated long-term memory [17] [18] [19] [20] [21] [22] [23] [24] [25]. It could be expected that a large number of the pathways associated with neuronal activity, could be integrated into the complex process of memory formation and consolidation, by an “in common” coupling by the interdependence of signal and ATP-energy transduction mechanisms [26] [27] [28] [29]. Up-regulation of AC by ionic equilibrium in dependence of changes in ATP concentrations, leaded to investigate possible metabolic-ionic feedbacks related to the energy requirement of the NA-AC system. Stimulated neurons require O2 to maintain the supply of ATP for the Na+-pump. O2 increases turnover of both electron transport chain and Krebs cycle, which occurs through the increase in dicarboxylic and tricarboxylic acids concentrations in mitochondria. Effect which increases Ca2+ retention in mitochondria and upregulate the Na+/K+-ATPase-AC system. The relationship between the rate of aerobic glycolysis and neuronal work was therefore examined, in the context of the signal transduction of tissue networking. The latter, allows a large increment in the rate glucose and O2 uptake by stimulated brain areas, even when homeostatically sustaining glucose levels in blood.

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METHODS Preparations

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Female Sprague-Dawley rats weighing 210-300g were ovariectomized to decrease interference by estrus cycles [8] and decapitated at 4°C two weeks after surgery. The brains were dissected in the cold (4°C) to isolate and separately collect the hypothalamus, cortex and striatum from groups of 10 to 15 animals [8] [30]. The total wet weight of each pool of tissues was colected in 20 ml of ice-cold 2 mM Tris-HCI containing 10 mM NaCl, 10 mM KCl and 5µM EDTA, buffer at pH=7.4. These were rinsed twice with 15 ml/g wet weight of tissue and homogenized using a motor-driven (Inframo, Wayne, New Jersey) Teflon homogenizer at 400 rpm for 2 min. Each homogenate was centrifuged at 4°C and 6,000 gav in a RCA Sorvall, head SS-34 for 20 min. The resulting supernatants were discarded and the pellet washed, resuspended and centrifuged again once. Aliquots stored at -70°C. Protein concentration for each membrane preparation was determined by the method of Lowry (Lowry et al., 1951) using bovine serum albumin as the standard. Particle suspensions and reaction mixtures were tested, with a calcium ion electrode for Ca2+ contamination with negative results.

ASSAY OF ADENYLYL CYCLASE ACTIVITY Adenylyl cyclase (AC), systematic name: ATP diphosphate-lyase (cyclizing, 3´, 5´-cyclic-AMP-forming), EC 4.6.1.1, in the membrane preparations of cortex, corpus striatum and hypothalamus were kinetically characterized. The tissues were selected in expectation of finding differential kinetic properties, which could be related to specific areas of rat´s brain [8]. Defrosted particulate preparations (from l.7 to 6 g protein/assay) were resuspended in a standard reaction mixture, containing 20 mM Tris-HCl buffer (pH 7.4), 5 mM theophylline, MgCl2, plus ATP disodium salt, from equine muscle, with GTP content of less than 2% and traces of other nucleotides from Sigma. This ATP/GTP mixture was used when neurotransmitter dependent activation was desirable as in the experiments reported in figure 1 a) and b) and table 1. The ATP disodium salt from P-L Biochemicals, Inc (Milwaukee, Wisconsin) was used for the assay of AC basal activity. Other additions indicated in the figures legends.

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After incubating in a shaking bath at 37°C for 15 min the reaction was terminated by heating in a boiling water bath for 5 min. Time-dependence studies using the membrane preparations, showed linear responses for no less than 30 min. Cyclic AMP formed by adenylyl cyclase activity was assay by the Gilman method [31] modified by Brown et al. [32] according to publish specifications [33] but with cyclic [3H]AMP increased to 20,000 c.p.m./assay. These membrane preparations show an ATPase activity [34] of only 0.1 nmol Pi/h/g-protein [8]. Results were calculated by formula, (C- b)/(T- b), C: c.p.m. from the assay of the enzyme activity, T: c.p.m. from total binding (zero-time blanks) and b: c.p.m. in the millipore filters (moisture blanks). Calibration curves are linear for % of C- b/T- b versus log cAMP without interfering ATP or other reagents. Results mean values of triplicates or more: nanomoles of cAMP formed per hour per milligram of protein. The standard error of the mean (S.E.M.) did not exceed 6% [35].

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RESULTS Previous kinetic studies of the noradrenalin-activated adenylyl cyclase (AC), evidenced that the neurotransmitter response could be differentiated from a basal response, according to their different Vmax, which could explain memory at two differentiable levels. The noradrenalin dependent activity of AC, could be induced by events with strong emotional connections, related to the fight or flight reaction. The activity of the neurotransmitter-enzyme complex correlates to a reinforcement over the basal activity one elicited by free Mg2+- or Mn2+-dependent activation of the basal state of the enzyme [9] [10]. However, prolonged effects may elicit a CNS excited or anxious state which prolonged results in a depressive state [9] [36]. The latter, could be related to memory deficiencies so emotional potentiation of memory capability became limited because under stress condition collapses. The present studies were directed: I) To elucidate the kinetic properties of AC relevant to modeling mechanism: a) for NA-dependent emotional eliciting and reinforcement of AC-dependent long-term memory, b) for continuous memory processing by activation of the basal AC activity by free divalent metals; II) Characterization of hemoglobin red cell-system as a sensor of glucose levels in blood, and responsiveness for O2-release matching glucose uptake III) To detect a metabolic feedbacks of a tissue network, capable to

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shift the rates of O2 and glucose consumption, between hormonal activated and the basal memory processing stage. The matching of the rates of brain glucose consumption and tissue oxygenation requires “in common” molecular signaling. This one should operate within a network, by a healthy physiology resolving through the sensor role of the red cell-Hb system, the matching of brain metabolism with the homeostatic need of balancing the metabolic flows, within the liver fat-cell subsystem.

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RELEVANT BRAIN AC KINETIC PROPERTIES FOR EMOTIONAL REINFORCEMENT AND BASAL MEMORY PROCESSES Purified enzymes preparations from rat’s tissues were obtained for adenylyl cyclase of fat cells, brain and insulin receptor tyrosine kinase (IRTK) from liver. This preserved their allotopically associated receptors by not extend purification to an extraction procedure to separate the enzyme from the membrane [34]. These enzyme-membrane systems were shown to preserve modulatory properties required for their physiological role. The preparations of AC [ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1)] obtained from corpus striatum, hypothalamus and cortex of the brains of rats show that noradrenalin (nor-epinephrine) activation is dependent of the concentration of magnesium ion (Mg2+). Table 1. Kinetic characterization of adenylyl cyclase obtained from specifics brain areas. The values obtained do not allow inferring differences between the responsiveness to the substrate of the basal adenylyl cyclase activity Particulate Membrane preparations Cortex Striatum Hypothalamus

Km(MgATP) [mM] 2.2 2.0 2.0

Cooperativity (n) 1.4 1.1 1.1

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Figure 1. a) and b). Control by MgT of the responsiveness of noradrenaline activated adenylyl cyclase. Samples of an EDTA-particles preparation from cortex containing 100g of protein were incubated at 37oC for 1hour, in 40mM-Tricine/Tris buffer, pH=7.4, 6.67mM-caffeine, constant 1mM-ATP(Sigma purified from horse muscle), in a final 0.5ml with the indicated concentration of MgCl2 (MgT). Vmax for each curve was determined at the indicated MgCl2 concentration. Activity reported: nano-moles of cAMP formed/h per mg of membrane protein. Figure 1.a). MgCl2 saturation curves: o) +1mM-adrenaline, at 30mM [MgCl2] were determined Vmax values, 21214(4);) basal, 787(4). Figure 1.b) MgCl2 saturation curves: basal, at 15 mM [MgCl2], 787(4); ) +1mM-adrenaline +0.3 mM CaCl2 at 25mM [MgCl2], 272(4).

Impulse transmission and/or noradrenaline binding the receptor induce the depolarization of the plasma membrane. Neuronal transmission is terminated by the neurotransmitter reuptake linked to Na+ entry. The β-receptor is present in the hormone-binding subunits of the enzyme AC. When noradrenaline binds to its receptor, this one becomes activated to bind G-protein, which is activated by GTP. As has been reported elsewhere, the neurotransmitter activated enzyme, increases Vmax. The activated enzyme generates endogenous cAMP, which rather than acting as a messenger between one neuron and another, has the capability to induce changes in the function of ion channels within the membrane. The cAMP has effect on gene expression in the nucleus of the cell and in the processes of growth and development. Changes on the genetic material of cells may lead to long-term alterations of behavior. Second messengers play a role in the manufacture and release of neurotransmitters, intracellular movements, and a carbohydrate metabolism in the brain. Hence, Table 1 shows that allotopic enzyme-membrane properties are similar for the brain regions tested.

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Figure 1.a) shows the activatory effect of noradrenaline versus basal on the AC of cortex increasing by 300% basal Vmax. The cortex enzyme shows cooperativity for total-magnesium in the presence of 0.1mM-noradrenaline with a value n(MgT)=3.9. Figure 1.b) shows that the AC basal cooperativity decreases from n(MgT)=2.9 and by addition of noradrenaline plus CaCl2 decreasing to n(MgT)=2.5 and decreased Vmax to 1/3. Comparative results on the effect of noradrenaline in adenylyl cyclase of corpus striatum are reported in table 2.

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Table 2. Effects of Mg2+ and ATP4 on the response to noradrenaline and calcium of adenylyl cyclase of corpus striatum. To investigate the individual and separate effects of free Mg2+ and of ATP4, their concentration were determined by multiple-equilibrium equations Kinetic Constants MgT MgT Mg2+ Mg2+ ATP4 ATP4

Basal

0.1mM-Noradrenaline

n=2.7 S0.5=8mM n= 1.1 S0.5=4.7mM n=1.1 S0.5=0.1mM

n=4.4 S0.5=9mM n=1.9 S0.5=4.6mM n=1.9 S0.5=0.1mM

0.1mM-noradrenaline +0.3mM CaCl2 n=2.3 S0.5=9.4mM n=1.3 S0.5=4.6mM n=1.3 S0.5=0.1mM

Figure 2. Lineweaver-Burke plot. Effect of ATP4 on the substrate saturation curve of adenylyl cyclase particulate preparation from corpus striatum. Portions of an AC particulate preparation containing 2.5 g of protein were incubated. Basal, MgATP saturation curve, at 5mM MgATP, Vmax: 9nmol cAMP/h/per mg of protein; o) basal+2.5 mM ATP4 at 9mM MgATP, Vmaxapp: 7nmol cAMP/h/per mg of protein constant equilibrium concentration adjusted according to table 3.

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In table 2, the AC of corpus striatum at basal conditions and for 0.1mMnoradrenaline and +0.1mM-noradrenaline+0.3mM CaCl2, show similar affinity S0.5(MgT) and S0.5(Mg2+) of about 9mM. The affinity for the inhibitory specie S0.5 (ATP4-)=0,1mM. Hence, noradrenalin mainly increases Vmax but does not change by much the enzyme affinity for either Mg2+ or ATP4-. The AC of corpus striatum cooperativity for 0.1mM-noradrenaline n=4.4. Cooperativity for basal n=2.7, since for basal n(Mg2+), n(ATP4-) and n(MgATP) show values close to 1. Hence, the values obtained for basal activity n(Mg2+), are influenced by the summation to the activatory effect of Mg2+, and the disappearance of the inhibitory effect of ATP4-. The enzyme saturation system, shows a n(adrenaline) values of about 4, because the neurotransmitter potentiates Mg2+ mediated binding to the membrane. The hormonal receptor for the expression of properties dependent of its position in the membrane, allotopic, may require Mg2+ [34] for the activatory binding of G-protein. The enzyme shows a single active site, therefore, requires Mg2+ and MgATP for basal activity, and a separate for the binding of an adrenaline receptor. The applicability of the random of the RARE BiBi model is confirmed by the ability of the model to predict a change of Hill cooperativity from a basal value of n=2.6 to 4.0 in the presence of noradrenaline (Figure 1 and Table 2). This indicates that excess ions Mg2+ over that required for the saturation of sites, for substrate formation and for the Mg2+ regulatory activation. Hence, an additional saturation by ionic Mg2+ is an obligatory requirement for the manifestation of third site linked to the responsiveness to hormonal dependent activation [8] [33] [35]. Lineweaver-Burk plots of adenylyl cyclase activity as a function of substrate concentration at different concentrations of Mg2TOTAL show a noncompetitive behavior, indicating that Mg2+ interacts with a distinct regulatory site differentiable from the active site [11]. If the data of Figure 5 is plotted according to Lineweaver-Burk, the addition of CaATP shows Ki=0.015mM. However, Mg2+ (MgT) can easily displace Ca2+ from ATP. The equilibrium favors that Mg2+ can easily displace Ca2+ from ATP, and reduce the concentration of CaATP. Since Ka (MgATP)=-20×103M-1 versus Ka(CaATP)=-9×103M-1. Evaluation of the responsiveness of the enzyme indicates that there is a requirement that the concentration of free Mg2+ exceeds that of MgATP to activate the enzyme. The AC response appear coupled to the operation of the Na+ gates augmenting free ionic Mg2+. The gates associated ATPase reaction

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releases free Mg2+ because since ADP3 has a much weaker association constant for the divalent metal than ATP4. The Hill Plot in Figure 3 was obtained from saturation curves with a Vmax(MnATP): 19.2nmol cAMP/h/mg, a considerable higher activity than the corresponding Vmax(MgATP): 8.2nmol cAMP/h/mg. Table 3. Equilibrium concentrations of MgATP, ATP4 and Mg2+ under experimental conditions where ATP4 is kept constant and MgATP is increased. Equilibrium concentrations, adjusted from the calculated ionic equilibrium [37] [38]

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Mg2+ (mM) 0.0058 0.0229 0.0455 0.0896 0.1110 0.1320 0.2000

ATP4(mM) 2.20 2.23 2.25 2.29 2.31 2.33 2.33

MgATP (mM) 0.244 0.976 1.950 3.910 4.880 5.860 8.900

[MgATP]/[Mg2+] 42.1 42.6 43.4 43.4 43.9 44.0 44.0

Figure 3. a) MnATP substrate saturation curve and 3.b) MgATP substrate saturation curve. Portions of adenylyl cyclase particulate preparation from corpus striatum containing 6µg proteins were incubated in the conditions for assay of basal activity described for Figure 1. Activity reported: nmol cAMP formed/h per mg of membrane protein. Figure 3.a), MnATP saturation curve: o) At 1mM MnATP reached Vmax value: 19.2nmol cAMP, cooperativity value n(MnATP)=1.4, Km(MnATP)=0.05mM. Figure 3.b), MgATP saturation curve: At 3.6mM MgATP reached Vmax: 8.2nmol cAMP, cooperativity value n(MgATP)=1.6, Km(MgATP)=0.84mM.

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Figure 4. Effect of inhibitor ATP4 on the substrate MnATP saturation curve of corpus striatum adenylyl cyclase. 6g protein portions of an adenylyl cyclase-membrane preparation were incubated at 37oC as indicated in the legend to Figure 1 o) Basal; with the indicated increasing equilibrium concentration of MnATP at 1mM MnATP reached Vmax: 19.2nmol cAMP/h/per mg; basal+inhibitor 5.5mM ATP4at 4mM reached Vmax: 15.2nmol cAMP/h/per mg.

Comparing both Km, it could be shown a marked increase for the basal activity of adenylyl cyclase in the presence of MnATP, with an affinity increment of over 17 times than the Km MgATP. Moreover, Vmax was increased for the substrate MnATP 2.3 times over that of Vmax MgATP. Equilibrium conditions for MnATP (log Ka=4.6) allowed to increase the concentration of MnATP at negligible concentration of free ionic Mn2+. Hence, it could be infer that if both substrates are separately compartmentalized the adenylyl cyclase consuming MnATP would be able to produce cAMP at much higher rates than in the presence of MgATP alone. Additionally, the adenylyl cyclase in the presence of substrate MnATP manifests generation of cAMP independently of the already discussed free Mg2+ activatory requirement (Figure 1), controlling responsiveness at the neurotransmitter and basal levels. Hence, it could be infer the existence of neurons or synaptic structures able to orchestrate the response of adenylyl cyclase utilizing MgATP, with a control role in the generation of neuronal memory nets. The increasing concentration of [MnATP]/[Mn2+][ATP4-]=4104, implies that equilibrium maintains negligible concentration of free Mn2+. Hence, the addition to the basal curve of ATP4- maintains its equilibrium concentration well above that in the basal curve. However, the ATP4- inhibitory-curve

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approaches the basal at the higher maximal concentration, showing unchanged Vmax. Kmapp(MnATP) changes from 0.075 to 1.5 mM (Table I). The calculated Ki(ATP4)=0.25mM similar to the value obtained for substrate MgATP, Ki(ATP4)=0.27mM. Hence, the presence of MnATP does not alter the affinity for adenylyl cyclase for ATP4, ruling out, indirect modulation by Mn2+ on the affinity of the enzyme for ATP4. Figure 5 shows that addition of CaATP produce competitive inhibition decreasing affinity from 2mM to 20mM Kmapp(MgATP) [11]. Energy charge [39] is described as the intracellular relationship of [ATP]+1/2[ADP]/([ATP]+[ADP]+[AMP]), can have a value ranging from 0 (all AMP) to 1(all ATP). This index could be given a role in ionic equilibrium by its relationship to the control of the availability of free divalent metals. The latter, related to the stronger binding constants of ATP4− for Mg2+ and Ca2+ than for ADP3− and AMP2−.

Figure 5. Effect of inhibitor CaATP on the substrate MgATP saturation curve of hypothalamic adenylyl cyclase. 3.3g protein portions of a hypothalamic-membrane preparation were incubated at 37oC as described in the legend to Figure 1 with increasing equilibrium concentrations of 0.41 to 9.3 mM MgATP: MgATP saturation curve: o) Basal at 9.3mM MgATP Vmax: 13.2nmol cAMP/h/mg; □) basal+inhibitor 0.37±0.05mM CaATP at 9.3mM MgATP Vmax:5.8nmol cAMP/h/mg. Cooperativity value: n, computational obtained were included in the figure.

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Table 4. Kinetic values from Hill and Lineweaver and thermodynamics constant from Figure 2, 4 and 5 for the indicated E-Complexes. Kd dissociation constant; Kf formation constant for enzyme complexes; Go, Gibbs free energy. Kd values [37] [38] change by only 9% when ionic strength changes 100%. Hence, Kd values where affected by considerably lower deviations

---

0.10

1.1

4.1

0.015

6.67

0.9

6.5

0.02 ---

--0.27

5.0 0.37

--1.49

6.4 4.8

---

4.7

---

0.021

1.1

3.2

---

3.8

---

0.026

---

3.3

Enzyme Km Kd Ki Complex (mM) (mM) (mM)

Kf104 (M-1)

MnATP

---

---

MgATP

---

MgATP

+0.5mM CaATP --+2.26mM ATP4 +2.2mM Mg2+ +5.5mM ATP4

E0.065 --MnATP E1.05 --MgATP E-CaATP ----E-Ca E-ATP4-

-----

E-Mg E-Mn

--MgATP MgATP MnATP

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Go (Kcal)

1.43

Cooperativity (n) 0.8

Substrate Additions Saturation

5.6

In table 4 is reported that Km(MnATP)=0.065mM versus Km(MgATP)=1.05mM, correspond to 16 times lower affinity of the basal enzyme for the substrate MgATP than for MnATP. Table 4 dissociation constants, show an ionic equilibrium order Kd(Mg2+)=4.7mM>Kd(Mn2+)=3.8, indicating that Mg2+ can only at higher concentrations than Mn2+ displace the latter from the divalent metal activatory site. The activity order MnATP>MgATP and both ATP4- and CaATP act as inhibitors. Since Ki(ATP4-)=0.27 and Ki(CaATP)=0.015mM the competitive affinity order CaATP>MnATP>ATP4->MgATP. Table 4, indicates that at lower concentration of excess ATP4- the greater Ka of the nucleotide for either Mn2+ or Mg2+ than for Ca2+ could maintain the enzyme active. However, at higher concentration of excess ATP4- the greater affinity (Ki=0.01) of the enzyme for CaATP, could predominate reducing the availability of cAMP for sustainable memory persistence. Hence, eradication of particular memory, confer to the system plasticity to re-conform synaptic connectivity for acquisition of a new memory or reinterpretation of the preceding one.

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Scheme 1. Equilibriums for the RARE BiBi kinetic mechanism of the basal adenylyl cyclase. Study of initial velocity patterns gave evidence, that at the active site the substrates MgATP and MnATP by group transfer exchange PPi between ATP4- and a pyrophosphate (PPi) accepting group, releasing cAMP and PPi. Mn2+ substitutes for Mg2+ for substrate formation and as an enzyme modulator. The catalytic rates and dissociation constants for Mn2+ and MnATP differ from Mg2+ and MgATP (Table 4), with active site binding by inhibitor CaATP and ATP4- resulting in dead-end inhibition

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(

).

However, Mg2+ can easily displace Ca2+ from ATP and reduce the concentration of CaATP, because the association constant: Ka(MgATP) =20×103 M-1 is greater than Ka (CaATP)=9×103 M-1 [37] [38]. The mutual competitive inhibition of ATP4 and CaATP on the substrates, MgATP and MnATP, and a constant cooperative value of about one indicate that the ionic and divalent metal complexes of ATP, bind to the same site on basal adenylyl cyclase. A requirement of cAMP leading to increment neuronal connectivity implies that the level of AC activity may play a partial role of neuronal plasticity [40] [41]. This process results from a perdurable lack of cAMP to drive the creation and/or reinforcement required to maintain synaptic connectivity. The requirement to control cAMP levels indicates the applicability of AC kinetics, for modeling the versatility of AC responsiveness. Enzyme plasticity is implied in the scheme 1, showing a random BiBi sequential group transfer mechanism (RARE BiBi) with deadend inhibition, since the binding at the active site of CaATP or ATP4- results in dead-end inhibitions. In the intracellular fluid at 1mM [Mg2+] is considerable higher than 1M 2+ Ca [42]. The ratio is practically constant, except for certain pathological

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states [43]. However, the equilibrium dynamics of divalent metal existing as Mg2+ and MgATP, as well as Mn2+ and MnATP fluctuate. The latter, due to accumulation of endogenous metabolite intermediates like ATP4, amino acids, dicarboxylic acids, and organic phosphates, which can complex Mg2+ or Mn2+. The cytoplasmic concentration of Ca2+ is in a dynamic state by the same metabolic variables. Additionally, 1mM extracellular and 10mM mitochondrial concentration of Ca2+ allow import and export of Ca2+ across structural compartmentalization [44] [45]. A strong restriction to the adenylyl cyclase activation, became evident from S0.5(ATP4) of about 0.1mM (table 4). Hence, any decrease in the rate of ATP4 consumption by the Na+ gate activity and others functions, should turn off the adenylyl cyclase at both the neurotransmitter and basal levels. It is suggested that a matching the rate of ATP production and consumption should be optimal because the brain lacks storage capability. If the rate of ATP production overcomes consumption and endogenous accumulation occurs, ATP4 may compete for the divalent metal structurally present for binding receptors and enzyme to the membrane [34]. This requirement may have important implications, since neurons may need to adjust endogenous aerobic glycolysis to prevent ATP4- accumulation, overcoming the stability of receptors in the membrane.

CHARACTERIZATION OF THE DYNAMICS OF THE QUATERNARY STRUCTURE OF HEMOGLOBIN AS A FUNCTION OF A SENSOR ROLE OF GLUCOSE LEVELS IN BLOOD BY THE ERYTHROCYTE The insulin-regulated transporter GLUT4 is found in adipose tissue and striated muscle. GLUT1 is present in erythrocytes and endothelial cells of barrier tissues like the Blood brain barrier (BBB), is also expressed in liver cells which require bidirectionality to uptake glucose and by having glucose 6phosphatase to release it from glucose-6-phosphate (G6P). GLUT3 is expressed mostly in neurons. Hence, brain and liver use transporters which do not require insulin for efficient uptake of glucose. Cerebrum cells, lack of storage pathways are nutritionally dependent of a continuous uptake of glucose and oxygenation, to support aerobic glycolysis. Under starvation ketone bodies can only partially substitute for glucose. Hence, the need for signal transduction between tissues involved, required for

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matching the rates of O2 and glucose consumption. This capability allows cerebral areas to increase by 10 time’s the rate of aerobic glycolysis, without overcoming the homeostatic controls of glucose level in blood. Heterotropic allosteric modulation was shown for: 2,3-diphosphoglycerate (2,3-DPG) [46]. 2,3-DPG cross-links subunits 1 with 2 of deoxyHb, at the tissues low pH a central cavity of the Hb tetramer became structured as an binding site for 2,3-DPG [47]. The latter, became surrounded by the terminal amino acid, Lys 82, and the protonated imidazole rings of histidines His 143 and His 2, in the 1 and 2 chains, which bear two NH bonds and show overall positive charge. The 2,3-DPG-Hb complex of much lower affinity for O2 than isolated Hb could be regarded as the physiological T structure [48] [49]. Upon deoxygenation increase the concentration of free Mg2+ ion [50] showing that deoxyHb and Mg2+ were competitors binding reciprocally with organic phosphates [50]. At the pH of lungs, the protons dissociate from R-histidines and the imidazole ring changes from overall positive to manifest the reactivity of negatively charged nitrogen atoms. Thus, now negatively charged groups like His 143 [51] repels the similarly charged 2,3-DPG. The R-groups participants in H+ release or Bohr effect have been widly reported and allows to molecular characterization of proton disociated Hb as the R configuration within oxyHb. Interconvertion of deoxyHb pKa=80.2 to oxyHb pKa=6.40.2 releases Bohr protons from R-groups, within the two  chains: from Cys1 and 293, plus that from Asp1 and 294 [52] [53] [54]. Treatment to obtain human Hb stripped-out of 2,3-DPG and other ligands increases Hb affinity for O2 decreasing P50(O2) 2.8mmHg [53]. The Figure 6.a) illustrates a Hill plot showing that addition of 2,3-DPG [55] decreases Hb affinity for O2 by increasing P50, but without affecting Hb cooperativity for O2. A modified WoolfAugustinssonHofstee (WAH) plot of %saturation on the ordinate versus %saturation/(pO2)2.3 on abscissa, Figure 6.b) allowed to obtain an straight lines intersection at the theoretical 100%saturation on the ordinate. The plot indicates that under the ideal condition of increasing pO2 until reaching a theoretical maximum O2-saturation, the presence of a constant concentration of 2,3-DPG may not prevent the homotropic Hb response of forming oxyHb.

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Hence, a mutual exclusion between pO2 and 2,3-DPG could lead to predict, that a theoretical increase in pO2, could reduce an inhibitory effect of a constant 2,3-DPG concentration [55].

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Figure 6. a) b). Graphic of the 2,3-DPG effect on the %-Oxygenation of stripped Hb. Adapted data [53] for the control: absence of 2,3-DPG (____); addition: +410mM of 2,3-DPG (----) and +610mM of 2,3-DPG (. . . .) were plotted according to: a) to Hill equation and b) a modified Woolf-Augustinsson-Hofstee plot in where the abscissa shows pO2 at the exponential 2.3, value obtained from the Hill Plot. Introduction of the exponential is a solution to include cooperativity in order to transform curves into straight lines. Control values: (____) P50=2.8mmHg, cooperativity n=2.5; in presence of +410mM of 2,3-DPG; (----) values: P50=8mmHg, cooperativity n=2.46 and +610mM of 2,3-DPG (

....

) P50=10mmHg cooperativity n=2.2.

However, under physiological conditions Hb binds into a specific bind 2,3-DPG and forms a complex in which the Hb quaternary structure, became stabilized as the deoxyHb form by the release of O2. Lineweaver-Burk a double reciprocal plot adapted to subtract cooperativity, by introducing the cooperativity n-value for each curve in the abscissa 1/(pO2)n. The plot becomes 1/%Oxygenation versus 1/(pO2)n and allowed to obtain intersection of straight lines for a theoretical maximal 1/(pO2)1.5 and 1/(pO2)2.3. Hence, at a theoretical maximal pO2 (1/pO2=1/∞→0) saturation of Hb by O2 would be reached even in the absence of Zn2+. Hence, the activatory effect was to decrease the control value S0.5=1.64mmHg by addition of Zn2+ to S0.5=0.5mmHg [53] [55]. The strip method to free of ligands the Hb molecule appears to reduce steric hindrance. Even so Zn2+ becomes a strong enhancer of striped Hb affinity for O2. The effects of 2,3-DPG decreasing affinity (Figure 6) and Zn2+ increasing affinity (Figure 7) of Hb for O2, suggest by allosteric

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considerations alone, that a single site for both actions, should be discarded. However, even if solving by classical competitive interaction could not be applied, other treatments were tested. The nullification of 2,3-DPG inhibitory (Figure 6.b) and of the activatory Zn2+ actions (Figure 7.b), became evident when the plottings allow to assume a tendency for pO2 to reach the equivalent of infinite concentration. The figures 6 and 7 illustrate that because these ligands were at an invariant concentration, could be displaced from Hb by a competitive mechanism involving pO2 increment. Therefore, competition between O2 with inhibitor 2,3-DPG (Figure 6.b) do not ocurr at the same site but may result from this heterotropic ligand tendency to bind and stabilize one or more R-groups restricting O2-saturation of the Heme site of Hb. Accordingly, increasing pO2 would eventurally release the hindrance preventing O2-binding to the Heme. The activatory effect by Zn2+ (Figure 7.b) implicates that the effect of divalent metal is to decrease the concentration of O2 required to remove the hindrance effect. Hence, a model (figures 8 and 9) was develop that shows that O2 per se has full homotropic capability for the interconversion of deoxy- to oxyHb. Hence, competition for a modulatory site, which control hindrance, could explain Hb heterotropic reactivity with ligands decreasing (Figure 6.b) or increasing (Figure 7.b) affinity for O2. These dynamics imply that the modulatory sites could not be fully covalently structured, but are rather structures supported by coordinative bonding. The affinity of Hb for the ligands Mg2+ and/or Zn2+ supports the tendency to conform two coordinative structures of Hb, which fit the theoretical and physiological findings. The plasticity of this divalent metal or sugar phosphates modulated sites becames evident in term of a glucose sensor role for the Hb-erythrocyte system. This one, adding the physiological regulation by the endogenous metabolic state of the red cell, expressed by endogenous 2,3-DPG concentration, to the physiological pO2 between lung and CSF. Hence, when Hb becomes characterized in the sole presence of O2 shows the quaternary changes in the structural links between T- to R- forms, which characterize homotropic interconversion. However, the Hb potential to acquire metal chelating sites allows the molecular quaternary structures T- and R-, to participate in 2,3-DPG-Hb and divalent metal-Hb complexes stabilizing changes in the O2 affinity. These ones induced on Hb by endogenous heterotropic ligands within the red cells, allowing the addaptative response to the O2 needs of tissues and fullfiling in brain the matching of the rates of glucose and O2 uptake.

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Figure 7. Graphic of the effect of Zn2+ on the %-Oxygenation of stripped Hb. Adapted data [53] for the control: absence of Zn2+ (----), values: P50=1.6mmHg; and addition ____

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of 4.4710mM Zn2+ ( ), values: P50=0.5mmHg was plotted according to a) Hill equation and b) a modified doble reciprocal Lineweaver-Burk plot in were the abscissa shows pO2 at the exponentials values obtained for the individual curves plotted according to Hill. Thus, the –Zn2+, 1/(pO2)2.3 and +Zn2+1/(pO2)1.4.

The homotropic tendency of isolated Hb shows that during oxygenation a link between His146 and Asp94 breaks up and the imidazole ring at above pH 6 takes up a proton (alkaline Bohr Effect), which may be able to trigger the changes from T to R as a sole function of O2 through a G=3Kcal [56] [57]. The erythrocyte concentrations: 65g/g for Mg2+ and 11g/g for Zn2+, lead to physiological significant forms of the coordinative complexes: (Mg2+)2Hb, (Zn2+)2Hb and (Mg2+or Zn2+) Hb stabilizing complexes of higher affinity for O2 than the R-form of Hb [53] [55] [58] [59]. In Figure 6 b) and Figure 7 b), were reported plots [55] [59], both directed to use the cooperativity value n as exponential of the pressure of oxygen: (pO2)n. This treatment allows obtaining straight curves. These predicts that the extrapolation to a maximal (pO2)n, renders an homotropic dependence on the transition between T and R forms of Hb. However, it could be predicted that the physiological ligand-Hb equilibriums, within the red cell, would lead to an heterotropic mutual inclusion of O2 and divalent metals Zn2+ or Mg2+, with a mutual exclusion of 2,3-DPG and H+. The exergonic association per atom of Zn2+ to Hb: G=9.7Kcal [53], the tetramer has two binding sites, for either Zn2+ or Mg2+. In the direction of mutual inclusion of divalent metal and oxygen the exergonic binding of Zn2+ of 19.4Kcal plus pH dependent neutralization of Bohr protons, exceeds the

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Molecular Mechanisms Integrating Adenylyl Cyclase …

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endergonic dissociation of 2,3-DPG from Hb of only: G=5.7Kcal. Hence, in the lungs this thermodynamic coupling integrates pO2 with other contributions for a decrease in free energy (G.05). One - way ANOVA was employed in order to find significant differences between males and females, “young” elderly” (M < 68.13) and “old elderly” (M > 68.13). Finally, we created a new variable (mean of total mistakes in the three navigational test) and we compared subjects with higher scores and lower scores. Table 4 and table 5 show results about neuropsychological standardized test and experimental tasks. With regard to navigational subtest, every participant saw the first route for one time, and errors decreased in the three paths Route F1 (M = 2.13, SD = 1.35), Route B1 (M = 1.47, SD = 1.47), Route F2 (M = 1.03, SD = 0.89); along with time Route F1(M = 49.93, SD = 39.24), time Route B1(M = 36.73, SD = 30.26), time Route F2 (M = 21.47, SD = 12.32). Even if participants had seen the plastic city, they were able to remember more met landmarks (M = 5.63, SD = 2.04 ) in the first path (forward and back) than those not met (M = 1.87, SD = 1.43). Subjects show more difficulty to recall and replace landmarks in a blank map (subtest 8): anybody was able to put back all 6 landmarks (M = 2.47, min = 0, max 5); on the contrary, they performed better in replacing 5 landmarks on plastic city (subtest 4: M = 2.87, min 0.5, max 5). Besides, photo replacement test resulted to be more difficult (M = 3.6, SD = 2.45) than the recognition task (M = 6.53, SD = 1.71): subjects were always not able to return correctly on map the photos they had recognized before as true or false. In Map Drawing (M = 21. 44, SD = 10.19) we noticed that anyone draw the name of streets excepts in a few cases (Corso del Popolo). Finally, subjects performed better in driving directions, both in performance and in time, when they were watching the city (subtest 10) rather than they had to use a mental map (subtest 9). As regard Cognitive reserve index and Bidimensional Stimuli task, results are shown in Table 6 and Table 7. In order to verify correlation between navigational test and neuropsychological tasks, we used Pearson’s correlation (p< .05) to compare spatial and memory neuropsychological test. As expected, we found that Elithorn’s test was correlated to route F1 r (-.55), p< .05, route B1 r (-.56), p< .05, route

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planning r (.55), p< .05 and short route planning r (.52), p< .05. An interesting observation regards Corsi span that resulted to be not associated to visuo– spatial navigation test while it was correlated with time for the backward path r (-0.38), p< .05 and with time for short route planning r (-0.47), p< .05. As it could be seen in table 8, Trail Making Test was one of the neuropsychological testing that most correlated to experimental ones. Table 6. CRI index M

SD

CRI_school

93.17

11.52

CRI_work

97.73

15.71

CRI_freetime

91.13

18.29

CRI_tot

92

15.88

M = mean, SD = Standard deviation.

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Table 7. Mean and Standard deviation of experimental bidimensional stimuli test

Bidimensional stimuli

M

SD

Min

Max

6.17

2.45

1

10

M = mean, SD = Standard deviation.

In particular, TMTB and TMT B-A were negatively correlated to route recall r (-.44), p< .05; r (-.44), p< .05 (respectively) and total recall r (-.38), p< .05; r (-.36), p< .05. Recall replacement on map showed association with Rey’s complex figure copy r (.51), p< .05, Rey’s complex figure- immediate r (.49), p< .05 and Rey’s complex figure- delayed r (.51), p< .05. Rey’s complex figureimmediate was also correlated to bidimensional stimuli r (.41), p< .05. Manikin test showed association with route B1 r (-.44), p< .05, route recall r (.45), p< .05, total recall r (.45), p< .05, map drawing r (.58), p< .05 and map replacement r (.51), p< .05. An interesting result regards short story (MODA) that was correlated with route F1 r (-.43), p< .05, route recall r (.34), p< .05, city landmark r (.67), p< .05, recall replacement on map r (.40), p< .05 and map drawing r (.39), p< .05

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Maria Luisa Rusconi, Claudia Zamin, and Laura Carelli Table 8. Significant correlations between navigational subtest and executive functions

Route F1 Time F1 Route B1 Time B1 Route recall Tot recall City landmark Map drawing Map replacement Recall replacement on map Route planning Short route planning

TMTA

TMTB

TMTB-A

.66* .41* . 73* .46* -.20 -.19 -.37* -.45* -.45* -.40* -.40* -.57*

.53* .35* .60* .54* - .44* - .38* - .58* - .47* - .52* - .57* - .41* - .61*

.36* .24 .41* .47* - .44* - .36* - .54* - .34 - .42* - .52* - .36* - .52*

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* Pearson’s correlation, p 68.13). Among the neuropsychological tasks, ANOVA showed that only Manikin test was discriminatory between the two groups F(1,28) = 5.94, p = .05 (“young elderly” had higher scores). On the contrary, there were many differences in navigational subtest (see Figure 1). In particular, “young elderly” showed significant better results in the first part of navigational test: Route F1, F(1,28) = 4.09, p = .05; time F1, F(1,28) = 5.16, p = .03; route B1, F(1,28) = 8.41, p = .01; time B1, F(1,28) = 6.44, p = .02; route recall, F(1,28) = 6.28, p = .02; other recall, F(1,28) = 6.47, p = .02; total recall, F(1,28) = 9.418, p = .02; map drawing, F(1,28) = 9.23, p = .01 and in time route planning, F(1,28) = 10.51, p = .01.

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With regard to cognitive reserve index, we have not found significant differences in CRI school between old and young elderly although the “younger” group was different in CRI work, free time and total respectively, F(1,28) = 9.69, p = .00, F(1,28) = 9.38, p = .00 and F(1,28) = 10.25, p = .00.

□ 68.13

Figure 1. Mean and SD of young elderly and old elderly.

Mean of mistakes during the three paths (see Figure 2) was used to have a global index about orientation navigation errors and it resulted to be M = 4.63 (SD = 2.96). We divided subjects between those ones who made mistakes higher and lower than mean: ANOVA revealed that the youngest participants were more oriented than the oldest ones, F(1,28) = 18.76, p = .00. Moreover, “bad oriented subjects" showed a worse performance also in Elithorn’s test, F(1,28) = 5.98, p = .02 and TMT A and TMT B (respectively, F(1,28) = 12.07, p = .00 and F(1,28) = 5.5, p = .03). None of the other neuropsychological test discriminated the two groups. Further, “good oriented subjects” performed

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Maria Luisa Rusconi, Claudia Zamin, and Laura Carelli

62

better than the others in map drawing, F(1,28) = 5.47, p = .03; map replacement, F(1,28) = 6.27, p = .02 and recall replacement on map, F(1,28) = 5.69, p = .02.

8

Frequenza

6

4

2

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Media =4,63 Dev. stand. =2,965 N =30 0 -5

0

5

10

15

errori_tot_orientamento M = 4.63, SD = 2.96

Figure 2. Mean and Standard deviation of orientation total mistakes.

Since map drawing involves egocentric and allocentric prospective and it requires several visuo-spatial abilities, we have divided our sample into subjects with low and high scores in map drawing (M = 21.44, SD = 10.19; see Figure 3). In this case, demographic indexes and many neuropsychological tasks differed between the two groups (see figure 3); in particular, subjects who performed map drawing at higher level of mean were different in age, F(1,28) = 15.91, p = .00; education, F(1,28) = 4.75, p = .04; MODA, F(1,28) = 15.29, p = .00; short story MODA, F(1,28) = 4.51, p = .04; Rey’s copy complex figure, F(1,28) = 5.55, p =.03 and Manikin test, F(1,28) = 9.8, p = .00). On the contrary, only few experimental tasks differentiated two groups: route B1, F(1,28) = 6.64, p = .02; other recall, F(1,28) = 6.02, p = .02; total recall, F(1,28) = 6.47, p = .02; map replacement, F(1,28) = 10.06, p = .00.

Long-Term Memory : Mechanisms, Types and Disorders, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook

Long-Term Memory and Topographical Disorientation …

21.44

0.79 62.71

9.64 93.35 5.67 34.14 25.57 104.07 98.71

2.50

8.86

4.93

72.88 6.88 90.46 4.38 29.39 19.69 92.19 86.13 2.06 1.31

6.31

2.44

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Figure 3. Significant differences in map drawing.

With regard to bidimensional stimuli test, we found no significant correlation about gender, age, total errors, map drawing while this experimental test showed some significant correlations with neuropsychological evaluation and navigational test as reported below (table 9). Finally, cognitive reserve index showed interesting results. First of all, no clear differences were observed between “young elderly” and “old elderly” in CRI school; the same was observed for gender, total errors in orientation and map drawing. Besides, CRI school did not have any correlation with neuropsychological test while it showed significant association with city landmark r (.36), p