How the Vertebrate Brain Regulates Behavior: Direct from the Lab 9780674978751

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H O W T H E V E R T E B R AT E B R A I N R EG U L AT E S B E H AV I O R

HOW THE VERTEBR ATE BR AIN REGU L ATES BEHAVIOR DI REC T FROM TH E L A B DONALD PFAFF

Cambridge, Massachusetts & London, England 2017

Copyright © 2017 by the President and Fellows of Harvard College All rights reserved Printed in the United States of Amer ica First printing Library of Congress Cataloging- in-Publication Data Names: Pfaff, Donald W., 1939– author. Title: How the vertebrate brain regulates behav ior : direct from the lab / Donald Pfaff. Description: Cambridge, Massa chusetts : Harvard University Press, 2017. | Includes bibliographical references and index. Identifiers: LCCN 2016042682 | ISBN 9780674660311 (alk. paper) Subjects: LCSH: Brain. | Mammals—Behav ior. | Neurophysiology. | Neuroendocrinology. | Molecular neurobiology. Classification: LCC QP376 .P447 2017 | DDC 573.8/619— dc23 LC record available at https:// lccn.loc.gov/2016042682

CO NTENTS

Introduction 1. Hormone Receptors

1 8

2. Discovering the Neural Circuit for a Vertebrate Behavior Essential to Reproduction

40

3. Hormonal Regulation of Gene Expression in the Brain

79

4. Genes Regulating Behavior

128

5. Neuropeptide: Gonadotropin-Releasing Hormone

158

6. Neuropeptide: Oxytocin

179

7. Brain–Body Relations

195

8. Central Ner vous System Arousal Fueling Instinctive Behaviors

215

9. Sex Difference

227

10. Summary

238

Acknowledgments

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Index

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H O W T H E V E R T E B R AT E B R A I N R EG U L AT E S B E H AV I O R

I N T RO D U C T I O N

When I began in neurobiology, there was a longstanding problem: most scientists felt that it was necessary to work with extremely simple animals to understand how ner vous systems govern behavior. Vertebrates and especially mammals were considered to be too hard, too complex. A new paradigm was needed to attack the scientific problems of how the vertebrate brain regulates behav ior. To fi ll that need, I analyzed and then exploited the effects of hormones on behavior. In doing so I reaped the analytic advantages of chemical specificity (steroid hormones) and neuronal specificity (circuitry for a simple mammalian behavior) so as to understand how a biologically important mammalian behavior is regulated. To put it another way, a neuroscientist might win by studying complex functions in a simple organism or by studying a simple behavior in a complex organism. I chose the latter. Having done so, I succeeded in linking molecular chemistry to physiology and ultimately to the causation of behavior. In recounting my own laboratory’s work I am going through all the steps that any laboratory must to solve problems as our science of the vertebrate brain develops. In the first several chapters of this scientific account, a review of some of my laboratory’s accomplishments, I will show that we produced

the first demonstrations of specific molecular changes in particu lar neurons that drive a chain of social behaviors of extraordinary biologic importance. That is, I proved that the female’s behavioral response to the male’s mount, which is essential for fertilization (lordosis behavior), is driven by specific estrogen-dependent molecular events in ventromedial hypothalamic neurons, which regulate the neuronal circuit that we subsequently worked out. As mentioned, the first creative step was to find a problem worth solving that, in fact, would turn out to be solvable: how the chain of behaviors essential for reproduction is orga nized and regulated. My findings, starting 50 years ago, are related here, together with the work of others who have enlarged the field of neuroendocrinology to the point where it presents the best opportunities for relating molecular genetics to neuroscience and behavior. Some people think that mechanistic explanations for behav ior should be set up against evolutionary explanations. No! I note that my type of work— discovering brain mechanisms for behav ior— does not  contradict those who emphasize how behavioral regulation has evolved, an aspect of biology and medicine led by E. O. Wilson, at Harvard (Wilson 1975). My field of neuroscience is complementary to Wilson’s field, including his seminal work on sociobiology. Wilson thinks about how biologically important behaviors have evolved through time, while I have discovered mechanisms that are the result of that evolutionary process and drive behaviors right now. In fact, it is those very mechanisms that we neuroscientists study, which actually evolve! To put it another way, I figured out how the ner vous system works by solving the problem posed by a specific behavioral function. Rather than asking “How does the brain work?” I worked out how a specific function is accomplished. I had the advantage of working with a hormone-controlled behavior, so I could “triangulate” brain mechanisms, viewing them from one angle as hormone targets and from another as behavioral response producers. By Chapter 5 in this book, you will see that we achieved a realization of these brain mechanisms in physical terms. I also note that although traditionally neuroscientists considered the central ner vous system to be an isolated entity and studied it as

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such, in the course of my analyses of lordosis behavior mechanisms I was studying a behavior that is necessarily and essentially social.

At MIT Again, the first and most important issue a scientist faces is to decide on the topic to investigate. What questions are deeply interesting yet solvable? As the great immunologist Sir Peter Medawar said, “Science is the art of the soluble.” That is, when I started laboratory research the core problem was to figure out how to solve the mysteries of how higher, vertebrate brains regulate entire normal behav iors. For a long time people could study simple ner vous systems. So the choice was between examining complex behavior in simple animals and trying to unravel mechanisms for simple behaviors in complex animals. Eric Kandel (1965, 1976), a Nobel Prize–winning professor at Columbia, did the former. I figured out how to do the latter. A seven-word sentence, uttered more than 50  years ago, combined with study in organic chemistry did the trick. As a Harvard undergraduate I had volunteered to serve on the back ward of a state mental hospital. I wanted to be a psychiatrist or neurologist. But the medical care in that ward was terrible. My surgeon father would not have approved. Then when I went to the Massachusetts Institute of Technology (MIT) for graduate school I was helping my thesis advisor, Joseph Altman (who had discovered postnatal neurogenesis), trace how newly divided neurons migrate to their final positions in the rat brain. Thinking back to my medical intentions, I said, “Joe, I’d like to do something more closely related to brain function”—that is, to behavior. He answered in seven words: “I’d like to do something with hormones.” Well, I had been a good student in organic chemistry and knew quite well that steroids are not such complex molecules! I could not resist once I realized that some important hormones are steroids, and I found that some steroid hormones control simple, biologically crucial animal behav iors. The core problem to be solved was how two major communication systems in the body—in all vertebrate bodies, including humans—signal to each other to regulate biologically crucial behaviors

INTRODUCTION

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in biologically adaptive ways. This book gives the essence of the solution.

This Book The findings recounted in this book redefined what neuroscientists studying the mammalian brain can accomplish. Using different levels of investigation—single genes, ion channels, single nerve cell physiology, neural circuits, and an entire social behavior—we built a behavioral mechanism. Taken together, these sets of discoveries proved for the first time exactly how specific chemicals (hormones) acting on specific nerve cell groups can determine a complete, natural mammalian behavioral response. Chapter 1 summarizes my discovery about 50 years ago of hormone receptors in the brain. The limbic-hypothalamic system of neurons with nuclear receptors for sex hormones was discovered in the rat brain but turned out to be universal among vertebrate brains—“from fish to philosopher.” Chapter  2 explains how the hormone-sensitive hypothalamic neurons summarized in Chapter 1 proved to be one of three entry points for working out the first neural circuit for a vertebrate behavior. The other two were the sensory inputs (followed neuroanatomically and neurophysiologically) and the motor outputs (from muscles back to motoneurons and so forth). Applying the logic of Sherringtonian physiology (Sherrington, 1947) but using modern methods and working with a large laboratory group over several years, we were able to demonstrate a spinal-midbrain-spinal circuit for lordosis behavior, a circuit that is regulated by an estrogen-dependent output from the hypothalamus: the first circuit for a vertebrate behavior and a social behavior at that. Principle: the hierarchically orga nized circuit features modules, each of which contributes its unique physiological regulation. Then we got lucky. The hormone receptors I had discovered in the brain turned out to be ligand-activated transcription factors. Thus, I could use molecular techniques to study hormone-facilitated gene transcription and, in turn, link the eventual products of such transcription to reproductive behavior. Chapter  3 covers this work. The product of this work, showing how transcription of specific genes in specific neurons

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leads to an estrogen-dependent hypothalamic output that regulates a defined neural circuit—thus producing a biologically crucial behavior—has been called a high-water mark in neuroscience. Chapter 4 deals with gene expression causally linked to the reproductive behav iors mentioned previously. Taken together, these four advances—hormone receptors, neural circuitry for female behavior, hormone effects on gene expression, and gene / behavior causal relations— proved for the first time exactly how specific chemicals acting in specific parts of the  brain could determine a complete (vertebrate) behavioral response. The behav ior explained is a social behav ior essential for reproduction. Chapters 5 and 6 deal with specific genes involved in lordosis behavior regulation, genes expressing gonadotropin-releasing hormone (GnRH) and oxytocin, respectively. The GnRH discoveries revealed, surprisingly, how a simple decapeptide regulates reproduction body-wide. Then Chapter 7 wraps up the story of the unity of the body, explaining neural and behavioral controls of reproduction and discussing seven principles of hormone / behavior relations. Neural and endocrine systems are united. I have argued elsewhere that, deep to the sexual arousal that fosters mating behavior, there operates a fundamental neuronal “force,” a concept called generalized arousal. In Chapter  8, I carry that argument forward because I feel that elevated generalized arousal is probably essential for initiating all motivated behaviors. Sex differences in brain and behavior (Chapter 9) are crucial, obviously, for reproductive physiology; for the neurobiology of the normal brain, remote from reproduction, they are of minimal importance. For certain maladies, though, such as autism, a new science of the importance of sex difference is emerging. Arthur Arnold, at the University of California–Los Angeles, works on these subjects, examining the implications of reproductive biology for sexually differentiated phenomena, all significant for medicine and public health. The final chapter, Chapter 10, summarizes discoveries that turned out to be universal among vertebrates.

* * * The subtitle of this book, “Direct from the Laboratory,” reflects the vibrancy and immediacy of my field of work. Actually two fields of

INTRODUCTION

5

science developed as recounted here. First, the neuroscience of the vertebrate brain and behavior developed over the last 50  years. Torsten Wiesel and David Hubel opened up the visual cortex. Sten Grillner (2005, 2007) and Anders Lundberg elucidated motor mechanisms. Eric Kandel (2012) and Paul Greengard explained mechanisms of learning and reward. In my case developing the science of neurobiology consisted of applying a Sherringtonian ([1906] 1947) type of logic and work to the long- standing problem of solving a mammalian behav ior. Second, within neuroscience, neuroendocrinology used to be thought of as a “boutique” field, amazingly, despite its role to unite two major signaling systems in the body, neural and hormonal. Now we recognize that hormone actions on brain and behav ior offer an “intellectual interstate highway” to the linkage of modern molecular biology and genomics to neuroscience by virtue of the types of nuclear hormone receptors (ligand activated transcription factors) that I discovered in brain tissue (Chapter 1). So, putting it another way, 50 years ago I decided to understand how the ner vous system works by solving the problem posed by a specific behavioral function. I had the advantage of working with a hormonecontrolled behav ior— detailed in Chapter 1—so I could “triangulate” brain mechanisms, viewing them from one angle as hormone targets and (Chapter 2) from another angle as behavioral response producers. Chapters 3 and 4 add the genetics and genomics. By Chapter 5 in this book you will see that we achieved a realization of these brain mechanisms for a reproductive behavior in physical terms. Yes, it is possible to analyze fully the mechanisms for a vertebrate behavior.

Further Reading Grillner, S., A. Kozlov, P. Dario, C. Stefanini, A. Menciassi, A. Lansner, and J. Hellgren Kotaleski. 2007. “Modeling a Vertebrate Motor System: Pattern Generation, Steering and Control of Body Orientation.” Progress in Brain Research 165: 221–34. Grillner, S., H. Markram, E. De Schutter, G. Silberberg, and F. E. LeBeau. 2005. “Microcircuits in Action—From CPGs to Neocortex.” Trends in Neurosciences 28 (10): 525–33. Kandel, E. R. 1976. Cellular Basis of Behavior. San Francisco: Freeman.

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Kandel, E. R., J. H. Schwartz, T. M. Jessell, S. A. Siegelbaum, and A. J. Hudspeth, eds. 2012. Principles of Neural Science. 5th edition. New York: McGraw-Hill. Kandel, E. R., and L. Tauc. 1965. “Heterosynaptic Facilitation in Neurones of the Abdominal Ganglion of Aplysia depilans.” Journal of Physiology 181 (1): 1–27. Sherrington, C.  S. (1906) 1947. The Integrative Action of the Nervous System. Reprint, New Haven, CT: Yale University Press. Wilson, E.  O. 1975. Sociobiology: The New Synthesis. Cambridge, MA: Harvard University Press.

INTRODUCTION

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1 H O R M O N E R ECEP TO R S

Problem: Two major signaling systems in the body are the endocrine system and ner vous system. How do they communicate? Does this question have anything to do with the brain’s regulation of behavior?

We all have heard physicists say something like this: “You guys in biology and medicine don’t have laws or principles. You have piles of phenomena accompanied by lots of Latin names.” In this chapter, you will see, once I had made the discovery of hormone receptors in the brain, to answer the question above, I plowed on with brains representing every major vertebrate class, using every species for which I could find excellent collaborators—people who knew that species well. By doing that, we established a lawfulness, a massive degree of reliability for my initial finding. Here is the historical backdrop. The British physiologist Geoffrey Harris had, during the late 1930s, established that neurons in the hypothalamus regulate secretions from the anterior pituitary gland, which in turn controlled the release of protein hormones that would circulate to the ovaries, testes, thyroid, and adrenal glands. Thus, communication of the ner vous system to the endocrine system. But what about the reverse? Hormone effects on brain and behavior?

First, based on my interests, I had to establish the existence and specificity of sex hormone actions on behavior (Pfaff 1970b). This was an exercise in “endocrine engineering.” The subjects were ovariectomized female rats and castrated male rats. Indeed, estradiol injections turned on the ability of the females for lordosis behavior, the primary and essential female mating behavior, especially if they were supplemented by a progesterone injection about four hours before the lordosis behavioral assay. I noted that testosterone injections supplemented by progesterone also could elevate lordosis behav ior per for mance, probably (as later shown) because aromatase enzymes in the brain can transform testosterone into estrogen. Likewise, in the males either testosterone or estradiol significantly increased all masculine mating behaviors (such as mounts). Thus, the existence and strength of steroid hormone effects on sex behaviors were established, and an additional fact popped out of the data. The same females that showed the greatest amount of feminine behavior under one hormonal condition also showed the most under other hormonal conditions. The correlations were as high as 0.88 and 0.90. This indicated that in addition to straightforward hormonal determination of sex behavior, there must have been a tissue sensitivity factor that varied from animal to animal. The level and efficiency of estrogen receptor (ER) binding, transport, and affinity for estrogen response elements on the promoters of estrogen-responsive genes would later be seen as providing the reason for these tissue sensitivity phenomena. Then for the purposes of behavioral control, I asked, are direct actions of hormones on the brain really necessary? Perhaps estrogenic actions on the pituitary would be of primary importance, and pituitary hormones feeding back upon the brain would provide the main controls over behavior. I addressed these questions using animals in which the pituitary gland had been surgically removed (Pfaff 1970a). The answer was clear. Estrogen and progesterone injections increased female sex behavior (lordosis) performance in hypophysectomized ovariectomized female rats as well as in animals with an intact pituitary gland. Likewise, testosterone injections increased male sex behavior vigor in hypophysectomized castrated male rats as well as in males with intact pituitaries. Thus, direct actions of sex hormones on neurons were indicated. How does that happen?

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9

Early ideas about how steroid hormones could influence cells began with the knowledge that steroids are flat, planar, rigid molecules. Arriving at the cell, they could influence the fluidity of the cell membrane. Adding to that idea, biochemists such as Claude Villee at Harvard Medical School studied the effect of steroid sex hormones on the activity of cytoplasmic enzymes, especially those that had to do with steroid metabolism. That was the situation when I entered the field. Those lines of research were shelved when organic chemist Elwood Jensen synthesized the first carrier-free radioactively labeled steroid hormone: tritiated estradiol. He used biochemical techniques to show that after a systemic injection of labeled estradiol, the hormone at first flooded uterine cells then quickly subsided (within minutes); however, in uterine cell nuclei the hormone was retained much longer—for hours. I obtained tritiated estradiol from the first batch produced by New England Nuclear and took Jensen’s developments to the brain.

Hormone Receptors in the Brain As mentioned in the introduction, as a first-year graduate student at MIT in 1961, I was helping Joseph Altman (the discoverer of postnatal neurogenesis) trace the migration of newly divided neurons around the developing ner vous system. But as a young researcher who wanted to contribute to the science underlying psychiatry and neurology, I said, “Joe, I’d like to do something more closely related to brain function.” His seven-word response, “I’d like to do something with hormones,” set up my discovery of hormone receptors in the brain. The limbic-hypothalamic system of sex hormone receptors that I found in the rat brain turned out to be universal among vertebrates—“from the fish to the philosopher.” As well, since these are nuclear receptors, they offered the unique advantage of studying hormone-dependent transcription factors. Behavioral and neural biology were linked in the early days to molecular biology (see Chapter 3). Joe Altman was a true artist at the laboratory bench. His feeling for  beautiful histochemistry emerged most dramatically when he responded to the illustrations published by Tomas Hokfelt and Kjell Fuxe, their histochemistry demonstrating aminergic systems in the brain. Joe was a really tall and muscular guy, and his sharp intake of

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HOW THE VERTEBRATE BRAIN REGULATES BEHAVIOR

breath when he saw those illustrations, and then explained them to me, said it all. So he was a great guy from whom to learn histochemistry. For almost four years I worked to optimize histochemical techniques for discovering hormone-binding neurons in rat brain tissue. Repeated attempts to optimize the methodology were necessary for three reasons: the low specific activity of labeled steroids in those days, the difficulty of keeping the bound hormone exactly in place in the target neurons, and the necessity of avoiding histochemical artifacts due to the pressure sensitivity of the Kodak NTB-3 nuclear emulsion, which was best for detecting the β particle that emanated from the bound tritiated hormone. The tritium was great for my purpose because the β particle has such low energy that it can only travel into the nuclear emulsion directly over the target neuron, thus offering good spatial resolution at the light microscope level. Eventually I succeeded, and I published my preliminary results (Pfaff 1965). But a tremendous amount of quantification remained to be done: counting grains over labeled hormone-binding neurons. The highest numbers of estradiol-binding neurons were found in a subset of limbic / telencephalic and hypothalamic / diencephalic cell groups (Pfaff 1968) that formed a limbic / hypothalamic system with an extension into the central grey. For the purpose of facilitating female mating behavior, the most important concentration of estradiol-binding neurons was in the ventrolateral corner of the ventromedial nucleus of the hypothalamus (Figure 1.1), because this is where estrogens act to facilitate lordosis (as I’ll discuss more later). In general, the grouping of estrogen-receptive neurons did not always respect classic neuroanatomical boundaries; for example, a few such neurons were just outside the ventromedial nucleus. Elsewhere in the hypothalamus, tritiated estradiol retention was strong in the arcuate nucleus (of importance for controls over the pituitary) and the ventral premammillary nucleus (of importance for pheromonal effects on reproduction). I found labeled cells as well in the anterior hypothalamic area and at the lateral caudal tip of the magnocellular portion of the paraventricular nucleus of the hypothalamus. Forward of the hypothalamus, large numbers of estrogen-binding neurons were seen in the medial preoptic nucleus—heavily labeled— especially near the midline at levels under the anterior commissure and

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Plane of Figure 1.1 bottom.

Figure 1.1. Locations of estrogen receptor- expressing cells in the female rat brain (symbolized by black dots). Top: Drawing of a sagittal section through the rat brain, looking from the left side. Each black dot represents clusters of neurons expressing estrogen receptors. The vertical black line approximates the coronal section. Bottom: Looking at the cross section indicated at the top, the nerve cells expressing the estrogen receptors crucial for lordosis behavior are in the blackened area (very densely packed cells) in the ventrolateral corner of the ventromedial nucleus of the hypothalamus (vm, arrow). (Adapted from Pfaff and Keiner 1973.)

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in the suprachiasmatic portion of the preoptic area. The former turned out to be important for the estrogen dependence of maternal behaviors (see Chapters 4 and 7). In the limbic forebrain I found impressive groups of estrogen receptive neurons in the medial nucleus of the amygdala, particularly in its dorsal posterior subdivision, and in a cell group named the bed nucleus of the stria terminalis (which fosters communication between the amygdala and the diencephalon). Certain cells in the lateral septum were well labeled. Following up my brief 1965 report, I found labeled cells at all levels of the hippocampus, both among the small granule cells of the dentate gyrus and the large pyramidal cells of Ammon’s horn. Also important for lordosis behavior regulation, because it fits into the neural circuit for lordosis (see Chapter  2), the central grey of the mesencephalon contains labeled cells throughout its extent, from the diencephalic / mesencephalic junction all the way back to the transition from the cerebral aqueduct and the fourth ventricle. I found labeled cells most easily directly lateral or ventrolateral to the aqueduct. The number of such cells was not as great as in the most densely labeled hypothalamic nuclei. After I moved from MIT to Rockefeller, and because I had explored a number of histochemical approaches, I was able to follow up the original report with a dif ferent procedure involving frozen sections and direct mounting onto the nuclear emulsion (Pfaff and Keiner 1973). All the major features of the original paper were replicated in the atlas I published. Later, using biochemical techniques and working with Bruce McEwen and Richard Zigmond, we confirmed my histochemical / neuroanatomical results. We exploited my cellular findings to design dissection schemes that could give us clear results. The major features of our findings, besides confirming my findings, were that prior injections of unlabeled estradiol significantly reduced tritiated estradiol uptake in brain regions—indicating limited capacity binding as is true for the uterus—and that hypophysectomy reduced uptake in all brain structures. Similar results were found for tritiated testosterone uptake in the rat brain. For the estrogen findings, the results from my laboratory and Richard Whalen’s (at the University of California–Riverside) were confirmed by James Clark at Baylor using biochemical methods, by John

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Cidlowski at the University of North Carolina, and by Fred Naftolin working with Jack Fishman at Yale. A whole new field of work was initiated when a second estrogen receptor, ER-β, was cloned by Jan-Ake Gustafsson and his team at the Karolinska Institutet. The behavioral consequences of ER-β expression are interesting and are significantly dif ferent from the ER- α we have concentrated on (see Chapter 4). Strong evidence for the specificity of the estrogen-binding neurons I have reviewed here comes from other results of Bruce McEwen and his team at Rockefeller. By far the most impressive binding of corticosterone, a glucocorticoid hormone, was found in the hippocampus, not in the hypothalamic and preoptic neurons just emphasized here, so important for mating and maternal behaviors. Likewise, the thyroid hormones that are so crucial for the regulation of metabolism are bound in the brain by the gene product from thyroid hormone receptor α1 (TR- α 1), as discovered by Karolinska professor Björn Vennström. TR- α1 is found expressed in an extremely wide neuroanatomical pattern, giving further indication of the specificity of our results with labeled estrogens (Wallis et  al. 2010). It was known that thyroid hormone is essential for brain development, but the potential for the hormone to act in adult neurons needed molecular markers in order to be proven. In Vennström’s results in the adult brain, TR- α1 expression was detected in essentially all neurons. So a new field of work on thyroid hormones in the brain was opened up. Always we have tried for accurate quantification of our results, with the desire that precision in our area of neuroscience would bring it one step closer to the precision typical of the physical sciences. This underlying desire has animated all my work, from neuroanatomy to electrophysiology, molecular biology, and ultimately behavior. With this in mind, Joan Morrell and Monica Krieger in my laboratory used 2-micron sections and cell-by- cell grain counting in four areas of the forebrain that feature high levels of estrogen binding: the ventromedial nucleus of the hypothalamus, preoptic area, arcuate nucleus of the hypothalamus, and the medial nucleus of the amygdala (Krieger, Morrell, and Pfaff 1986). The null hypothesis was that the frequency distribution of grains-per- cell would fit a random process, a simple Poisson distri-

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bution. Impressively, the frequency distribution in all four brain regions deviated markedly from the Poisson. Then, when the labeled estrogen was accompanied in other animals by a tenfold excess of nonradioactive estradiol to wipe out the specific binding sites in the brain, the results in all four brain regions, indeed, collapsed onto frequency distributions indistinguishable from the random, Poisson curve. Later, considerable work was started to identify the neurochemical contents of neurons in the hypothalamus that bind high levels of radioactive estrogen. For example, Andrea Gore, now at the University of Texas, reported that expression of ER-α is colocalized with the obligatory glutamatergic NMDAR (N-methyl-D -aspartate receptor) subunit NR1 in neurons of the anterior hypothalamus and medial preoptic nucleus, showing how hormonal and glutamate neurotransmitter influences might synergize. Another example: our student Harker Rhodes, working with professor Joan Morrell, found paraventricular hypothalamic neurons that would produce oxytocin or vasopressin, which retained radioactive estradiol (Rhodes, Morrell, and Pfaff 1981). This type of work, characterizing ER-α receiving neurons in neurochemical terms, is still ongoing. As we began to put together the entire circuit for lordosis behavior (Chapter  2), we needed to know where estrogen-sensitive signals were going after the uptake of estradiol by ventromedial hypothalamic neurons. Joan Morrell at Rockefeller invented a technique by which she could characterize estrogen-concentrating hypothalamic neurons according to their axonal projections (Morrell and Pfaff 1982). Most importantly, retrograde-transported fluorescent dye primulin, injected into the dorsal midbrain by the central gray, identified estrogen-binding neurons in the ventrolateral portion of the ventromedial nucleus of the hypothalamus. This discovery provided part of the evidence for an estrogen-sensitive link at the top of the lordosis behav ior circuit, later verified by other techniques. Thus, with neuroanatomical precision and biochemical sophistication we established a hormone–brain link that would provide one crucial entry point to working out the female reproductive behav ior circuit (Chapter 2) and to introducing the tools of molecular biology (Chapter 3) to our program of research.

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Hormone Binding in a System Universal among Vertebrates I doubted that all the data discovered in rat brains were particular to  that one species. The overall neuroanatomical pattern seemed too solid, too integral for that, and made too much physiological sense to be so biologically restricted. So we branched out toward other species. Of course, at first we simply used other mammals to extend the discoveries from rats to other species. But the long-term aim was always to establish an endocrine-neural link from “fish to philosopher”—across all vertebrate species. Here, after summarizing results from other rodent species, I will review our hormone-binding fi ndings from other classes of vertebrate species: fish, amphibian, reptiles, and birds. Then things will get very interesting with results from the monkey brain and human brain. The bottom line of the hundreds of studies from a lot of laboratories, mine included, is that the limbic / hypothalamic system of sex hormone-binding neurons I discovered in rat brain is universal among vertebrates.

Hamster Brain The experimental skill of postdoctoral researcher Monica Krieger came to the fore in her work at Rockefel ler with Professor Joan Morrell in my laboratory. Female hamster brains were a natu ral for study because of their striking lordosis displays upon mounting by the male. The autoradiographic results mirrored my results with the rat brain. There were heavily labeled cells in the medial preoptic area posterior to the lamina terminalis, especially in the medial portion of the preoptic area. The anterior hypothalamus had large numbers of estrogen-binding neurons, particularly in the distribution of the stria terminalis. Important for the female hamster’s lordosis, the ventrolateral subdivision of the ventromedial hypothalamus showed estrogen-binding neurons while the dorsomedial nucleus was virtually unlabeled. As expected, because of the neuroendocrine functions of the arcuate nucleus of rat hypothalamus, it had heavily labeled neurons.

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In the limbic system, the medial nucleus of the amygdala showed some of the heaviest labeling in the brain whereas other amygdaloid subregions were almost unlabeled. Interestingly, the bed nucleus of the stria terminalis had strongly estrogen-retaining neurons. Throughout the hippocampus we saw some labeled pyramidal neurons, plus some well-labeled cells in the subiculum lateral to the tip of the ventral hippocampus. As expected from my rat brain findings, Krieger showed well-labeled neurons in the lateral portion of the midbrain central gray.

Mouse Brain It was natu ral to extend my fi ndings in the rat brain to mice because of the importance of mouse studies for behav ior genetics. The most comprehensive study was published by Marie Warembourg and Eduard Milgrom working in the INSERM unit in Lille, France. Their immunocytochemical work replicated my findings in the preoptic area and medial hypothalamus of the mouse brain. These results were confirmed by James Clark at Baylor using biochemical methods, and by endocrine chemist John Cidlowski at the University of North Carolina.

A Wide Range of Vertebrate Species Fish Not everyone understands how much of the vertebrate body plan has been conserved from fish to humans. We studied sex steroid-binding neurons in paradise fish with the leadership of visiting University of Michigan professor Richard Davis (Davis, Morrell, and Pfaff 1977). Starting our summary with the binding of tritiated estradiol in the hypothalamus, we saw excellent hormone retention in the medial hypothalamus, with the greatest intensity of binding in a cell group called nucleus lateral tuberis. Farther posterior we saw estrogen binding in the nucleus recessus lateralis, and some binding in the nucleus posterioris periventricularis. No labeled cells were found in the mesencephalon, rhombencephalon, or spinal cord.

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I am not trying to match the fish brain with mammalian brain nuclear group for nuclear group, but the principle that there are a high number of estrogen-binding cells in the hypothalamus remains true in the fish. We were especially pleased about the tightly backed estrogenbinding cells in the preoptic area, nucleus preopticus parvocellularis— most of these are very small cells, and they are found dorsal and posterior to nucleus preopticus periventricularis, also labeled. What can we say about the limbic system in fish? Considering the limbic system as phylogenetically ancient telencephalon, it was gratifying that we could find estrogen-labeled cells in the area ventralis telencephali pars ventralis. For many of these cells, just rostral to the anterior commissure and extending dorsally, one could imagine a homology to the mammalian bed nucleus of the hypothalamus. For testosterone-labeled cells, locations in the brain of the paradise fish were, in general, similar to the estrogen-labeled cells. The “limbic” (telencephali pars ventralis) neurons were essentially identical to the estrogen story, as was the case in the hypothalamus. Overall, there tended to be fewer testosterone-binding neurons than estrogen-binding neurons. We also confirmed these findings with the green sunfish, a second teleost species, and other laboratories got convergent data from the brain and pituitary of the brown trout.

Amphibians Rockefeller graduate student Darcy Kelley, a brilliant young woman who went on to become the first female head of the department of biology at Columbia University, worked with Joan Morrell in my laboratory on two species of amphibians, Xenopus laevis and Rana pipiens. Xenopus are amazing. Their copulatory response lasts longer than I had ever seen. Darcy injected them systemically with small doses of tritiated estradiol (0.1 millicurie) and sacrificed them two hours later to prepare the brain tissue for the steroid autoradiographic procedures I had used for the rat brain. Starting the description of her results with the diencephalic nerve cell groups that “stand in for” the hypothalamus, Darcy discovered estrogen-binding cells in the ventral infundibular nucleus (Morrell,

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Figure 1.2. Black dots show locations of neurons expressing estrogen receptors in the ventral infundibular nucleus (VIN) in the hypothalamus of Xenopus. The expression is bilateral, plotted here on one side. (Adapted from Morrell, Kelley, and Pfaff, 1975.)

Kelley, and Pfaff 1975). Especially impressive are the compacted labeled cells along the border of the ventricle. As expected from my work with rats, estrogen-concentrating cells are seen throughout the entire extent of the anterior preoptic area. In the preoptic area slightly more posterior they were still found throughout the dorsal-ventral extent of the preoptic area, but still more caudally they were clustered close to the ventricle, ventrally. Considering the telencephalon (Morrell, Kelley, and Pfaff 1975), the ancient forebrain, the labeled cells were found in the septum, the striatum, and the amygdala. For example, in the ventral lateral septum, they were curved ventrally around the tip of the ventricle. Likewise, the amygdala’s labeled neurons were close to the posterior tip of the ventricle, seen most easily in horizontal section (Figure 1.2). As is the case with rats and mice, estrogen-labeled cells extended posterior into a specific part of the midbrain. In amphibians, the structure lateral and dorsolateral to the cerebral aqueduct is called the torus semicircularis. Comparing estrogen-binding patterns of neurons in neuroanatomical and systems terms, we found no apparent differences between the female brain and the male brain. Even the intensity of labeling (grains per cell) was about the same. Darcy Kelley also studied testosterone binding in the brain (Kelley, Morrell, and Pfaff 1975). As with estrogens, brains were harvested two

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hours after systemic injection of tritiated testosterone. Again, beginning with the “hypothalamus equivalent,” she found well-labeled neurons in the ventral infundibular nucleus, especially posterior in this nucleus. Whether these are “exactly the same neurons” as can bind estrogen is open to conjecture. Such a question depends on how you define a “cell type,” a developmental biological problem we recently have argued in the light of modern epigenetics (Tabansky, Stern, and Pfaff 2015). In the preoptic area as well, the anterior subdivision contains large numbers of labeled cells following systemic tritiated testosterone. As you move caudally in the preoptic area, the density of labeled cells declines, especially in its dorsal portion. We were surprised by the labeled cells in the dorsal tegmental area of the medulla, and in a columnar nucleus in the hindbrain (Kelley, Morrell, and Pfaff 1975). We speculated (p. 57) that based on a comparison with the literature on androgen-modulated vocalizations, these more posterior testosterone-binding neurons are involved in the regulation of mating calls, a speculation that Kelley followed up effectively at Columbia. Thin-layer chromatography of radioactive hormone in the brain showed that the great majority of hormones in the brain were still in the form of testosterone itself rather than a metabolite. Another amphibian, Rana pipiens, was used for cell fractionation studies of hormone binding in the brain after systemic injections of radioactive estradiol or radioactive testosterone (Kelley et al. 1978). In the block of tissue that included preoptic area, infundibulum, and amygdala, radioactivity from the labeled estradiol was about three times that found in other brain tissue, but less than that found in the pituitary. After testosterone injections, the radioactivity was about the same as in other brain tissue, presumably due to the hindbrain groups of labeled cells discussed previously, and a substantial fraction had been converted into metabolites such as dihydrotestosterone. In fact, the hypothalamic / limbic block of tissue just mentioned had five times as much radioactive dihydrotestosterone in cell nuclei as in the rest of the brain. Rana was also used for autoradiographic studies of hormonebinding neurons. For both estradiol and testosterone, the labeled cells

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were impressive in the infundibular nuclear groups, in the preoptic area, and in the ancient telencephalon around the ventromedial tip of the telencephalic ventricle. Labeled cells were seen in the septum and the amygdala. Hormone-receiving neuronal distributions were, by and large, very similar to the results for Xenopus. However, Rana lacked the hindbrain testosterone-concentrating cells seen the Xenopus brain. Thus, we referred to literature that indicated regulation of mate calling might be under the control of the preoptic area and infundibulum. It was in this study that we extended my earlier argument (Pfaff and  Keiner 1973) that sex steroid-binding nerve cells are connected in systems—that is, that they project to each other (Kelley et al. 1978). I explore the implications of this phenomenon later.

Reptiles Joan Morrell at Rockefeller worked with reptile neuroendocrinology expert David Crews (now at the University of Texas) to study sex hormone receptors in the brain of the lizard Anolis carolinensis (Morrell et  al. 1979). Females and males were injected, and the brains were harvested two hours later. For tritiated estradiol, we concentrated first on the hypothalamus. Although few labeled cells were seen in the anterior hypothalamus, the ventromedial nucleus of the hypothalamus was well labeled. In fact, in its posterior lateral segment it was plastered with heavily labeled cells. This is reminiscent of what we originally saw in the rat brain. The periventricular nucleus of the hypothalamus had many heavily labeled cells throughout its extent. In commonsense terms, this nucleus looks like the reptilian version of the mammalian arcuate nucleus. The reptilian preoptic area as well showed results similar to those in mammals. A large number of heavily labeled cells was found throughout its extent. As with rat brains, estrogen-binding cells were particularly densely concentrated near the preoptic recess of the third ventricle. The reptilian forebrain comprises an early version of the limbic system in mammals. We found estrogen-labeled neurons in the septum, particularly in the ventral and lateral septum. Amygdaloid subnuclei in the lizard brain are divided into cell groups named A1, A2, and A3. A1, the most lateral cell group, and A2 had a large number of labeled cells.

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A3 was surrounded by labeled cells at its dorsal and medial edges but internally was not labeled. A smaller number of estrogen-concentrating neurons was found in the midbrain, but they were found in regions that seem to match the pattern for the mammalian brain. That is, we saw them in the midbrain central grey lateral to the cerebral aqueduct, and in a structure called the torus semicircularis, again lateral to the aqueduct. In other animals we injected labeled testosterone or labeled dihydrotestosterone. In general, the neuroanatomical pattern of the labeled cells was very similar to that just described for estradiol. Notably, as predicted from our work with rats and from the notion of a universal vertebrate pattern of sex hormone-binding neurons, androgen hormone binding was strong in the medial hypothalamus and in the preoptic area. Also, large numbers of androgen- concentrating neurons were seen in the amygdala and the septum. An impor tant difference from the estrogen results, after labeled androgenic hormone administration, lay in the significant number of neurons seen in the mesencephalic tegmentum. Estrogen binding there was rare. We have not been able to link these androgen-binding cells to male sex behavior, but some investigators have suggested that these may be involved in species-specific aggressive displays. Throughout our work with lizard brains, the patterns of hormonebinding cells were essentially identical between male and female. This was true both after labeled estradiol administration and testosterone administration. Another reptile project used garter snakes and benefited from a collaboration with the skilled neuroanatomist Mimi Halpern, professor and later dean at the Downstate Medical Center of the State University of New York (Halpern, Morrell, and Pfaff 1982). Brains were harvested two or three hours after labeled hormone injection. In considering the labeled estrogens, the anterior hypothalamus had a rostral and dorsally located cell group that contained large numbers of very densely labeled cells. The ventromedial hypothalamic nucleus is large and spherical and densely packed with nerve cell bodies. Labeled cells could be found there in specific subregions: a dorsal subgroup and a posteromedial subgroup. As expected, the periventricular nucleus was well labeled, especially in its ventral aspect. Farther poste-

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rior a cell group close to the ventricle that is called the arcuate nucleus had the largest proportion of labeled neurons and the densest labeling of any area in the snake brain. Similar to other species studied, the thalamus rarely had any labeled cells. In the snake brain, the preoptic area is similar to that in mammals, cell-dense in its medial nucleus and cell-poor laterally (containing the medial forebrain bundle). The medial preoptic neurons are extremely densely labeled, and there are a large number of them. Regarding the limbic system, cells in the septum are lightly labeled. In the snake brain we do not talk about the amygdala; instead, in the lateral telencephalon, we have the nucleus sphericus. Cells in this nucleus are labeled, especially on the medial side, and there is excellent labeling in the bed nucleus of the stria terminalis. In the midbrain, estrogen-binding neurons could be seen in the central gray and just lateral to it. The distribution was similar to the mammalian brain, but there are fewer cells. Were testosterone-labeled cells significantly dif ferent than the estrogen-labeled cells? Overall, animals injected with tritiated estradiol had more densely labeled cells and a greater number of them. But the overall neuroanatomical pattern was similar. As with the lizard brain, we saw no consistent differences in the patterns of intensities of labeling in the brains of males and females receiving a given treatment of labeled hormone.

Birds It was natural enough for us to collaborate with our friend and Rutgers University neighbor, professor of biology Ronald Barfield, and the bird of choice was the domestic fowl—chickens (Barfield, Ronay, and Pfaff 1978). Even before the intravenous injections of radioactive testosterone I was instructed on how to hypnotize a chicken by a rapid finger movement in front of the bird’s face. This way the axial vein could be viewed directly while the bird was restrained with its wing extended. Brains were harvested one hour after injection because of the rapidity of the intravenous route. The most densely labeled cells were, as expected, in the hypothalamus and preoptic area. The posterior portion of the medial hypothalamic

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nucleus displayed many testosterone-binding cells, but the lateral hypothalamus was not labeled at all. The preoptic area, especially in its medial and suprachiasmatic subdivisions, had a large number of labeled cells. As far as the limbic system is concerned, in the bird we must look to the archistriatum, which had testosterone-binding cells ranging from lightly to quite heavily labeled. The most impressive were on its medial side in the so-called nucleus taeniae, which looks to me as though it occupies a neuroanatomical position analogous to the medial amygdala. In the midbrain, the central grey itself had no labeled cells, but a structure lateral to it, the nucleus intercollicularis, retained as much tritiated testosterone as any neurons in the chicken brain. Thus, results with chicken brains conformed to the vertebrate-wide pattern of limbic / hypothalamic sex steroid-concentrating cells. Arthur Arnold, now a professor at the University of California–Los Angeles, worked with Rockefeller professor Fernando Nottebohm and me to localize testosterone or its metabolites in the brain of the zebra finch, a songbird that Fernando had made extremely popu lar for studying the mechanisms of song learning. Finches were doubleinjected 1.5 and 1.0 hours before sacrifice, and the brains, accompanied by a large number of control groups, were given autoradiographic exposures under our usual dark / dry / cold / lead box conditions for periods of 5 to 13 months (Arnold, Nottebohm, and Pfaff 1976). I describe the results in two parts: the first part fits the usual limbic / hypothalamic pattern I began with in the rat brain. The second part is particular to the regulation of androgen-dependent singing in the zebra finch. In the hypothalamus, an anterior field of heavily labeled cells extends from the back of the preoptic area all the way to the magnocellular periventricular nucleus. A more posterior hypothalamic field of  labeled cells near the midline paraventricular organ. This heavy labeling in cells of the infundibular nucleus looks most like the ventromedial hypothalamic and arcuate nucleus labeling in rats and mice. More labeled fields were lightly labeled or not labeled at all. Large numbers of testosterone binding cells were detected just anterior to the anterior commissure and also directly underneath it. These are exactly in the position to match our rat brain preoptic distribution

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of cells. The extension of labeling back into the midbrain was impressive. The most prominent labeling, in the dorsomedial region of the nucleus intercollicularis, matched what I had reported for the chicken brain. Limbic system labeling was strong. A far-anterior cell group named the magnocellular nucleus of the anterior neostriatum was impressive, as was the labeling in the septum. The medial side of the archistriatum had testosterone-binding cells, including the nucleus taeniae (as also seen in the chicken brain). As with other species, some of the most beautiful labeling occurred near the ventral trip of the lateral ventricle. Thus, the general “limbic / hypothalamic system” formula adopted from the rodent brain can be seen here in the zebra finch brain. The second emphasis in the results concerns what is needed for the hormonal facilitation of song in this bird. Back in the midbrain and hindbrain, in addition to labeled cells in the nucleus intercollicularis, heavily labeled cells were found in the tracheosyringeal portion of the hypoglossal nucleus in the medulla. Recognizing this as the motor nucleus of the hypoglossus gave us adequate reason to believe that it is centrally involved in the androgen-dependence of song in these animals. More than that, Art Arnold did calculations that suggested that all the neurons in this nucleus might accumulate testosterone and further proved by retrograde degeneration studies that they actually supply the syringeal musculature that produces birdsong. Moreover, a telencephalic cell group named the hyperstriatum ventrale, pars caudale (HVc), where lesions disrupt singing, also had well-labeled cells. Thus, in the song-control system, the HVc, nucleus intercollicularis, and motor nucleus of the hypoglossus all can work together, seriatum, to account for the testosterone-dependence of song production. A third avian species we studied was the chaffinch. Richard Zigmond had begun the autoradiographic work by focusing on testosterone accumulation by neurons in an area of the midbrain known to be involved in the control of testosterone-dependent birdsong, the nucleus intercollicularis. Indeed, he found heavily labeled cells there so dramatic that he wanted to submit an article to Science. Because the format of Science was so dif ferent from that of other journals and because the journal was so exclusive, I advised him that it was too much trouble to submit there. But by then he already had submitted—and his article was

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published without even a request for change (Zigmond, Nottebohm, and Pfaff 1973). In a later more comprehensive study (Zigmond, Detrick, and Pfaff 1980) that I will summarize only briefly, Richard found large numbers of testosterone-accumulating cells in the hypothalamus anteriorly in the periventricularis magnocellularis and also in the infundibular nucleus (like the mammalian arcuate nucleus). Well-labeled cells were discovered, as expected, in the medial preoptic area. Regarding the limbic system, testosterone-binding neurons were found in the lateral septum and the nucleus magnocellularis neostriatalis anterioris. As usual, a large number of labeled cells is found in the medial part of the forebrain near the ventricle. In addition to the limbic / hypothalamic distribution, we paid attention to cells that might have to do with song control. In addition to the nucleus intercollicularis, motor neurons, likely important for song, were labeled in the nucleus of the hypoglossal nerve. Thus, throughout our studies with bird brains we carried forward two themes: replication of the vertebrate-wide limbic hypothalamic system, and the addressing by testosterone of neurons important for testosterone-facilitated song.

* * * Returning to mammals, we came to a carnivore, the mink Mustela vison (Morrell, Ballin, and Pfaff 1977). These were scary to study because they could bite your hand off. Estradiol binding was studied in the brains of females that, on the mink ranch, were proven breeders, and that, in our laboratory, were housed in cages identical to those on the mink ranch. These are seasonal animals, and we studied them both in their estrus and anestrous conditions. Neuroanatomical results were the same for the two groups. As expected, in the hypothalamus the arcuate nucleus and the ventrolateral section of the ventromedial nucleus were plastered with labeled cells. Estrogen-binding neurons were also seen in the anterior hypothalamic area and were scattered just outside the ventromedial nucleus. In the medial preoptic area, labeled neurons were packed especially densely in its medial and suprachiasmatic portions. In the limbic system, starting from the anterior and working posteriorly, we found estrogen- concentrating cells in the lateral septum

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and in the bed nucleus of the stria terminalis. Very few labeled cells were in the medial septum. The medial nucleus of the amygdala had a substantial number of labeled neurons, and some were also seen in the cortical nucleus. The labeled neurons in the hippocampus were the large pyramidal cells in the ventral hippocampus and cells in the granular layer of the dentate gyrus. As in the other species we had studied, estrogen-binding neurons were found in the mesencephalic central grey and lateral to it in the deep layers of the superior colliculus.

Primate Brains By collaborating with medical doctors Michel Ferin, Peter Carmel, and Earl Zimmerman from Columbia College of Physicians and Surgeons, we gained the exciting opportunity to study estrogen-binding neurons in the brain of the female rhesus monkey (Pfaff et al. 1976). The same limbic / hypothalamic system I had seen in the rat brain some years earlier showed up again in the primate brain. In the hypothalamus, we saw heavy labeling throughout the extent of the arcuate (infundibular) nucleus (Figure 1.3) and by the lateral-placed neurons within the ventromedial nucleus of the hypothalamus. Estrogenbinding cells were also found in the anterior hypothalamic area and posteriorly near the borders of the medial mammillary nucleus. Large numbers of well-labeled cells were in the medial preoptic area. The estrogen concentration was especially strong on its medial side and in the suprachiasmatic portion of the preoptic area. In the limbic system, we could see estrogen binding in the ventrolateral septum and, to a much lesser extent, in the medial septum. The bed nucleus of the stria terminalis was well labeled, as were neurons in the medial nucleus of the amygdala. All the tissue from the basal ganglia was not labeled. In the anterior mesencephalon, lightly labeled cells were seen behind the mammillary bodies in the periventricular stratum leading to the central grey. There was a small number of labeled cells in the central grey near the dorsolateral and ventrolateral borders. Other mesencephalic tissue was unlabeled. The neuroanatomical pattern of cell nuclear estrogen binding in this limbic / hypothalamic system was seen to be specific because the

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Figure 1.3. Black dots show the locations of neurons expressing estrogen receptors in the ventromedial hypothalamus (VM) of the female rhesus monkey. The expression is bilateral, plotted here on one side. (Adapted from Pfaff et al. 1976).

corticosterone binding was by far highest in the hippocampus, as Bruce McEwen had found for the rat brain, and some corticosterone-labeled cells were even seen among the cerebellar granule cells.

Human Brain Finally, of course, we approached the most important species for medical approaches to the brain: humans. Dick Swaab, a leader at the National Institute for Brain Research in Amsterdam, used an antibody against ER- α to study nuclear ER in material from the Netherlands brain bank. His distribution of limbic and hypothalamic neurons showing ER immunoreactivity matches what I originally reported in the rat brain. Likewise, Jeffrey Blaustein, a professor at the University of Massachusetts, not only extended my findings in the diencephalon of the rat to the human brain but also documented statistical sex differences: men’s brains showed somewhat more ER immunoreactivity in the medial preoptic area whereas women’s brains showed somewhat more in the ventromedial hypothalamus. Overall, this was a long saga. During the first couple of years after my articles were published I had to fight off a challenge from the late Walter Stumpf, whose technique led to artifacts and whose neuroanatomical charting was oversimplified, which confused things for a while. But my work enjoyed a massive amount of replication, as I’ll discuss later. In summary, from fi sh through amphibians through reptiles, through birds and several mammalian brains including monkeys and

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humans, we discovered a common neuroanatomical pattern of sex hormone-binding neurons. Hormone binding in limbic structures is strong. Medial preoptic neurons and medial hypothalamic neurons show high levels of sex hormone concentration in the cell nucleus, especially in the arcuate nucleus and the ventromedial nucleus (as will be crucial for lordosis behavior). A frequent posterior extension of this system is the mesencephalic central grey.

Replication at the mRNA Level The canonical sequence of biochemical and physiological events that we addressed includes 1) gene expression for ER, 2) translation to the ER protein, 3) binding of estrogens by the ER protein, and 4) consequent estrogenic facilitation of female reproductive behavior. This last step I established in the work I discussed at the beginning of this chapter, and I will continue that discussion here. First, let’s address step 1. By studying hormone binding in neurons, I was addressing the actual function of an ER gene and its protein products. Nevertheless, it was comforting to see in situ hybridization results from my own and other laboratories demonstrating ER gene expression in the same nerve cell groups I have emphasized previously. The earliest confirmation came from the in situ work of the always upbeat Richard Simerly, who had shown his brilliance early by choosing to work with neuroendocrine pioneer Roger Gorski (at the University of California–Los Angeles), and then with Larry Swanson (now at the University of Southern California), one of the premier neuroanatomists of his generation. Simerly’s results matched our binding results so well that we decided to follow them up. Andrea Lauber not only showed ER messenger RNA (mRNA) expression in the ventrolateral corner of the ventromedial nucleus of the hypothalamus (the most important for lordosis behavior regulation) and arcuate nucleus of the hypothalamus, but she also demonstrated that estrogen administration down-regulates the mRNA for its own ER receptor there (Lauber et al. 1990). As mea sured by grain counting over individual cells in those hypothalamic structures and via in situ hybridization, the decline in ER mRNA reached its lowest point 18 hours after a subcutaneous injection of estradiol, having knocked

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down gene expression to less than half its initial rate. After that, by 24 hours the mRNA levels were recovering. Further, this endocrine feedback dynamic was sexually differentiated (Lauber et al. 1991). To begin with, there is a clear sex difference in initial ER mRNA levels in these hypothalamic neuronal groups, notably the ventromedial nucleus. Males exhibited 52 percent less ER message than females. On top of that finding, the decline in ER mRNA levels in males was small and not statistically significant in the same experiment where the large (>50  percent) decline was replicated in the female hypothalamus. Interestingly, these endocrine dynamics and sex differences did not show up in the medial amygdala. Although there was a trend toward a decline in the female amygdala and a trend toward a sex difference, those trends were not statistically significant. Molecular endocrinologists were surprised when the laboratory of Jan-Ake Gustafsson, at the Karolinska Institutet, cloned a second ER, now called ER- β. As a result of that, the ER highly expressed in the hypothalamus and important for lordosis is now called ER- α. ER-β binding is not crucial for lordosis; its import in the brain, highly significant, is sometimes the opposite of ER-α. Its impact on estrogen-dependent cancers is of great interest.

Replication at the Protein Level The most popu lar way to study ER- α protein in the brain has been to use specific antibodies for immunocytochemistry. Mapping studies doing exactly this have repeated the neuroanatomical distribution I reported for estrogen binding. Jeffrey Blaustein at the University of Massachusetts was the first to show ER- α protein in the same preoptic, hypothalamic, and limbic areas as I had observed estradiol binding. In addition, he reported that progesterone receptor (PR) immunoreactivity tended to appear only in ER-expressing neurons. This is important because estradiol working through ER- α strongly induces expression of the progesterone receptor gene (Chapter 3). Blaustein’s work in the guinea pig was replicated by Marie Warembourg and Eduard Milgrom in the INSERM Unit in Lille, France, and also by Joan Morrell, now at Rutgers. The same limbic /

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hypothalamic distribution of ER protein was reported for the rat brain, with an emphasis on high levels of immunocytochemical product in the medial preoptic area and ventromedial hypothalamus. And for our later work on behavior genetics, it was important that these ICC results were also repeated in the mouse brain. Mitsuhiro Kawata, in Kyoto, did an immunocytochemical experiment that replicated with an amazing degree of precision Andrea Lauber’s previously mentioned work at Rockefeller. He not only reported the distribution of ER identically to the Lauber’s studies, but he also looked at the negative feedback effects of circulating estrogen on ER protein in the brain. In his results, there were more ER-immunoreactivity cells with higher immunoreactivities in ovariectomized rats than in estradiol-treated rats. The number of ER-immunoreactivity cells in estradiol-treated rats compared with ovariectomized rats was reduced by 43  percent. In particu lar, the number of ER-immunoreactivity cells in the central part of the MPN was largely decreased. These data quantitatively match Lauber’s results with in situ hybridization. Results with other species fell into line. Carol Jacobson, at Iowa State University, saw the identical limbic / hypothalamic distribution of ER protein in the Brazilian opossum. In the brain of the Japa nese quail, Jacques Balthazart, at the University of Liege in Belgium, reported a high percentage of labeled cells was observed in the lateral septum, the nucleus accumbens, the preoptic medial nucleus, the supraoptic nuclei, the anterior medial hypothalamus, the paraventricular magnocellular nucleus, the caudal parts of the lateral hypothalamus, and the whole tuberal and infundibular area. A small number of labeled cells was also observed in the ventromedial nucleus of the hypothalamus Primate brain. Most importantly, Alan Herbison, then at the University of Cambridge, studied monkey brains and showed that ERimmunoreactive cells in the monkey hypothalamus are distributed in a manner similar to that observed in other mammalian species. His double-labeling experiments provide further evidence that luteinizing hormone-releasing hormone (aka gonadotropin-releasing hormone) neurons do not possess ERs, thus replicating a somewhat controversial finding I had reported for rat brain some years before.

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Summary to This Point Studies of ER- α gene expression, protein levels, and actual estrogen binding all tell us of high levels of hormone binding in the medial hypothalamus, the medial preoptic area, and specific portions of the limbic system, the phylogenetically ancient portions of the forebrain. Expression in the ventromedial nucleus of the hypothalamus is most important for the estrogenic facilitation of lordosis behavior.

Importance for Behavior Paula Davis, in my laboratory, used an extremely discriminating approach to hormone implantation into the brain to demonstrate that ventromedial hypothalamic neurons are sensitive to estrogens in a manner that facilitates lordosis behavior. First, she implanted extremely small amounts of tritiated estradiol directly into the ventromedial hypothalamus and showed that it could facilitate lordosis behavior of female rats (Davis, McEwen, and Pfaff 1979). Although significant amounts of radioactivity were concentrated in the nuclei of ventromedial hypothalamic neurons, no leakage to other sites—such as the lateral hypothalamus, the preoptic area, the amygdala, or the cortex, or even high-affinity sites such as the pituitary and the uterus— could be detected. Thus, estrogenic binding in ventromedial hypothalamic neurons is sufficient for facilitating lordosis behavior. Davis followed up those experiments by using autoradiography to prove, in quantitative terms (grains per unit area), the absence of significant diffusion of estrogen from the ventromedial hypothalamus implantation site, and further to prove that estrogen exposure in the central and lateral subdivisions of the ventromedial hypothalamus (not the anterior pole) acts to promote lordosis (Davis et al. 1982). Estrogen binding to ER- α in the ventromedial hypothalamus is also necessary for lordosis. Implantation of an antiestrogen, an ER blocker, in the ventromedial hypothalamus but not in other parts of the brain could significantly reduce the effects of systemic estrogen administration on female reproductive behavior (Meisel et al. 1987).

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Necessary and Sufficient Thus, estrogen binding in the nuclei of ventromedial hypothalamic neurons is necessary and sufficient for elevated levels of female reproductive behavior. Estradiol in the ventromedial hypothalamus can permit lordosis responses to the male’s mounting even when there is no estrogen elsewhere in the body. Conversely, estrogens can be flooding tissues elsewhere in the body but if an antiestrogen implantation in the ventromedial hypothalamus prevents its nuclear binding there, then lordosis behavior will not occur. Paula Davis’s and Bob Meisel’s work in my laboratory was complemented by elegant results from Ron Barfield’s laboratory at Rutgers. He found that in the ventromedial hypothalamus dilute estrogen implants facilitated lordosis and an ER antagonist implant blocked lordosis. Because ER-α is a ligand-activated transcription factor (see Chapter 3) we can expect to see a train of events that leads from nuclear binding of estradiol in the ventromedial hypothalamus to the promotion of lordosis behavior: new mRNA synthesis, followed by new protein synthesis, followed by axoplasmic flow from the ventromedial hypothalamic cell body toward the midbrain (Chapter  2), accompanied by action potentials. All these are necessary for estrogen-stimulated sex behavior. In the 1970s David Quadagno working with Roger Gorski at the University of California–Los Angeles showed that intrahypothalamic infusion of the RNA synthesis inhibitor actinomycin D blocked hormone-induced lordosis behav ior. Brenda Shivers and Richard Harlan, working at the University of Texas before they came to my laboratory, as well as Tom Rainbow who was working with Bruce McEwen at Rockefeller, replicated the Quadagno fi nding. In turn, Quadagno infused the protein synthesis inhibitor cycloheximide and blocked lordosis. Our own work (Harlan et al. 1982), led by Harlan and Shivers, concentrated on the axoplasmic flow of newly synthesized protein from the hypothalamus toward the midbrain (see the circuitry information in Chapter 2). Colchicine is a drug well-known to disrupt axoplasmic transport by inhibiting microtubule polymerization by binding to

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tubulin, one of the main constituents of microtubules. It was infused into or near the ventromedial hypothalamus, and its effect on lordosis was compared with vehicle controls. In a dose-dependent manner, colchicine rapidly and significantly reduced behavioral sensitivity to estrogen treatment. In later experiments, Harlan and Shivers studied the requirement for hypothalamic action potentials in the maintenance of estrogensupported action potentials (Harlan et al. 1983). Local anesthetics such as procaine or Marcaine infused into the hypothalamus were not very effective. However, infusion of tetrodotoxin for blocking voltage-dependent sodium currents was very effective, reducing estrogen-dependent lordosis in a dose-dependent manner for eight hours. Electrophysiological recordings confirmed the expected abolition of electrical activity by the affected hypothalamic neurons, the basis of the reproductive behavioral effect. Joel Rothfeld in our laboratory extended this work by microinfusing tetrodotoxin into the midbrain central grey, where ventromedial hypothalamic axons that are relevant for lordosis terminate (see Chapter 2). For a time period from 10 minutes to four hours, tetrodotoxin in the central grey virtually abolished lordosis (Rothfeld et  al. 1986). Taking this all together we can summarize that after estrogen binding to ER in the ventromedial hypothalamus, estrogen-responsive gene expression is necessary for lordosis behavioral regulation, followed canonically by protein synthesis and flow down the axon, accompanied by estrogen-sensitive electrical activity.

Hormone-Binding Neurons Projecting to Form Networks This chapter is not an appropriate venue for a comprehensive review of limbic system, preoptic, or hypothalamic neuroanatomy. Examples of where such reviews can be found include an overview by Swanson (2013), with behavioral interpretations by Kringelbach (2016). The oldest ideas about close limbic / hypothalamic relations began with the Papez circuit, which related hippocampal outputs to preoptic and hypothalamic function (with a return through the anterior thalamic nuclei). Swanson and Cowan detailed hippocampal efferents to the hypothalamus. Later they described dense and multiple interconnections

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between the medial and lateral septum, on the one hand, and the preoptic area and hypothalamus, on the other. David Amaral, when he was at the Salk Institute, extended our knowledge of hypothalamoamygdaloid relations to the monkey brain. In his words, all these projections connect the amygdala to the hypothalamus by way of the so-called ventral amygdalofugal pathway, but at least some of the fibers that arise in the ventromedial nucleus run in the stria terminalis. These are some of the leading neuroanatomical scholars and fi ndings that gave me the courage to theorize as well. Starting about 1971, it became obvious to me that sex hormoneconcentrating nerve cell groups tend to project to other sex hormone concentrating nerve cell groups, and our first chart was published as figure 4 in Pfaff and Keiner (1973). Here are just a few examples: 1) strong medial preoptic projections to the medial amygdala, 2) medial preoptic projections to the ventromedial hypothalamus, 3) medial preoptic efferents to the septum, 4) anterior hypothalamic projections to the preoptic area, 5) anterior hypothalamic efferents to the septum, 6) anterior hypothalamic connections to the ventromedial hypothalamus, 7) anterior hypothalamic projections to the medial amygdala, 8) amygdala projections to the preoptic area, 9) amygdala projections to the ventromedial hypothalamus, 10) septal projections to the hippocampus, 11) hippocampal projections to the preoptic area, 12) hippocampal projections to the ventromedial hypothalamus, 13) ventromedial hypothalamic projections to the preoptic area, 14) ventromedial hypothalamic projections to the septum, 15) ventromedial hypothalamic projections to the amygdala, and 16) ventromedial hypothalamic projections to the central grey. By no means does this dense web of connections follow a pattern of laminar flow, analogous to some parts of the visual system. However, Rockefeller graduate student Lily Conrad and I noticed a greater trend toward spatial orderliness than is usually claimed for the hypothalamus and basal forebrain (Pfaff and Conrad 1978, see figures 8 and 9). That is, more medially placed nerve cell bodies gave rise to axons that tended to descend medially—and laterally placed neurons, to axons more laterally, and dorsally placed neurons, to axons more dorsally. The implications of this neuroanatomical trend for reproductive behavior are not clear, but the interconnectedness of sex hormone-binding nerve cell groups does have a theoretical implication.

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We derived the theory that networks of hormone-sensitive neurons offer possibilities of systems that effectively multiply hormone effects. One reason for pointing out the neuroanatomical interconnectedness of sex hormonebinding neuronal cell groups is that it offers the possibility of hypothesizing interesting hormone-sensitive mathematical functions that could result from such interconnectedness. These interactions could be important for the regulation of behavior. The simplest theory would be that such interconnectedness could magnify effects of hormones on strings of neurons important for mating behavior. For example, if estrogen exposure multiplies physiological activity by some constant k in neurons named a, b, and c, and if neuron a projects to neuron b which in turn projects to neuron c, then it seems possible that system output following estrogen is multiplied as follows: ka × kb × kc. Of course, large numbers of other such theories are possible, and they can be tested with modern electrophysiological and optogenetic techniques.

* * * In summary, this tremendous amount of work told us how endocrine signaling systems impact the brain. In Chapters 2 through 4 we will go on with physiological and genomic techniques to work out how hormone-dependent neurons work through circuitry to produce an entire vertebrate behavior. That is, we understand how the ner vous system works by taking a specific behavioral function and solving it as an analytic problem. Principle inferred: The brain needs to be continually informed about the state of the body. For the purpose of successful reproduction, that is accomplished by systems of specific hormone receptor proteins in neurons in selected parts of the brain. The system of hormone-binding neurons that I discovered appears to be universal among vertebrates.

In the mammalian brain, specific nuclear receptors for estrogens offer a launching point, one of three, for working out the first neural circuit for a vertebrate behavior, as described in Chapter 2. And these nuclear receptors, being ligand-activated transcription factors, allowed

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us to apply the techniques of molecular biology to our developing field, as described in Chapter 3.

Further Reading Arnold, A., F. Nottebohm, and D.  W. Pfaff. 1976. “Hormone- Concentrating Cells in Vocal Control and Other Areas of the Brain of the Zebra Finch (Poephila guttata).” Journal of Comparative Neurology 165: 487–512. Barfield, R. J., G. Ronay, and D. W. Pfaff. 1978. “Autoradiographic Localization of Androgen- Concentrating Cells in the Brain of the Male Domestic Fowl.” Neuroendocrinology 26: 297–311. Davis, P. G., M. S. Krieger, R. J. Barfield, B. S. McEwen, and D. W. Pfaff. 1982. “The Site of Action of Intrahypothalamic Estrogen Implants in Feminine Sexual Behavior: An Autoradiographic Analysis.” Endocrinology 111: 1581–1586. Davis, P. G., B. McEwen, and D. W. Pfaff. 1979. “Localized Behavioral Effects of Tritiated Estradiol Implants in the Ventromedial Hypothalamus of Female Rats.” Endocrinology 104: 898–903. Davis, R. E., J. I. Morrell, and D. W. Pfaff. 1977. “Autoradiography Localization of Sex Steroid- Concentrating Cells in the Brain of the Teleost Macropodus opercularis (Osteichthyes: Belonttidae).” General and Comparative Endocrinology 33: 496–505. Halpern, M., J. I. Morrell, and D. W. Pfaff. 1982. “Cellular 3H-Estradiol and 3 H-Testosterone Localization in the Brains of Garter Snakes: An Autoradiographic Study.” General and Comparative Endocrinology 46: 211–224. Harlan, R. E., B. D. Shivers, L.-M. Kow, and D. W. Pfaff. 1982. “Intrahypothalamic Colchicine Infusions Disrupt Lordotic Responsiveness in EstrogenTreated Female Rats.” Brain Research 238: 153–167. ———. 1983. “Estrogenic Maintenance of Lordotic Responsiveness: Requirement for Hypothalamic Action Potentials.” Brain Research 268: 67–78. Kelley, D. B., I. Lieberburg, B. S. McEwen, and D. W. Pfaff. 1978. “Autoradiographic and Biochemical Studies of Steroid Hormone- Concentrating Cells in the Brain of Rana pipiens.” Brain Research 140: 287–305. Kelley, D. B., J. I. Morrell, and D. W. Pfaff. 1975. “Autoradiographic Localization of Hormone- Concentrating Cells in the Brain of an Amphibian, Xenopus laevis. I. Testosterone.” Journal of Comparative Neurology 164: 47–62. Krieger, M. S., J. I. Morrell, and D. W. Pfaff. 1976. “Autoradiographic Localization of Estradiol- Concentrating Cells in the Female Hamster Brain.” Neuroendocrinology 22: 193–205. Kringelbach, M. L. 2016. “Limbic Forebrain: The Functional Neuroanatomy of Emotion and Hedonic Processing.” In Neuroscience in the 21st  Century. 2nd edition. Edited by D. W. Pfaff and N. D. Volkow. New York: Springer, 1335–1363.

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Lauber, A. H., C. V. Mobbs, M. Muramatsu, and D. W. Pfaff. 1991. “Estrogen Receptor mRNA Expression in Rat Hypothalamus as a Function of Genetic Sex and Estrogen Dose.” Endocrinology 129 (6): 3180–3186. Lauber, A.  H., G.  J. Romano, C.  V. Mobbs, and D.  W. Pfaff. 1990. “Estradiol Regulation of Estrogen Receptor Messenger Ribonucleic Acid in Rat Mediobasal Hypothalamus: An in Situ Hybridization Study.” Journal of Neuroendocrinology 2 (5): 605–611. Meisel, R., G. Dohanich, B. McEwen, and D.  W. Pfaff. 1987. “Antagonism of Sexual Behavior in Female Rats by Ventromedial Hypothalamic Implants of Antiestrogen.” Neuroendocrinology 45: 201–207. Morrell, J. I., A. Ballin, and D. W. Pfaff. 1977. “Autoradiographic Demonstration of the Pattern of 3H-Estradiol Concentrating Cells in the Brain of a Carnivore, the Mink, Mustela vison.” Anatomical Record 189 (4): 609–624. Morrell, J. I., D. Crews, A. Ballin, A. Morgentaler, and D. W. Pfaff. 1979. “3HEstradiol, 3H-Testosterone and 3H-Dihydrotestosterone Localization in the Brain of the Lizard Anolis carolinensis: An Autoradiographic Study.” Journal of Comparative Neurology 188: 201–224. Morrell, J. I., D. B. Kelley, and D. W. Pfaff. 1975. “Autoradiographic Localization of Hormone- Concentrating Cells in the Brain of an Amphibian, Xenopus laevis. II. Estradiol.” Journal of Comparative Neurology 164: 63–78. Morrell, J.  I., M.  S. Krieger, and D.  W. Pfaff. 1986. “Quantitative Autoradiographic Analysis of Estradiol Retention by Cells in the Preoptic Area, Hypothalamus and Amygdala.” Experimental Brain Research 62: 343–354. Morrell, J.  I., and D.  W. Pfaff. 1978. “A Neuroendocrine Approach to Brain Function: Localization of Sex Steroid Concentrating Cells in Vertebrate Brains.” American Zoologist 18: 447–460. ———. 1982. “Characterization of Estrogen- Concentrating Hypothalamic Neurons by Their Axonal Projections.” Science 217: 1273–1276. Pfaff, D. W. 1965. “Cerebral Implantation and Autoradiographic Studies of Sex Hormones.” In Sex Research: New Developments. Edited by J. Money. New York: Holt, Rinehart & Winston, 219–234. ———. 1968. “Autoradiographic Localization of Radioactivity in Rat Brain after Injection of Tritiated Sex Hormones.” Science 161: 1355–1356. ———. 1970a. “Mating Behav ior of Hypophysectomized Rats.” Journal of Comparative and Physiological Psychology 72: 45–50. ———. 1970b. “Nature of Sex Hormone Effects on Rat Sex Behav ior: Specificity of Effects and Individual Patterns of Response.” Journal of Comparative and Physiological Psychology 73: 349–358. Pfaff, D. W., and L. C. A. Conrad. 1978. “Hypothalamic Neuroanatomy: Steroid Hormone Binding and Patterns of Axonal Projections.” In International Review of Cytology, vol. 54. Edited by G. Bourne. New York: Academic, 245–265.

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Pfaff, D. W., J. Gerlach, B. S. McEwen, M. Ferin, P. Carmel, and E. Zimmerman. 1976. “Autoradiographic Localization of Hormone- Concentrating Cells in the Brain of the Female Rhesus Monkey.” Journal of Comparative Neurology 170: 279–294. Pfaff, D. W., and M. Keiner. 1973. “Atlas of Estradiol- Concentrating Cells in the Central Ner vous System of the Female Rat.” Journal of Comparative Neurology 151: 121–158. Rhodes, C. H., J. I. Morrell, and D. W. Pfaff. 1981. “Distribution of EstrogenConcentrating, Neurophysin- Containing Magnocellular Neurons in the Rat Hypothalamus as Demonstrated by a Technique Combining Steroid Autoradiography and Immunohistology in the Same Tissue.” Neuroendocrinology 33: 18–23. Rothfeld, J. M., R. E. Harlan, B. D. Shivers, and D. W. Pfaff. 1986. “Reversible Disruption of Lordosis via Midbrain Infusions of Procaine and Tetrodotoxin.” Pharmacology Biochemistry and Behavior 25: 857–863. Swanson, L.  W. 2013. “Basic Principles of Ner vous System Organ ization.” In Neuroscience in the 21st  Century, vol. 3. Edited by D.  W. Pfaff. New York: Springer, 3: 1255–1288. Tabansky, I., J. N. H. Stern, and D. W. Pfaff. 2015. “Implications of Epigenetic Variability within a Cell Population for ‘Cell Type’ Classification.” Frontiers in Behavioral Neuroscience 9: 342. Wallis  K., S. Dudazy, M. van Hogerlinden, K. Nordström, J. Mittag, and B. Vennström. 2010. “The Thyroid Hormone Receptor α1 Protein Is Expressed in Embryonic Postmitotic Neurons and Persists in Most Adult Neurons.” Molecular Endocrinology 24: 1904–1916. Zigmond, R.  E., R.  A. Detrick, and D.  W. Pfaff. 1980. “An Autoradiographic Study of the Localization of Androgen Concentrating Cells in the Chaffi nch.” Brain Research 182: 369–381. Zigmond, R. E., F. Nottebohm, and D. W. Pfaff. 1973. “Androgen- Concentrating Cells in the Midbrain of a Songbird.” Science 179: 1005–1007.

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2 D ISCOV ER I N G T H E N EU R A L CIR CU IT FOR A V ERTEBR ATE BE H AV IOR E SSEN T I A L TO R EPRO D U C T I O N

Problem: No one had put together the mechanisms for a complete vertebrate behavior. It had been thought that for ease of methodology and conceptual clarity, simpler animals such as Aplysia and fruit flies must be used to study the neural basis of behavior. For a mammalian behavior, we cover the subject from ion channels through neuroanatomy through electrophysiology to the logic of the social behavior itself.

You cannot state seriously that you have the mechanisms for a behavior unless you have figured out the entire neural circuit. So that is what we did. From the work discussed in Chapter  1, we had the information about how hormones impact the brain. In Chapters 3 and 4 we will be able to link these hormones to gene expression and genomic influences on behavior. But now, in Chapter 2, we are looking for a manifestation of lordosis behavior mechanisms in physical terms. Lordosis behavior involves the vertebral dorsiflexion by the female in response to the male’s mount, a behavior essential for fertilization and therefore reproduction. Working out the entire neural circuit for producing lordosis took the combined efforts of many smart young scien-

tists over a period of many years. Throughout this chapter I will recognize their contributions. So there were three points of entry that allowed discovery of the first neural circuit for producing a vertebrate behavior: First: the neurons expressing the hormone receptors whose action in hypothalamic neurons facilitate the behavior (Chapter 1). Second: the cutaneous stimuli (from mounting by the male) that triggers the behavior. Third: working our way in backward from the muscles that execute the behavior, through the motor neurons, and backward up the motor control pathways. A large number of analytic experiments revealed a spinal–midbrain–spinal loop whose activity is regulated by an estrogen-dependent output from the hypothalamus. The estrogen-dependent output relies on hormoneregulated gene expression (Chapter 3). The flow of the sections in this chapter will take us from the hypothalamus out to the midbrain, at the top of the spinal–midbrain circuit we discovered. In subsequent sections we start from the relevant sensory surface, work our way up ascending pathways to the midbrain (to meet the hypothalamic outflow), and then come back down descending pathways to the relevant motor neurons. Unless other wise stated, female animals were treated with estrogen whenever appropriate to the experimental aim.

Hypothalamic Outflow Adds the Hormone Dependence Neuroanatomy of Ventromedial and Other Hypothalamic Neurons’ Projections From the point of view of lordosis behavior control, projections from the ventromedial nucleus of the hypothalamus (VMH) were the most important to determine. As will be noted later, in lesions of VMH abolish lordosis, stimulation of VMH triggers lordosis and estrogen exposure in VMH is necessary and sufficient for lordosis (Chapter 1). The task of VMH projection discovery fell to the Rockefeller graduate student Lily C. A. Conrad, a young neuroanatomist so skilled that I told her, “You could be the next century’s Ramón y Cajal!” Instead, after receiving her Ph.D. she went to medical school. Lily and I used autoradiographic techniques

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with long exposure times to chart all the efferents of VMH (Conrad and Pfaff 1976a). Ascending VMH axons swept through the anterior hypothalamus and preoptic area into the diagonal band and septum. Projections were prominent in the preoptic area itself and the lateral ventral septum. Some fibers ran into the stria terminalis and its bed nucleus. Most terminations were ipsilateral to the isotope’s microinjection in VMH, but scattered contralateral projections were also detected. Descending projections, most impor tant for lordosis behav ior, formed two systems. One group of labeled axons ran laterally and posteriorly, distributing through the zona incerta and fields of Forel to terminate in the midbrain reticular formation lateral to the mesencephalic central grey. These axons formed a sheet-shaped projection that twisted ninety degrees as it curved posteriorly to the midbrain. Some fibers, following the supraoptic commissure, made it into the amygdala. The second group ran medially, turning through the posterior hypothalamus and following a periventricular route to the central grey itself. These fibers were scattered in bundles posterior to VMH as they swept past premammillary regions, projecting to ventral and dorsal premammillary nuclei on the way. Then, from a supramammillary projection they turned dorsally and projected to all nuclei of the mesencephalic central grey. Postdoctoral researcher Monica Krieger added more detail to this description of VMH efferents by microinjecting the tritiated leucine more discretely into dif ferent components of this behaviorally crucial hypothalamic nucleus (Krieger, Conrad, and Pfaff 1979). For example, axons emanating from the ventrolateral subdivision of VMH, the neuronal group with most intense estrogen binding, emphasized projections to the medial preoptic area, axons sneaking laterally beneath the cerebral peduncle to arrive at the medial amygdala, and ascending periventricular projections that would innervate the most rostral pole of the dorsolateral central grey. No axons made it all the way to the pons, a fact that helps define the midbrain module of the lordosis behavior circuit. In addition, as covered in Chapter 1, Joan Morrell’s combination of retrograde tracing using the fluorescent dye primuline with estrogen binding defi ned by steroid autoradiography proved that estrogen-

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concentrating VMH neurons really do project to the mesencephalic central grey and surrounding dorsal midbrain (Morrell and Pfaff 1982). These descending projections from VMH would later be shown physiologically to be the essential link from the hypothalamic module to the midbrain module of the newly discovered lordosis behav ior neural circuit.

U LT R A S T R U C T U R E

Having defined VMH projections that would be shown as the output of the hypothalamic module of the lordosis circuit, we wanted to take a look at the nature of the relevant synapses in the midbrain central grey (MCG) (Chung, Pfaff, and Cohen 1990a). Following electrolytic lesions in the VMH, we saw a type of degenerative pattern in the central grey known as “watery degeneration”: both in the presynaptic processes and processes (identified by the presence of a postsynaptic density) there was a swollen appearance, electron lucent. Dendrites were devoid of microtubules. In animals surviving longer after the VMH lesion (eight days), presynaptic vesicles were clumped and misshapen, there are abnormally large and dark mitochondria, and the postsynaptic processes are surrounded by reactive glia. We followed up that work with chemical (kainic acid and N-methyl aspartate) lesions of the VMH to make sure that the foregoing results were not due to fibers of passage (Chung, Pfaff, and Cohen 1990b). Once again, in the MCG, we could easily see axonal, presynaptic and postsynaptic degeneration. Presynaptic endings were shrunken and dense, and contained clumped synaptic vesicles and abnormally large dark mitochondria. Postsynaptic processes were swollen and showed watery degeneration. Dendrites were devoid of microtubules. Midbrain central grey synapses were engulfed by glial processes. These experiments relieved us by showing the central grey fine structural phenomena were really due to VMH projections, not to fibers of passage. As a side point, I note that communication between the hypothalamic module of the female reproductive behavioral neural circuit and the midbrain module issue is due to descending fibers from the VMH, not ascending fibers from the MCG. That is, Jerry Eberhart, during his year in the laboratory, saw many ascending projections from the MCG,

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but they did not go to the VMH. Another side point: Lily Conrad’s subsequent results dealt with efferents from the medial preoptic area and gave strong neuroanatomical support to what I said in Chapter  1 (Conrad and Pfaff 1976b). Neurons in regions with estrogen-binding cells tend to project to others that do the same. Electrophysiology. Yasuo Sakuma’s plane arrived from Japan on a Monday night, and he started his first experiment at 7:30 AM on Tuesday. Armed with and an M.D. and a Ph.D. (in neurophysiology) from the Yokohama City University School of Medicine, he was the most brilliant protégé of one of the founders of modern neuroendocrinology, Masami Kawakami. I was not surprised at the quality and volume of Yasuo’s accomplishments at Rockefeller nor have I been surprised at his subsequent, highly successful career as a professor at Tokyo’s Nippon Medical School (and now president of a school for allied medical sciences). I was not surprised because, before Yasuo came to New York, I had made a lecture trip to Japan, and, walking in and around Yasuo’s electrophysiology rig, I thought, “This is a guy who really knows what he is doing.” Yasuo used microelectrode recordings to characterize the properties of lordosis-critical neurons with axons to the MCG. These VMH neurons were identified by “back firing” them from the MCG, that is, by recording antidromic potentials in the VMH neuronal cell body having stimulated their axon in the MCG. Further confirmation of the crucial VMH / MCG connection was shown when an orthodromic spike emanating from the VMH cell body canceled the antidromic spike coming from the MCG (Sakuma and Pfaff 1981, 1982). Latencies for the antidromic spike to reach the cell body varied widely, from 1.4 to 41.5 milliseconds. Amazingly, a few VMH neurons with projections to MCG were also identified, using the same technique, as projecting to the amygdala. These various electrophysiological defined connections were not altered by estrogen. However, it can be said that there were detailed differences between males and females. The latencies of the VMH spikes to the antidromic stimulation were significantly different—females were faster—and one entire latency class was absent in the male. The apparent orderliness of flow of hypothalamic and preoptic axons to the midbrain was already noted in Chapter 1.

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Estrogenic Facilitation of Ventromedial Hypothalamic Neurons’ Electrical Activity A long series of electrophysiological experiments led by my long-time colleague Lee-Ming Kow led to the following conclusion: VMH neuronal responses to virtually every neurotransmitter effective for triggering VMH firing would, in turn, be amplified by estrogen treatment. Lee had gotten his Ph.D. in biophysics at the California Institute of Technology (“Cal Tech”) and wanted to come to my laboratory for his postdoctoral work; and his wife, the best dentist on the planet, was pleased to come to New York City as well. Fortunately for a generation of young scientists in my laboratory, Lee simply did not want to do what a typical American professor does—he has been pleased to stay in my laboratory as a pure neurobiological scholar. The reference list of this chapter hardly could do justice to his scientific productivity. The easiest way to summarize parts of Lee’s massive accomplishments will be to handle them neurochemical system by system. First cholinergic, then noradrenergic, then histamine, then glutamate. To describe the explanatory power of the results most clearly, I would like to use the idea of a logical syllogism, that is, reasoning in this form: “If (a) John is a scientist and (b) all scientists are good, then (c) John is good.”

AC E T Y L C H O L I N E

Lee’s electrophysiology was motivated by others’ behavioral results. Gary Dohanich, a professor at Tulane University, and Lynn Clemens at Michigan State had shown that agonists that stimulate the muscarinic type of cholinergic receptor would increase lordosis behavior and, conversely, that antagonists would decrease it. Laura Kaufman, in our laboratory, restricted cholinergic agonist application to the VMH and got the same result: increased female reproductive behavior. How does that work? Tom Rainbow, working with Bruce McEwen, showed that estrogens increase muscarinic receptor levels in VMH. Previously, Lee-Ming Kow and I had reported two results: that acetylcholine increases VMH electrical activity and that estrogen administration increases VMH responses to acetylcholine (Kow and Pfaff 1985, 1995).

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To summarize this work, (a) estrogens heighten lordosis behavior, and (b) estrogens also increase muscarinic receptors and electrophysiological responses to acetylcholine in VMH neurons. Therefore, it follows that (c) one way in which estrogens facilitate reproductive behavior is to increase acetylcholine receptors and electrical responses to acetylcholine by VMH neurons.

NOR EPI N EPH R I N E

Bundles of noradrenergic fibers, ascending to the hypothalamus from their sources, the locus coeruleus and medullary cell groups A1 and A2, innervate the VMH. The late Robert Moss at the University of Texas reported that damage to the ventral bundle as well as the administration of noradrenergic α1 receptor blockers decrease lordosis, whereas receptor agonists applied specifically to the VMH increase it. How does that work? Anne Etgen, a professor at the Albert Einstein School of Medicine, showed that estrogens increase the amount of noradrenergic α1b receptors in hypothalamus. In addition, Victoria Luine, at Hunter College, reported that estrogens decrease the activity of monoaminoxidase, an enzyme that degrades norepinephrine. In parallel, when recording from individual neurons in the VMH, Lee-Ming Kow and I had two results: that an α1 receptor agonist would increase electrical activity, and the estrogens would increase the magnitude of responses to norepinephrine (Kow and Pfaff 1987, 1995; Kow, Weesner, and Pfaff 1992; and with patch clamp, Lee et al. 2008). Thus, to summarize, (a) estrogens heighten lordosis behavior, and (b) estrogens also increase noradrenergic α1b receptors and electrophysiological responses to noradrenaline in VMH neurons. Therefore, it follows that (c) one way in which estrogens facilitate reproductive behavior is to increase adrenergic receptors and electrical responses to noradrenaline by VMH neurons.

H I S TA M I N E

Stephano and Donoso had shown that bathing the hypothalamus in histamine (HA) would increase lordosis behavior, so we wanted to use electrophysiological techniques to mea sure responses of VMH neurons

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to HA. Years ago our laboratory was blessed with the arrival of Jin Zhou, M.D., who had placed fi rst in her class at the medical school in Beijing, People’s Republic of China. Jin used patch-clamp techniques to demonstrate that VMH neurons had excitatory responses to HA and that estrogens would potentiate HA actions in a way that would explain the positive effects of HA on lordosis (Zhou et  al. 2007). Christophe Dupré went farther to explore the ionic basis of the HA effect (Dupré et  al. 2010). Lee-Ming Kow would use the patch- clamp recording of VMH neurons with a variety of ion substitution and pharmacological maneuvers to explore in greater detail exactly how HA depolarizes these neurons in a way that would foster lordosis behav ior (Kow et al. 2016). First, as Christophe Dupré had reported, H1 receptors are involved. Second, neither sodium currents nor calcium currents were necessary. In detail, Chris’s results showed that HA acting through H1 receptors depolarizes these neurons. Further, acute administration of estradiol, an estrogen necessary for lordosis behavior to occur, heightens this effect. Hyperpolarization, which tends to decrease excitability and enhance inhibition, was not affected by acute estradiol or mediated by H1 receptors but was mediated by the other HA receptor subtypes H2 and H3. Sampling of messenger RNA (mRNA) from individual VMN neurons showed colocalization of expression of H1 receptor mRNA with estrogen receptor α (ER- α) mRNA but also revealed ER colocalization with the other HA receptor subtypes and colocalization of dif ferent subtypes with each other. The latter finding provides the molecular basis for complex push-pull regulation of VMN neuronal excitability by HA. Thus, in the simplest causal route, HA, acting on VMN neurons through H1 receptors, provides a mechanism by which elevated states of generalized central ner vous system arousal can foster a specific estrogen-dependent, aroused, sexual behavior. Ion channels. To explore potassium (K+) ion channels using a blocking approach, cesium (Cs+, 2 mM), and 4-aminopyridine (5 mM), which block fast-acting channels such as the A-current, were added to the channel blocker tetraethylammonium. With this combination, HA depolarization was essentially abolished. This result shows that K+ currents are essential for mediating HA depolarization.

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Then, to determine whether K+ currents are also sufficient to support HA depolarization, VMH neurons were tested in a “K+ -only” environment, where K+ was the only permeable cation in both internal and external solutions. HA depolarized VMH neurons, demonstrating that K+ was sufficient. Thus, K+ currents are both necessary and sufficient for HA depolarization. A reasonable inference is that depolarization caused by HA is due mainly to the inhibition of K+ currents. Moreover, estrogens can rapidly potentiate depolarization induced by HA, applied either through the bath or by pico-spritzing. And hormonal specificity is further confirmed by the experiments using the estrogen agonists PPT [(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol] for ER- α and DPN [2,3-bis-(4-hydroxy-phenyl)-propionitrile] for ER-β. PPT acted like estradiol to potentiate depolarization in VMH neurons, whereas DPN had no significant effect. I inferred that the rapid potentiating action of estradiol was mediated via ER- α. To summarize, (a) estrogens heighten lordosis behav ior, and (b)  estrogens also increase electrophysiological responses to HA depolarizations in VMH neurons. Therefore, it follows that (c) one way in which estrogens facilitate reproductive behavior is to increase excitatory responses to HA by VMH neurons.

G L U TA M AT E AG O N I S T

NMDA. As the most common excitatory neurotransmitter in the brain, it would be expected that the glutamate receptor agonist Nmethyl-D -aspartate (NMDA) would depolarize VMH neurons. Indeed, Lee-Ming Kow demonstrated that NMDA, whenever effective, evoked only depolarizations in these neurons. NMDA’s evocation of depolarization also had five characteristics: 1) reduced the latency to action potential, 2) increased the action potential number, 3) increased relative depolarization, 4) increased the depolarization rate, and 5) lowered the action potential threshold (Kow et al. 2016). Estradiol can rapidly potentiate the depolarization induced directly by bath-applied NMDA by pico-spritzed NMDA applied at an appropriate holding potential. Under these circumstances, microinjections of NMDA should be able to increase lordosis behavior, but the initial data on this subject

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were mixed and complex. The most confusing aspects of NMDA would include the extremely rapid desensitization of its receptors and its ability to affect nearby inhibitory neurons. Finally, Jose Bueno, a Spanish neurologist in my laboratory for a year, showed that estrogenic treatment can increase the spontaneous activity of a subset of VMH neurons, those with extremely low firing rates. These neurons, likewise, would be expected to facilitate lordosis behavior.

Estrogen-Dependent Hypothalamic Outflow Regulation of Lordosis Behavior Early in Yasuo Sakuma’s work at Rockefeller, we decided to find out which of the estrogen-binding cell groups is necessary and sufficient for lordosis behavior. As noted previously, estrogen administration limited to the VMH permits lordosis behavior and antiestrogen administration limited to the VMH prevents lordosis behavior. To accompany those findings, Yasuo Sakuma electrically stimulated the VMH (Pfaff and Sakuma 1979b) and electrically destroyed the VMH (Pfaff and Sakuma 1979a). In the first study, electrical stimulation of the VMH significantly increased lordosis in response to the appropriate cutaneous stimuli (Figure 2.1). This effect was significant in that electrical stimulation in the preoptic area, another estrogenbinding cell group, had exactly the opposite effect. The stimulus currents were low (12.5 microamps), the optimal stimulus frequencies were low (between ten and thirty pulses per second), and the time course of behavioral facilitation featured latencies as little as 15 minutes with peak behavior occurring at about 1 hour. Pretreatment with estrogen was absolutely necessary for the behavioral effect of stimulation, and the best lordosis responses to electrical stimulation came from the part of the VMH that has a high concentration of estrogen-binding neurons. Second, what about destroying VMH neurons? Bilateral lesions led to a decline of lordosis to near-zero levels within forty- eight hours, and then partial recovery after about three weeks. Lesions never abolished all of the VMH. At Emory University, Ann Clark and David

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Lordosis behavior score Lordosis behavior score

Time (hr)

Dose of estrogen per animal ( μg)

Figure 2.1. Top: Electrical stimulation of neurons in the ventromedial nucleus of the hypothalamus (VMH) potentiated lordosis behavior, the duration of the effect depending on the length of stimulation. Bottom: Estrogen pretreatment potentiated the effect of VMH stimulation (Stim) on behavior compared with the unstimulated control (Pre) as a function of estradiol dose. (Adapted from Pfaff and Sakuma 1979b.)

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Edwards extended this type of work using not only electrolytic lesions but also knife cuts of outgoing VMH fibers. Using these approaches they showed massive decreases in the percentage of time lordosis occurred in response to the male (e.g., 94 to 16  percent, and 88 to 7 percent). Also, the lesions and knife cuts severely reduced the precopulatory courtship behaviors by the female. Kirk Manogue, a Rockefeller graduate student working with LeeMing Kow and myself, showed that knife cuts interrupting ascending and descending fibers at the intercollicular level could eliminate lordosis. Kirk followed that up by using knife cuts that interrupt those VMH fibers that converge with the ascending part of the lordosis circuit at the MCG (Manogue, Kow, and Pfaff 1980). If both the pathways that sweep lateral and dorsally as they head to the central grey and the medial, periventricular pathway were cut, lordosis behavior was abolished. Blocking only the medial pathway did not work. Kirk interrupted the lateral pathway using two dif ferent methods. Parasagittal transections at the level of the VMH showed that the lateral pathway was not required for lordosis if and only if the medial pathway to the central grey was intact. Selective parasagittal cuts farther posterior showed that among the lateral trajectories toward the central grey no particular subset was crucial for the behavior. As long as a sufficient percentage of the fibers remained unharmed, lordosis behavior could occur. The main features of the results lent themselves to the interpretation that the VMH provides a tonic estrogen-dependent facilitation of supraspinal mechanisms essential for producing lordosis, mechanisms located from the midbrain down to the medullary reticular formation. David Edwards and Jill Pfeifle at Emory University replicated and extended these results. Their extensive parasagittal knife cuts lateral to VMH abolished lordosis, as ours had. Then they used clever combinations of hypothalamic and midbrain cuts to show that the sweeping trajectory of VMH axons going toward MCG, earlier described by Lilly Conrad’s neuroanatomy studies, was the fiber trajectory required for normal lordosis behavior. What about the MCG itself? We used combinations of electrical stimulation and electrolytic lesions applied through the same electrodes to piece together the story (Sakuma and Pfaff 1979a,b). Electrical stimulation almost tripled lordosis per for mance (Figure  2.2). The

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Figure 2.2. Neurons in the midbrain central grey, having received estrogen- dependent signals from the ventromedial hypothalamus, facilitate lordosis behavior. Top: Stimulation of neurons in the midbrain central grey rapidly elevated lordosis behavior in the female rat. Greater electrical pulse amplitude was associated with higher levels of lordosis. Bottom: Estrogen pretreatment potentiated the ability of central grey (CG) electrical stimulation to facilitate lordosis, compared with the unstimulated control (Pre), as a function of estradiol dose. (Adapted from Sakuma and Pfaff 1979a.)

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maximum effect was evident in the very fi rst behavioral test after the beginning of stimulation, at about 5 minutes, and lasted as long as the stimulation was on. Lesions markedly interfered with lordosis, studied in three ways. First, they abolished lordosis, but after some hours behavior returned to about one-third its prelesion level. Second, lesions abolished the effect of electrical stimulation on the behavior, both applied through the same electrodes. Third, and most important, central grey lesions blocked the facilitatory effects of VMH stimulation, demonstrating the role of the central grey in linking estrogen-dependent signals to the rest of the lordosis behavior circuit. Lesion effects were specific in that destruction too far dorsal, posterior, or lateral were not effective. Pfeifle and Edwards followed our work with a dif ferent kind of behavioral assay and reached the same conclusion: lesions that interrupted fibers descending from the central grey toward the hindbrain reduced lordosis performance to zero. Thus, MCG neurons, especially those along the lateral border which are clearly competent to receive hypothalamic inputs as shown by Sakuma’s other studies, can facilitate lordosis; and their destruction severely reduces lordosis behavior. How do central grey neurons exert their behavioral influence? This analysis will be discussed later.

Sensory Inputs: How the Behavior Is Initiated Only somatosensory stimuli are involved. Vision, audition, olfaction, and taste are not necessary. Cathy Lewis, who worked in my laboratory for several years before she took up an academic career in chemistry, used frame-by-frame film analyses set against a history of ethological descriptions of reproductive behavior to determine the sensory stimuli necessary and sufficient for lordosis behavior (Pfaff and Lewis 1974). The entire chain of hormone-dependent behaviors will be covered in Chapter 7, but suffice it to say here that a competent male rat or mouse is almost always following the female, approaching from the rear. When he mounts, his forepaws contact her flanks, rump, and tail base. Then his initial pelvic thrusts will press against the skin of her perineum. The female’s vertebral

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dorsiflexion, the behavior essential for fertilization, starts about the time of the first thrust. The exact points of contact between the male and female were charted by coating the ventral surface of male rats with a dye. The flanks just in front of the female’s rear legs, the rump, the tail base, and the perineum of the female were most intensely marked (Pfaff, Montgomery, and Lewis 1977). Somatosensory, cutaneous inputs from these regions are most obviously involved. Other sensory modalities do not seem to be necessary. Subcutaneous injections of the local anesthetic procaine into these regions of the female’s skin virtually eliminate lordosis responses. However, the highest doses of estrogen could reduce the magnitude of the procaine effect. There was a hormone dose / sensory input trade-off. Likewise, surgical denervation of the female’s skin areas contacted by the male, most obviously the severing of the pudendal nerve (innervating the perineum) markedly reduced lordosis behavior (Kow and Pfaff 1976). Thus, cutaneous stimulation of the skin regions contacted by the male is necessary and sufficient for lordosis. Quantitative determination of cutaneous stimulus parameters that are sufficient showed that simple hair deflection is not sufficient but that light pressure on the female’s skin in the range of 50 to 450 millibars would lead to the behavior reliably (Kow, Montgomery, and Pfaff 1979). Increasing the dose of estrogen increased the probability of lordosis behavior to a given pressure of stimulus. The next step was to record from individual neurons in the dorsal root ganglia at lumbar levels, which cover the appropriate cutaneous area effective for lordosis (Kow and Pfaff 1979). Mapping the receptive fields of neurons in all lumbar dorsal roots revealed an orderly pattern proceeding posteriorly from L1 (fl anks) through L6 (tail base and perineum) (Kow and Pfaff 1975). The primary somatosensory neuron type responsible for triggering the onset of lordosis behavior was determined by a process of elimination. Noncutaneous units, unresponsive neurons, and neurons primarily responsive to hair deflection could not be crucial. Instead, dorsal root ganglion neurons responsive to very low pressure, especially sustained pressure (as opposed to punctuate skin deformation) should be involved, so-called type II neurons. These are associated with Ruffini endings in the skin. Thus, pressure in the L6

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receptive field of the female deforms Ruffini endings leading to an activation of type II neurons, which yield the input to the lumbar spinal cord effective for the sensory stimulation of lordosis behavior. In the spinal cord as well, neurons in the dorsal horn at lumbar levels showed strong responses to the types of cutaneous stimuli on the flanks and perineum that in the unanesthetized female rat would elicit lordosis. The big difference with dorsal root ganglion neurons was that, while the latter were absolutely silent in the absence of stimulation, dorsal horn neurons often had spontaneous activity (Kow, Zemlan, and Pfaff 1980). Among the dorsal horn neurons excited by lordosis-relevant cutaneous stimulation, some would show a sudden, stepwise increase in firing as soon as pressure was applied to the skin; others increased their firing gradually, tracking a steady increase of pressure on the skin. We were very pleased to see effects of estrogen treatment on sensory responses in this system. At the level of the primary sensory neuron, Lee-Ming Kow mea sured the receptive field size of the pudendal nerve, the electrical activity of which would obviously be important for causing the vertebral dorsiflexion of lordosis; it covers the perineal and hind leg skin area contacted by the male (Kow and Pfaff 1973). A large enough number of animals tested and the precise methodology used allowed the 22  percent increase caused by estrogen treatment (compared to estrogen-free, ovariectomized controls) to be highly statistically significant. At the level of the dorsal horn of the spinal cord as well there potentially lies the cellular basis for an estrogen effect on sensory processing as Joan Morrell found small numbers of estrogen-binding cells there (Morrell et al. 1982).

Ascending Pathways: How Behaviorally Relevant Sensory Information Gets Transmitted to the Brain Spinal circuitry is not adequate, not sufficient for lordosis behavior to be performed; however, sophisticated sensory-motor circuitry at each spinal cord level might be. We prepared female rats with complete transections of the spinal cord at low thoracic levels (Kow, Montgomery, and Pfaff 1977). Good postoperative care kept these animals in amazingly good physical condition (for example, they were voided, cleaned, and fed if necessary)— only eighteen of the ninety-two rats operated on

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showed a deteriorating condition. Even subtotal transections of the spinal cord could eliminate lordosis. For example, spinal operations that left the dorsal columns intact or the dorsolateral columns or the ventromedial columns intact did not permit lordosis behavior to occur. If the anterolateral columns, containing the ascending spinoreticular system, were left intact, then we could observe lordosis. In contrast, if much of cord was left intact but the anterolateral columns were badly damaged, then percentage of lordosis performance was low or zero. We concluded that supraspinal control is required for normal lordosis behavior, and that fibers necessary and sufficient for lordosis run in the anterolateral columns. For ascending sensory signaling, that would include spinoreticular fibers. (For descending systems that would mean vestibulospinal and reticulospinal systems.) The distribution of these fibers ascending from the spinal cord is quite clear. Anne Robbins, under the leadership of Susan SchwartzGiblin in my laboratory, microinjected the retrograde neuroanatomical tracer Fluoro- Gold among the cells in the medullary reticular formation, the large cells called nucleus gigantocellularis (NGC). Significant numbers of spinal cord cell bodies were backfi lled in the deep layers of the dorsal horn, near the central canal and even in the ventral horn. They were found both ipsilateral to the microinjection and contralateral. Control injections far lateral to NGC yielded only an extremely small number of backfi lled cells in the spinal cord (Robbins, SchwartzGiblin, and Pfaff 1990). This neuroanatomical result matched well the electrophysiological results from extracellular single neuron recordings by Lee-Ming Kow (Kow and Pfaff 1982). In anesthetized female rats we were able to fi nd a large number of NGC neurons (among the 742 cells studied) that responded to the type of cutaneous stimulation that elicits lordosis behavior—pressure on the flanks, rump, and tail base. Recording in unanesthetized animals was harder—the rats had to be surgically prepared with implanted “floating” wire electrodes. Nevertheless, of the hundred and eighteen cells we could study, forty-two responded. Estrogen pretreatment of the females studied under anesthesia tended to facilitate the excitability of these medullary reticular neurons and to increase the ratio of the number of cells excited by lordosis-relevant cutaneous stimulation to the number of cells inhibited.

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In turn, hindbrain reticular neurons project to the midbrain. Joan Morrell used the retrograde neuroanatomical horseradish peroxidase technique with initial injections in the dorsal midbrain, which covered the dorsal mesencephalic central grey and adjacent midbrain reticular formation including the neuronal areas contacted by ventromedial hypothalamic outflow (mentioned above and covered below) (Morrell and Pfaff 1983). Considerable numbers of cells in the medullary reticular formation were backfi lled, and even some of the axons of some neurons in the lumbar spinal cord made it up to the dorsal midbrain. This illustrates is the ladder-like character of the ascending side of the lordosis behavior neural circuit: spinal cord projects to the region of NGC, and NGC projects to dorsal MCG and reticular formation; some spinal neurons project all the way to the dorsal midbrain. Joan Morrell’s neuroanatomical results are complemented by the electrophysiological data of Yasuo Sakuma, the premier Japa nese neuroendocrine physiologist (Sakuma and Pfaff 1980a,b). Yasuo recorded neurons in the MCG and in the mesencephalic reticular formation just lateral to central grey. Large numbers of such neurons responded to lordosis-eliciting cutaneous stimulation (fifteen times as many excitatory responses as inhibitory responses) and also responded to electrical stimulation of medullary reticular NGC (seven times as many excitatory responses as inhibitory responses). What was most exciting was that many also responded to electrical stimulation of the ventromedial hypothalamus (four times as many excitatory responses as inhibitory responses). Thus, in terms of lordosis behavior-circuit building, we got convincing evidence of convergent effects of lordosis-relevant somatosensory and ventromedial hypothalamic influences on central grey cells in the female rat mesencephalon. A side point: at several points in the circuitry for lordosis behavior it is clear that not all sensory information is transmitted. As receptive fields grow larger, for example, from primary receptors to higher brain regions, spatial information is reduced. Apparently, for this type of neural circuitry, if a certain amount and form of sensory information is required for a behavior’s regulation, the nerve cell in question receives and responds to it. If not required, the sensory information is less precise or the nerve cell in question gets none at all. Clearly, this arrangement reflects an economy in the use of nerve cell signaling capacities.

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Thus, to meet the estrogen-dependent signals emerging from the hypothalamus, lordosis-relevant sensory information reaches neurons in and just lateral to the MCG (and its adjoining mesencephalic reticular formation). The central grey integrates hypothalamic neuroendocrine signals with brainstem sensory-motor physiology.

Descending Pathways How do central grey neurons exert their behavioral influence? Yasuo Sakuma used antidromic stimulation techniques while recording from cells of origin of descending projections (Sakuma and Pfaff 1980b,c). The stimulating electrodes were placed in the nucleus gigantocellularis (NGC) of the medullary reticular formation, while recording microelectrodes sampled the electrical activity of cell bodies in the MCG. Antidromic spike latencies ranged from 2 to 20 milliseconds, indicating a range of nerve fiber dia meters. Central grey neuronal cell bodies thus identified in these experiments (n = 82) were found near the lateral borders of the central grey in positions that appeared clearly to be in contact with axons descending from the hypothalamus. Most exciting, Yasuo discovered that the electrical activity of some of these central grey neurons is influenced by estrogen treatment and by VMH stimulation (Sakuma and Pfaff 1980b,c). Electrical responsiveness was mea sured by the ability of the antidromic action potential (these neurons project to the medullary reticular formation and thus are likely of a high degree of relevance to lordosis behavior) to propagate from the descending axon into the somatodendritic complex. This spike invasion happened almost twice as often in the estrogen-treated female rats compared with the ovariectomized, estrogen-free controls. Further, there were more neurons with high rates of resting discharge in the estrogen-treated group. Important for the regulation of excitability in the lordosis circuit, we studied the ability of low-intensity VMH stimulation to influence central grey cells that project to the NGC. Routinely, VMH stimulation markedly facilitated antidromic spike invasion to the cell body of central grey neurons, thus facilitating the excitability of those neurons that project down to the NGC (Sakuma and Pfaff 1980b).

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Studies I will describe show that the most important descending systems for regulating and stimulating lordosis are the reticulospinal system and the vestibulospinal system. Rockefeller doctoral student Sandra Cottingham discovered that central grey stimulation facilitates both systems. The most important features of both systems have to do with their elevation of excitability of the motor neurons that cause contraction of the deep back muscles that cause lordosis behavior. Cottingham placed monopolar electrodes in the lateral vestibular nucleus, the origin of a major vestibulospinal tract, and showed that stimulation there could cause electromyographic signals in the deep back muscles that run lordosis behavior (Cottingham and Pfaff 1987). Then, preparing for the critical experiment, she decreased the amount of current going into the lateral vestibular nucleus so that it could not activate deep back muscles. Superimposed upon that lower amount of current, stimulation of the MCG reinstated the ability of vestibulospinal to activate electrical responses in the deep back muscles. Effective stimulation points were in the lateral central grey; if we missed that midbrain target, no potentiation of the vestibulospinal system was possible. Ann Robbins, working with a long-time leader in the laboratory, Professor Susan Schwartz- Giblin, followed up Cottingham’s results with neuroanatomical studies (Robbins, Schwartz- Giblin, and Pfaff 1990). The retrograde fluorescent tracer Fluoro-Gold was microinjected into NGC; after a survival time of four days, Ann identified numerous cells in the central grey—both ipsilateral and contralateral to the NGC site of application—as projecting to NGC. These presumably supply the neuroanatomical basis of Cottingham’s findings. Thus, in a hierarchical fashion, the cell group in the MCG that receives estrogen-dependent input from the hypothalamus revs up the ability of a lower hindbrain center, the lateral vestibular nucleus, to activate the motor neurons that run lordosis behav ior. I further inferred that our reproductive behavioral system had co- opted a regular postural control system—the lateral vestibulospinal tract—in order to produce the female’s behav ior required for fertilization and reproduction. Then Cottingham used similar strategies for the medullary reticulospinal system, including those descending influences emanating from

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the NGC (Cottingham, Femano, and Pfaff 1987). In the medullary reticular formation, her best sites for stimulating the motor neurons of lordosis-relevant axial muscles were in and lateral to the NGC. Ineffective control sites in her work tended to be far lateral or at the very bottom of the brain. As earlier, she set up her experiments by calibrating the stimulus current strength. For example, in one experiment the mesencephalic central grey stimulation itself gave a very small activation of axial muscle motor neurons as mea sured by axial muscle electromyography, the key to lordosis behav ior. An NGC stimulus train of 50 microamps amplitude at a frequency of two hundred pulses per second yielded a larger axial muscle activation. The crucial experiments took place when she superimposed mesencephalic central grey stimulation on NGC stimulation and recorded a vast amplification of electromyographic activity. A key example would be when this combination central grey- on-NGC phenomenon worked on motor neurons for the axial muscle lateral longissimus, a key muscle in executing lordosis behav ior. Thus, the central grey of the midbrain plays an important role in relaying hormone-dependent hypothalamic information to the medullary reticular formation, which in turn has direct access to the motor neurons essential for lordosis behavior.

Lower Brainstem The reticulospinal link in the lordosis circuit was characterized extensively by Philip Femano, who had come to our laboratory from Rutgers and introduced us to computer analyses of neurophysiological data (Femano, Schwartz- Giblin, and Pfaff 1984a,b). Femano worked under the leadership of Susan Schwartz- Giblin who later became dean of the State University of New York (SUNY) medical school. Fermano anesthetized rats with urethane and used bipolar stimulation methods that were designed to minimize the spread of current, thus to define most precisely the NGC source of the effect on the deep back muscles that execute lordosis. Currents in the range of 15 to 40 microamps were typical. Under these conditions he discovered that all axial, deep back muscles could be activated by NGC stimulation: muscles named transversospinalis, medial longissimus, and lateral longissimus.

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The temporal aspects of the deep back muscle activation were interesting. With the low currents used, a single pulse in NGC was not effective, but a train of pulses always was. NGC circuitry could handle stimulation frequencies as high as two hundred pulses per second. The deep back muscles (and thus their motor neurons) could respond steadily to train after train after train of pulses; in fact, latencies to the onset of muscular activation tended to go down as train numbers increased. The minimal onset latency to the electromyographic activation of lateral longissimus was 12 milliseconds. The real-life physiological mode of this effect was analyzed by Rockefeller graduate student Mark Cohen (Cohen, Schwartz- Giblin, and Pfaff 1987a), working with Schwartz- Giblin. We knew that short trains of electrical stimuli to the pudendal nerve could evoke clear- cut responses in the axial muscle motor neurons essential for lordosis. The magnitude of the motor neuron response is drastically reduced by spinal transaction, and the fibers responsible for the supraspinal influence travel in the lateral columns, precisely as do the fibers responsible for facilitating lordosis behavior as a whole. Therefore, Mark asked the question, might NGC stimulation have its effect on the behavior primarily by enhancing the throughput from a lordosis-relevant sensory stimulation to the motor neurons essential for lordosis? Mark set up the experiment this way. Pudendal nerve stimulation intensity was adjusted so that, by itself, it gave only a small increase in the electrical response recorded in the lumbar-level nerves leading to the deep back muscle lateral longissimus. Likewise, NGC stimulation was calibrated such that hardly any electrophysiological response was seen in the nerve for this deep back muscle. The crucial experiment came when the two types of stimuli were combined (Figure  2.3). The answer was that concurrent stimulation of NGC combined with the pudendal nerve led to a very high motor nerve response, about ten times the number of spikes of either stimulus site alone. Thus, we understand how, in the lordosis circuit, NGC neurons (which themselves had received estrogen-dependent inputs from the ventromedial hypothalamus via the MCG) can greatly amplify lordosis-relevant motor neuron responses to lordosis-relevant stimulus inputs. Lateral vestibular nucleus stimulation (Modianos and Pfaff 1977) also facilitates lordosis. Low-intensity (10 microamps) stimulation was

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Figure 2.3. Electrical stimulation of fibers descending from the medullary reticular formation (horizontal black bars) greatly potentiates deep back muscle (for lordosis behavior) responses to segmental (sensory) inputs from the pudendal nerve (diagonally striped bar). (Adapted from Cohen, Schwartz- Giblin, and Pfaff 1987a.)

delivered bilaterally at two hundred pulses per second. The magnitude of the lordosis increase varied from female to female, but we knew we had a phenomenon; the increase during bilateral stimulation usually was a doubling of lordosis strength and in some cases much more. Unilateral stimulation also worked and yielded just as large a behavioral facilitation as bilateral stimulation. Follow-up tests showed that the magnitude of lordosis facilitation was an orderly increasing function of microamps per pulse (beginning at 0.5 microamps) and pulses per second (studied from ten through four hundred). When the lateral vestibular nucleus stimulation was turned off, lordosis mea sures returned to control levels. Conversely, lesions of the lateral vestibular nucleus led to marked loss of lordosis (Modianos and Pfaff 1976). The degree of behavioral decrement was an orderly (declining) function of the number of giant cell loss in the nucleus, and the effect was specific because lesions of the superior or the medial vestibular nucleus did not harm lordosis performance. Likewise, cerebellar lesions did not cause lordosis decrements. In turn, vestibulospinal and reticulospinal pathways (which we just covered here individually) interact with each other (Cottingham, Femano, and Pfaff 1988). They synergize. During these experiments, Cottingham had either monopolar or bipolar electrodes in the lateral

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vestibular nucleus as well as bipolar electrodes (twisted 75-micron tungsten wire) in the medullary reticular formation. Recording electrodes were in the deep back muscles essential for lordosis per formance ref lecting, obviously, the excitation of their respective motor neurons. As in previous experiments, Cottingham set up the experiments by using currents that were effective but not overwhelming. In a typical experiment, therefore, lateral vestibular nucleus stimulation would cause some motor neuron and muscle activation, but at a low level. Reticular formation stimulus, by itself, was the same. The combination of simultaneous vestibular and medullary (primarily NGC) stimulation vastly increased deep back muscle activity, for example, by more than ten times either of them by themselves. Thus, two major brainstem neuron groups, each of which receives descending inputs from central grey in the lordosis circuit, massively amplify each other’s actions to stimulate the motor neurons and thus the muscles involved in lordosis. What happens when we damage medullary / spinal pathways? The first experiments were very encouraging: NGC lesions reduced lordosis to about 20 percent of preoperative values (Modianos and Pfaff 1976, 1979). Then Frank Zemlan placed electrolytic lesions in NGC, the largest of which damaged virtually all of the NGC on both sides (Zemlan, Kow, and Pfaff 1983). Such lesions brought the occurrence of lordosis down to less than 40 percent of its prelesion values, and eventually, after about three weeks, lordosis recovered. Zemlan interpreted these data as demonstrating that large NGC lesions interrupted descending control mechanisms important both for the initiation and maintenance of lordosis behavior, descending tracts that he had shown run through the lateral columns of the spinal cord (Zemlan et al. 1979). So as we approach the motor neurons for lordosis, this summary of the evidence applies: Necessary and sufficient for lordosis are an estrogen-dependent signal coming from the VMH to the MCG and the reticular formation just lateral to it. Stimulation of central grey neurons potentiates both the NGC and the lateral vestibular nucleus to activate the motor neurons for the deep back muscles. Indeed, stimulation of each of these two cell groups increases lordosis, and lesions of each of these two cell groups decrease or abolish lordosis.

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Motor Mechanisms: How the Behavior Is Finally Produced Of course, the first step in piecing together the motor response side of the lordosis circuit was to record from the motor neurons whose excitation causes contraction in the deep back muscles that execute lordosis. Rockefeller graduate student Emily Brink did exactly that (Brink and Pfaff 1981). She identified the axons of the motor neurons that control the medial longissimus and lateral longissimus muscles, crucial for lordosis, and fi rst showed that they responded to segmental inputs, namely, to stimulation of the appropriate lumbar level dorsal roots. Then she used medullary reticular formation stimulation and systematically varied the time between its application and the dorsal root stimulation. If the dorsal root (segmental) stimulation preceded reticular (NGC) stimulation, there was no synergy. But in the series of intervals from simultaneity through 1.5 milliseconds, the NGC stimulation increased many-fold (Figure 2.4); the raw data look as though it is between five times and 10 times the facilitation. For example, if the NGC stimulation preceded the dorsal root stimulation by 1 millisecond, the lateral longissimus motor neurons had a much more amplified output to dorsal root stimulation than if the dorsal root stimulation alone was used. Similar data came from vestibular nucleus stimulation. If the vestibulospinal stimulation preceded the dorsal root stimulation by between 1 and 8 milliseconds, the motor neuronal response to segmental stimulation was greater, with maximal facilitation at the intervals 2 to 5 milliseconds. Then she put it all together—that is, Brink used the conditioning before segmental stimulation in the MCG which, of course, does not have axons projecting all the way to the lumbar deep back motor neurons and must (as we saw earlier) be working through NGC and the vestibular nuclei. For intervals of 10 milliseconds through 100 milliseconds, prestimulation in the MCG increased the motor neuronal response to segmental stimuli a bit more than three times (Brink and Pfaff, 1981). Thus we have the MCG (which receives the estrogen-dependent VMH signals) working through the NGC and vestibulospinal nuclei to facilitate the lordosisessential deep back motor neuronal responses to the segmental inputs.

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Figure 2.4. Conditioning medullary reticular stimulation, activating reticulospinal axons, greatly potentiated the ability of sensory, segmental stimulation to drive activity in axial muscle motoneurons essential for lordosis behavior. (Adapted from Brink and Pfaff 1981.)

These motor neurons are huge. Classically triangular shaped, they are found at or very near the most ventral extent of the ventral horn at lower lumbar and upper sacral levels (Brink, Morrell, and Pfaff 1979). Their locations were discovered using the horseradish peroxidase technique and confirmed by scanning for sites at which microstimulation produced visible twitches of the deep back muscles. Locations of the lowest threshold twitch sites were consistent with conclusions based on the horseradish peroxidase findings. These are the motor neurons whose activity produces lordosis behavior (Figure 2.5). As a side point, I note that the reticulospinal neurons later studied with extracellular single neuron recording by Lee-Ming Kow (Kow and Pfaff 1982) could respond to the type of cutaneous stimulation that produces lordosis. Thus, in the lordosis behav ior circuitry, one of the spinal-brainstem-spinal loops runs through NGC. If you take away the motor neuron-facilitating supraspinal descending tracts (Cohen, Schwartz-Giblin, and Pfaff 1987b), the pudendal nerve-evoked excitation of deep back motor neurons is severely reduced. For example, total transaction of the spinal cord at the fourth thoracic level almost abolished the motor neuronal response. After all of a variety of partial transaction surgeries (except those of the lateral columns) the pudendal nerve-evoked response was like that of the control, unoperated animals. However, if the lateral columns were transected, the motor neuron response was virtually abolished, just as with the total transaction. This makes sense because we know that the descending reticulospinal and vestibulospinal tracts run in the lateral column.

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Figure 2.5. Locations of cell bodies of motor neurons essential for generating lordosis behavior, in the ventromedial corner of the ventral horn in lumbar spinal cord. (Adapted from Brink, Morrell, and Pfaff 1979.)

Thus, descending facilitation originating in the brainstem’s NGC and vestibular nuclei are both sufficient and necessary to permit and  amplify the sensory-motor lordosis-producing connection in the lumbar spinal cord. Further, considering early (short latency) and later (long, 50- to 120-millisecond latency) deep back muscular responses to lordosis-eliciting cutaneous stimulation, estrogen pretreatment greatly (four times) increased the magnitude of the later component. I infer that the hypothalamic estrogen effect has been “read out” through central grey and thence reticulospinal and vestibulospinal systems to empower the sensory-motor lordosis machinery at lumbar levels.

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Behavior Quantified What about lordosis behavior itself, the female behavior essential to permit fertilization? First we used high-speed fi lms of lordosis behavior to time and define the female’s vertebral dorsiflexion—necessary for fertilization and all subsequent steps of reproduction—in response to cutaneous contact from the male’s mounting (Pfaff and Lewis, 1974). But because in conventional movie fi lming the hair, skin, fat, and other tissues reduce the precision with which the skeletal movements that constitute the physical basis for the behavioral response can be followed, we pursued the analysis of the behavior by collaborating with X-ray cinematographic expert Farish Jenkins at Harvard (Pfaff et  al. 1978). Of course, the most striking change at the beginning of the behavior is the rapid elevation of the rump from a convex-up posture toward horizontal and then, at the peak of lordosis, 30 degrees into a concave-up posture. This behavioral onset is triggered initially by the male’s paws on the receptive female’s flanks, but the behavior itself is strengthened in response to the male’s pelvic thrusts. To appreciate the muscles whose contractions constitute the lordosis response to male mounting, Susan Schwartz- Giblin worked with Michael Chen in the Electronics Laboratory at Rockefeller University to invent a strain gauge and electromyogram amplifier that could be used in unrestrained female rats (Schwartz- Giblin and Pfaff 1980). These recordings revealed that deep back muscles were contracting with high levels of electromyographic activity at the beginning of lordosis, but if the vertebral dorsiflexion of lordosis was sustained (e.g., for 4 seconds) the motor units were actually not showing high activity throughout (Schwartz- Giblin, Halpern, and Pfaff 1984). This method of recording confi rmed the effectiveness of light cutaneous stimulation on the flanks followed by pressure on the female’s perineum in triggering long and strong lordoses. The muscles involved are interest ing (Brink and Pfaff 1980). Not to be thought of as a single muscular “motor” that causes the vertebral dorsiflexion of lordosis behav ior, they are divided by region (thoraciclumbar and sacrocaudal) and by type. For example, the longissimus system includes the lateral longissimus and medial longissimus, both of which get their muscular power from their tendons’ insertions onto

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the vertebrae. Medial longissimus, as the name implies, lies between the lateral longissimus and the most medial system, the transversospinalis muscles. Further, these systems have short fiber components as well as long fibers. Most interest ing of all, when stimulated unilaterally they cause a lateral deflection to the side as well as  a dorsal movement. When stimulated bilaterally, the two lateral deflections cancel each other out, and we are left with a strong dorsiflexion. Thus, lordosis behav ior is a bilaterally balanced system in an obligatory manner. Lateral longissimus, the largest component of the dorsal vertebral musculature, was studied further using histochemical techniques to figure out the sources of metabolic energy used for a strong lordosis response that even supports the weight of the male (Schwartz- Giblin, Rosello, and Pfaff 1983). Among rat mating encounters, frame-byframe fi lm analyses could show a 225- gram female rat performing lordosis behavior and supporting the entire weight of a 500-gram stud male that was mounting with all four feet were raised off the substrate. Thus, we determined for lateral longissimus its fiber composition, fiber size, muscle spindle distribution, and, most importantly, the distribution of fast-twitch-glycolytic (FG) fibers, fast-twitch oxidative-glycolytic (FOG) fibers, and slow-twitch oxidative (SO) fibers. Lateral longissimus contains predominantly FG fibers; SO fibers were concentrated superficially in the L2–L6 region. We inferred that for the forceful, ballistic movement of the vertebral column during lordosis behavior, fast motor units with large tension strengths would be required, and precise control (as might be expected from slow muscle units with regulation by muscle spindles) would not be required. As an example of these deep back muscles, strong contraction of lateral longissimus is necessary and sufficient for lordosis. Dorsal root stimulation evoked a electromyographic response with latencies between 1 and 2.5 milliseconds, consistent with the latency of a monosynaptic reflex (Schwartz- Giblin, Femano, and Pfaff 1984), but polysynaptic responses predominated and had both early and late discharges (the latter enhanced by estrogen injections to the female). Conversely, as expected, surgical ablations of deep back muscles by clipping away the muscles from their tendinous attachments abolished lordosis (Brink, Modianos, and Pfaff 1980).

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Summary: The Circuitry Is Hierarchical and Modular Summarizing the discussion thus far, Figure 2.6 shows the lordosis behavior circuit, a social behavior essential for reproduction, the fi rst neural circuit determined for any vertebrate behavior (Pfaff and Schwartz- Giblin 1988). The most obvious property of the neural circuit is its modular structure.

Spinal Module The degree of sophistication of the circuitry within and between spinal segments (Pfaff and Schwartz- Giblin 1988) is not to be underestimated, as you would guess from the prodigious accomplishments of the previous generation, such as neurophysiologists Elzebieta Jankowska and Anders Lundberg. Sensory inputs from the behaviorally and electrophysiologically defined cutaneous receptive fields are processed through complex dorsal horn circuitry. As you move from the most superficial part of the dorsal horn, the substantia gelatinosa toward Rexed layer V, as determined by the British physiologist Patrick Wall, the receptive fields become larger, more multimodal, and more obviously related to behavioral regulation. While we have studied segmental regulation of lordosis-relevant motor neurons, we have also emphasized powerful descending influences from reticulospinal and vestibulospinal axons that facilitate motorneuronal activity both directly and through interneurons. Our data show that deep back muscle motor neurons for lordosis can be excited either directly by descending reticulospinal and vestibulospinal axons or indirectly by heightening throughput from the pudendal nerve. But spinal circuitry is not sufficient for lordosis. Transections of the lateral columns, especially the anterolateral columns, abolish lordosis. A brainstem module is required.

Lower Brainstem Module Although individual spinal segments primarily do local business, inputoutput relations among segments would tend to produce an amplification of response to a limited excitatory stimulus. Nevertheless, a lower

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Figure 2.6. Three experimental launching points were used to discover the complete working circuit for producing lordosis behavior. 1) The cutaneous stimuli that are adequate to start the behavior (lower left). 2) The motor response, dependent on deep back muscles (lower right). 3) The ventromedial hypothalamic neurons that express the estrogen receptor- α gene (top). The circuit is bilaterally symmetric, plotted here on one side for clarity. (Adapted from Pfaff and Schwartz- Giblin 1988.)

brainstem module is needed to coordinate the molar behavioral response, lordosis, across many spinal segments. The data cited above prove that descending signals in the reticulospinal and vestibulospinal tracts do the job. Of course, signals ascending from the spinal to the lower brainstem module play an important role. Neuroanatomical studies show cord neurons backfilled from the NGC, for instance. And Lee-Ming Kow recorded

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NGC neuronal responses to lordosis-relevant cutaneous stimulation both in anesthetized and free-moving unanesthetized female rats. The sensory summation is as follows. As part of our discussion of these ascending signals we can state a principle within a principle. Cutaneous pathways for lordosis behavior converge. As the signals ascend, the cutaneous receptive fields become larger, and the sensory submodality specificity becomes sharper. Then we find selective distribution. That is, as sensory information travels from the primary sensory surface into the lordosis circuitry, it is clear that not all sensory information is transferred. First, as part of the growth of receptive field size, spatial information is lost. Second, as the information ascends the neuraxis in the lordosis circuit, the magnitude of the sensory response compared with the background activity caused by other sources declines. Convergence from other parts of the circuit is required for high levels of activity. Pfaff and Schwartz- Giblin listed (1988) several properties that demonstrated a congruence between lower brainstem module neuronal properties and the requirements for lordosis behavior. Put briefly, they included the facilitation of deep back muscle activity by electrical stimulation of the NGC or lateral vestibular nucleus, the decrease in lordosis after lesions of either of these cell groups, the facilitation of responses to pudendal nerve inputs, bilateral symmetry, and the termination of reticulospinal and vestibulospinal axons across several spinal levels, as required for lordosis. Put briefly, the lower brainstem module integrates behavioral and postural changes across spinal segments. As mentioned previously, the hierarchical nature of lordosis motor control is clear. The hypothalamus energizes the MCG which, in turn, empowers reticulospinal and vestibulospinal descending signals to turn on lordosis-relevant motor neurons (Pfaff et al. 1990).

Midbrain Module The proof of existence of a midbrain module is neuroanatomical, in part. On the descending side of the lordosis circuit, crucial estrogen signal– carrying axons do not descend below the midbrain, so midbrain neurons as such must participate in the circuit. VMH neurons increase the excitability of MCG neurons that project in turn to the medulla.

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On the ascending side we have seen evidence of fibers from the anterolateral columns of the spinal cord ascending to reach the MCG; from Yasuo Sakuma’s work in the laboratory, central grey neurons respond to lordosis-relevant somatosensory input. From the lower brainstem module, axons from NGC to midbrain are dense and intense. My usual way of thinking about the midbrain module is that it acts as a neurophysiological version of an automobile transmission. That is, hypothalamic dynamics are slow. Endocrine signals to the hypothalamus change over hours or days. VMH neurons fire slowly—in fact, stimulation over thirty pulses per second blocks them. In contrast, many other neurons in the lordosis behav ior circuit— and in fact in most of the brain—are fast. Thus, analogous to an automobile transmission, MCG neurons take in slowly changing hypothalamic outputs and put out signals that work well with rapidly firing, rapidly reacting brainstem reticular and vestibular neurons. In summary, neurons in the central grey of the midbrain facilitate lordosis, and central grey neurons reduce lordosis. Peptides synthesized in the hypothalamus and preoptic area such as gonadotropin-releasing hormone (Chapter 5) and oxytocin (Chapter 6) play a major role.

Forebrain Module When forebrain systems have been studied for their roles in the regulation of lordosis behavior, clear-cut effects have been inhibitory. The most efficient way to sever forebrain outputs descending toward the diencephalon and midbrain is to sever those axons by what is called the roof deafferentation technique. This procedure greatly enhances lordosis. Decorticate females perform lordosis. Lesions of the septum and surgical removal of the olfactory bulb greatly increase lordosis. Not only are telencephalic cell groups not necessary for lordosis, but they have a net inhibitory effect. The lordosis circuit as presented in Figure 2.6 produces the behavior, effectively releasing the behavior from cortical inhibition.

Hypothalamic Module Obviously the main job of the hypothalamic module is to add hormone dependence to the behav ior, by virtue of which the hypothalamic 72

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module is necessary for the rest of the circuit to work. At the same time, hypothalamic neurons coordinate female reproductive behav ior with ovulation. The synchrony of lordosis with ovulation clearly is biologically adaptive, helping to ensure that mating events will be productive. We got lucky with respect to the properties of the VMH cells in the hypothalamic module. The estrogen receptors I had discovered in brain (Chapter  1) turned out to be ligand- activated transcription factors (estrogen-dependent genes transcribed consequent to estrogens binding to ER-α). This fact allowed us to use the techniques of molecular biology such as in situ hybridization and polymerase chain reaction to study gene expression related to lordosis in VMH cells, followed by epigenetic explorations of histone N terminus modifications (Chapter 3), followed by determination of gene / behavior relationships (Chapter 4). In summary, we started with the information I had discovered about how hormones impact the brain (Chapter  1). In Chapters  3 and 4 we will be able to link these hormones to gene expression and genomic influences on behavior. Here, in Chapter 2, I have detailed a manifestation of lordosis behavior mechanisms in physical terms. Principle inferred: Yes, it is possible to work out the circuit for producing a complete, normal vertebrate behavior. The circuit for this social behavior, essential for reproduction, is hierarchical, composed of modules. Each module adds a unique regulatory feature.

Further Reading Brink, E. E., D. T. Modianos, and D. W. Pfaff. 1980. “Ablations of Lumbar Epaxial Musculature: Effects on Lordosis Behav ior of Female Rats.” Brain, Behavior and Evolution 17: 67–88. Brink, E.  E., J.  I. Morrell, and D.  W. Pfaff. 1979. “Localization of Lumbar Epaxial Motoneurons in the Rat.” Brain Research 170: 23–41. Brink, E.  E., and D.  W. Pfaff. 1980. “Vertebral Muscles of the Back and Tail of the Albino Rat (Rattus norvegicus albinus).” Brain, Behavior and Evolution 17 (1): 1–47. ———. 1981. “Supraspinal and Segmental Input to Lumbar Epaxial Motoneurons in the Rat.” Brain Research 226: 43–60. Chung, S. R., D. W. Pfaff, and R. S. Cohen. 1990a. “Projections of Ventromedial Hypothalamic Neurons to the Midbrain Central Gray: An Ultrastructural Study.” Neuroscience 38: 395–407.

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———. 1990b. “Transneuronal Degeneration in the Midbrain Central Gray following Chemical Lesions in the Ventromedial Nucleus: A Qualitative and Quantitative Analysis.” Neuroscience 38 (2): 409–426. Cohen, M. S., S. Schwartz- Giblin, and D. W. Pfaff. 1985. “The Pudendal NerveEvoked Response in Axial Muscle.” Experimental Brain Research 61: 175–185. ———. 1987a. “Brainstem Reticular Stimulation Facilitates Back Muscle Motoneuronal Responses to Pudendal Nerve Input.” Brain Research 405: 155–158. ———. 1987b. “Effects of Total and Partial Spinal Transections on the Pudendal Nerve-Evoked Response in Rat Lumbar Axial Muscle.” Brain Research 401: 103–112. Conrad, L. C. A., and D. W. Pfaff. 1975. “Axonal Projections of Medial Preoptic and Anterior Hypothalamic Neurons.” Science 190: 1112–1114. ———. 1976a. “Efferents from Medial Basal Forebrain and Hypothalamus in the Rat. II. An Autoradiographic Study of the Anterior Hypothalamus.” Journal of Comparative Neurology 169 (2): 221–261. ———. 1976b. “Efferents from Medial Basal Forebrain and Hypothalamus in the Rat. I. An Autoradiographic Study of the Medial Preoptic Area.” Journal of Comparative Neurology 169 (2): 185–220. Cottingham, S. L., P. A. Femano, and D. W. Pfaff. 1987. “Electrical Stimulation of the Midbrain Central Gray Facilitates Reticulospinal Activation of Axial Muscle EMG.” Experimental Neurology 97: 704–724. ———. 1988. “Vestibulospinal and Reticulospinal Interactions in the Activation of Back Muscle EMG in the Rat.” Experimental Brain Research 73 (1): 198–208. Cottingham, S. L., and D. W. Pfaff. 1987. “Electrical Stimulation of the Midbrain Central Gray Facilitates Lateral Vestibulospinal Activation of Back Muscle EMG in the Rat.” Brain Research 421: 397–400. Dupré, C., M. Lovett-Barron, D. W. Pfaff, and L.-M. Kow. 2010. “Histaminergic Responses by Hypothalamic Neurons That Regulate Lordosis and Their Modulation by Estradiol.” Proceedings of the National Academy of Sciences of the United States of Amer ica 107 (27): 12311–12316. Femano, P. A., S. Schwartz- Giblin, and D. W. Pfaff. 1984a. “Brain Stem Reticular Influences on Lumbar Axial Muscle Activity. I. Effective Sites.” American Journal of Physiology 246: R389–R395. ———. 1984b. “Brain Stem Reticular Influences on Lumbar Axial Muscle Activity. II. Temporal Aspects.” American Journal of Physiology 46: R396–R401. Kow, L.-M., M. O. Montgomery, and D. W. Pfaff. 1977. “Effects of Spinal Cord Transections on Lordosis Reflex in Female Rats.” Brain Research 123: 75–88. ———. 1979. “Triggering of Lordosis Reflex in Female Rats with Somatosensory Stimulation: Quantitative Determination of Stimulus Parameters.” Journal of Neurophysiology 42: 195–202.

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Kow, L.-M., S. Pataky, C. Dupré, A. Phan, N. Martín-Alguacil, and D. W. Pfaff. 2016. “Analyses of Rapid Estrogen Actions on Rat Ventromedial Hypothalamic Neurons.” Steroids 111: 100–112. Kow, L.-M., and D. W. Pfaff. 1973. “Effects of Estrogen Treatment on the Size of Receptive Field and Response Threshold of Pudendal Nerve in the Female Rat.” Neuroendocrinology 13: 299–313. ———. 1975. “Dorsal Root Recording Relevant for Mating Reflexes in Female Rats: Identification of Receptive Fields and Effects of Peripheral Denervation.” Journal of Neurobiology 6: 23–37. ———. 1976. “Sensory Requirements for the Lordosis Reflex in Female Rats.” Brain Research 101: 47–66. ———. 1979. “Responses of Single Units in Sixth Lumbar Dorsal Root Ganglion of Female Rats to Mechanostimulation Relevant for Lordosis Reflex.” Journal of Neurophysiology 42: 203–213. ———. 1982. “Responses of Medullary Reticulospinal and Other Reticular Neurons to Somatosensory and Brainstem Stimulation in Anesthetized or Freely-Moving Ovariectomized Rats with or without Estrogen Treatment.” Experimental Brain Research 47: 191–202. ———. 1985. “Estrogen Effects on Neuronal Responsiveness to Electrical and Neurotransmitter Stimulation: An in Vitro Study on the Ventromedial Nucleus of the Hypothalamus.” Brain Research 347: 1–10. ———. 1987. “Responses of Ventromedial Hypothalamic Neurons in Vitro to Norepinephrine: Dependence on Dose and Receptor Type.” Brain Research 413 (2): 220–228. ———. 1995. “Functional Analyses of α1-Adrenoceptor Subtypes in Rat Hypothalamic Ventromedial Nucleus Neurons.” European Journal of Pharmacology 282: 199–206. Kow, L.-M., Y.-F. Tsai, N. G. Weiland, B. S. McEwen, and D. W. Pfaff. 1995. “In Vitro Electro-Pharmacological and Autoradiographic Analyses of Muscarinic Receptor Subtypes in Rat Hypothalamic Ventromedial Nucleus: Implications for Cholinergic Regulation of Lordosis.” Brain Research 694: 29–39. Kow, L.-M., G. D. Weesner, and D. W. Pfaff. 1992. “Adrenergic Agonists Act on Ventromedial Hypothalamic α-Receptors to Cause Neuronal Excitation and Lordosis Facilitation: Electrophysiological and Behavioral Evidence.” Brain Research 588: 237–245. Kow, L.-M., F.  P. Zemlan, and D.  W. Pfaff. 1980. “Responses of Lumbosacral Spinal Units to Mechanical Stimuli Related to Analysis of Lordosis Reflex in Female Rats.” Journal of Neurophysiology 43: 27–45. Krieger, M. S., L. C. A. Conrad, and D. W. Pfaff. 1979. “An Autoradiographic Study of the Efferent Connections of the Ventromedial Nucleus of the Hypothalamus.” Journal of Comparative Neurology 183: 785–816. Lee, A. W., A. Kyrozis, V. Chevaleyre, L. M. Kow, N. Devidze, Q. Zhang, A. M. Etgen, and D.  W. Pfaff. 2008. “Estradiol Modulation of Phenylephrine-Induced

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Excitatory Responses in Ventromedial Hypothalamic Neurons of Female Rats.” Proceedings of the National Academy of Sciences of the United States of Amer ica 105 (20): 7333–7338. Manogue, K., L.-M. Kow, and D. W. Pfaff. 1980. “Selective Brain Stem Transections Affecting Reproductive Behavior of Female Rats: The Role of Hypothalamic Output to the Midbrain.” Hormones and Behavior 14: 277–302. Modianos, D. T., and D. W. Pfaff. 1976. “Brain Stem and Cerebellar Lesions in Female Rats. II. Lordosis Reflex.” Brain Research 106: 47–56. ———. 1977. “Facilitation of the Lordosis Reflex in Female Rats by Electrical Stimulation of the Lateral Vestibular Nucleus.” Brain Research 134: 333–345. ———. 1979. “Medullary Reticular Formation Lesions and Lordosis Reflex in Female Rats.” Brain Research 171: 334–338. Morrell, J. I., and D. W. Pfaff. 1982. “Characterization of Estrogen- Concentrating Hypothalamic Neurons by Their Axonal Projections.” Science 217: 1273–1276. ———. 1983. “Retrograde HRP Identification of Neurons in the Rhombencephalon and Spinal Cord of the Rat That Project to the Dorsal Mesencephalon.” American Journal of Anatomy 167: 229–240. Morrell, J. I., T. D. Wolinsky, M. S. Krieger, and D. W. Pfaff. 1982. “Autoradiographic Identification of Estradiol- Concentrating Cells in the Spinal Cord of the Female Rat.” Experimental Brain Research 45: 144–150. Pfaff, D. W. 1980. Estrogens and Brain Function. Heidelberg: Springer. Pfaff, D. W., C. Diakow, M. Montgomery, and F. A. Jenkins. 1978. “X-ray Cinematographic Analysis of Lordosis in Female Rats.” Journal of Comparative and Physiological Psychology 92 (5): 937–941. Pfaff, D. W., A. Korotzer, S. Schwartz- Giblin, and S. L. Cottingham. 1990. “Hypothalamic Effects on Medullary Reticular Activation of Deep Back Muscle EMG.” Physiology and Behavior 47 (1): 185–196. Pfaff, D. W., and C. Lewis. 1974. “Film Analyses of Lordosis in Female Rats.” Hormones and Behavior 5 (4): 317–335. Pfaff, D.  W., M. Montgomery, and C. Lewis. 1977. “Somatosensory Determinants of Lordosis in Female Rats: Behavioral Defi nition of the Estrogen Effect.” Journal of Comparative and Physiological Psychology 91: 134–145. Pfaff, D. W., and Y. Sakuma. 1979a. “Deficit in the Lordosis Reflex of Female Rats Caused by Lesions in the Ventromedial Nucleus of the Hypothalamus.” Journal of Physiology 288: 203–210. ———. 1979b. “Facilitation of the Lordosis Reflex of Female Rats from the Ventromedial Nucleus of the Hypothalamus.” Journal of Physiology 288: 189–202. Pfaff, D.  W., and S. Schwartz- Giblin. 1988. “Cellular Mechanisms of Female Reproductive Behav iors.” In The Physiolog y of Reproduction. Edited by E. Knobil and J. Neill. New York: Raven, chapter 35, 1487–1568.

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Robbins, A., D.  W. Pfaff, and S. Schwartz- Giblin. 1992. “Reticulospinal and Reticuloreticular Pathways for Activating the Lumbar Back Muscles in the Rat.” Experimental Brain Research 92: 46–58. Robbins, A., S. Schwartz- Giblin, and D.  W. Pfaff. 1990. “Ascending and Descending Projections to Medullary Reticular Formation Sites Which Activate Deep Lumbar Back Muscles in the Rat.” Experimental Brain Research 80 (3): 463–474. Sakuma, Y., and D. W. Pfaff. 1979a. “Facilitation of Female Reproductive Behav ior from Mesencephalic Central Gray in the Rat.” American Journal of Physiology 237: R278–R284. ———. 1979b. “Mesencephalic Mechanisms for Integration of Female Reproductive Behav ior in the Rat.” American Journal of Physiolog y 237: R285–R290. ———. 1980a. “Cells of Origin of Medullary Projections in Central Gray of Rat Mesencephalon.” Journal of Neurophysiology 44: 1002–1011. ———. 1980b. “Convergent Effects of Lordosis-Relevant Somatosensory and Hypothalamic Influences on Central Gray Cells in the Rat Mesencephalon.” Experimental Neurology 70: 269–281. ———. 1980c. “Excitability of Female Rat Central Gray Cells with Medullary Projections: Changes Produced by Hypothalamic Stimulation and Estrogen Treatment.” Journal of Neurophysiology 44: 1012–1023. ———. 1981. “Electrophysiologic Determination of Projections from Ventromedial Hypothalamus to Midbrain Central Gray: Differences between Female and Male Rats.” Brain Research 225: 184–188. ———. 1982. “Properties of Ventromedial Hypothalamic Neurons with Axons to Midbrain Central Gray.” Experimental Brain Research 46: 292–300. Schwartz- Giblin, S., P. Femano, and D. W. Pfaff. 1984. “Axial Electromyogram and Intervertebral Length Gauge Responses during Lordosis Behavior in Rats.” Experimental Neurology 85: 297–315. Schwartz- Giblin, S., M. Halpern, and D.  W. Pfaff. 1984. “Segmental Organization of Rat Lateral Longissimus, a Muscle Involved in Lordosis Behavior: EMG and Muscle Nerve Recordings.” Brain Research 299: 247–257. Schwartz- Giblin, S., and D. W. Pfaff. 1980. “Implanted Strain Gauge and EMG Amplifier to Record Motor Behav ior in Unrestrained Rats.” Physiology and Behavior 25: 475–479. ———. 1990. “Ipsilateral and Contralateral Effects on Cutaneous Reflexes in a Back Muscle of the Female Rat: Modulation by Steroids Relevant for Reproductive Behav ior.” Journal of Neurophysiology 64 (3): 835–846. Schwartz- Giblin, S., L. Rosello, and D. W. Pfaff. 1983. “A Histochemical Study of Lateral Longissimus Muscle in Rat.” Experimental Neurology 79: 497–518. Zemlan, F., L.-M. Kow, J. I. Morrell, and D. W. Pfaff. 1979. “Descending Tracts of the Lateral Columns of the Rat Spinal Cord: A Study Using the

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Horseradish Peroxidase and Silver Impregnation Techniques.” Journal of Anatomy 128: 489–512. Zemlan, F. P., L.-M. Kow, and D. W. Pfaff. 1983. “Effect of Interruption of Bulbospinal Pathways on Lordosis, Posture, and Locomotion.” Experimental Neurology 81: 177–194. Zemlan, F. P., and D. W. Pfaff. 1979. “Topographical Organization in Medullary Reticulospinal Systems as Demonstrated by the Horseradish Peroxidase Technique.” Brain Research 174: 161–166. Zhou, J., A. W. Lee, Q. Zhang, L. M. Kow, and D. W. Pfaff. 2007. “HistamineInduced Excitatory Responses in Mouse Ventromedial Hypothalamic Neurons: Ionic Mechanisms and Estrogenic Regulation.” Journal of Neurophysiology 98 (6): 3143–3152.

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3 H O R M O N A L R EGU L AT I O N O F GEN E E XPR E SSIO N IN T H E BR A IN

Problem: The estrogen receptors studied in Chapter 1 are transcription factors. What does that have to do with brain function and behavior? Is it possible to make strong causal links from molecular chemistry to physiology (ion channels) and, ultimately, to a complete mammalian behavior?

In Chapter 1, I described our early work on hormone receptors in the brain, and in Chapter 2 how they helped in working out the neural circuit for producing a female reproductive behavior. After we worked out that circuit and were entering the era in which we could apply the techniques of molecular biology to the brain, we faced two challenges in discovering how estrogens, as they drive lordosis behavior, activate the expression of specific genes in specific neurons. First, at that time, gene sequences were not as fully described as they are now, so designing accurate molecular probes offered a challenge. Second, we had to devise assays of sufficient sensitivity and spatial resolution to achieve quantitative assays of gene expression in individual neurons. This chapter will show how, when we met these challenges, we drew an entire series of genes into the explanation of a mammalian behavior.

Lucky I got lucky. The hormone receptors I had discovered in the brain (Chapter 1) turned out to be “ligand-activated transcription factors”—proteins that, when the hormone was bound, would then bind to specific elements of DNA and facilitate the expression of hormone-dependent genes specifically in those neurons. My laboratory was effectively able to walk our scientific questions and problems into the arena of modern molecular biology. It was incredibly exciting to have the opportunity to link the neural circuitry we discovered (including specific neurotransmitters and ion channels) and reproductive behaviors to the chemistry of gene transcription. But there were challenges as well. Could we use in situ hybridization in a way that was sensitive enough to study messenger RNA (mRNA) levels in individual neurons? Could we achieve accurate quantification? The answers to both questions turned out to be yes. The reward for sticking with the program, gene after gene, year after year, was that we had the evidence to show, conceptually and factually, how these transcriptional systems overdetermine the occurrence of reproductive behavior and synchronize it with ovulation. In this chapter, I discuss, first, how we discovered several genes whose expression was turned on in reproductive behavior-controlling neurons by estrogen treatment. Second, we recently found protranscriptional chromatin changes caused by estrogens over the promoters of those genes. Third, in a slightly more sophisticated inquiry, we found interactions among transcription factors, estrogen receptors (ER), and thyroid hormone receptors (TR). All three are discussed here. Further, molecular changes in behavior-critical hypothalamic neurons were accompanied by morphological alterations associated with higher levels of synthetic activity. In this chapter, this will be discussed last.

Reproductive Behavior Requires Hormone-Dependent Synthetic Events in Hypothalamic Neurons Even with a lot of neuroanatomy, histochemistry, and electrophysiology behind me, I was scared to face the question of estrogen-stimulated

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newly synthesized proteins in the brain. My only background comprised courses in organic chemistry at Harvard and biochemistry at the Massachusetts Institute of Technology (MIT). I marshaled my courage to ask advice of Rockefeller University professor Stanford Moore, a famous protein chemist. Moore and his Rockefeller colleague William Stein had won the Nobel Prize for chemistry based on their first- ever determination of the amino acid sequence of an enzyme (RNAse). Thus, it was especially kind of Professor Moore to invite me to lunch in the Rockefeller dining room and explain the technique of protein chemical discovery that would be most likely to work in small amounts of hypothalamic tissue. More than that, he guaranteed the leadership of his most experienced laboratory member, Peter Blackburn, to help make this project succeed. Studying protein synthesis was logically required because I knew that new protein synthesis in hypothalamic tissue after estrogen treatment was required for lordosis (see Chapter  1). Following up those studies, Rockefeller graduate student Bruce Parsons showed the exact timing of the necessary protein synthetic events (Parsons et  al. 1982; Parsons, McEwan, and Pfaff 1982). Not only that, but from the work of Richard Harlan in the laboratory, it was clear that such proteins had to be axonally transported out of the hypothalamus, accompanied by action potentials (see Chapter 1). Which exact hypothalamic neurons were involved? Paula Davis’s localized implants showed that estrogen administration to the ventromedial nucleus of the hypothalamus (VMH) neurons was sufficient (Davis, McEwen, and Pfaff 1979), and Bob Meisel’s work with localized antiestrogen implants demonstrated that estrogen’s impact on VMH neurons was necessary for lordosis. Peter Blackburn, with Professor Moore’s encouragement, taught us to use liquid chromatography to separate radioactively labeled proteins after microinjections of carefully chosen labeled amino acids into the VMH (Pfaff, Rosello, and Blackburn 1984). In the experiments, we used matched pairs of female rats; in each matched pair, one had been ovariectomized (thus leaving the animal devoid of estrogen) and then given estradiol treatment for 7 days. All these rats exhibited lordosis behavior. The other of the matched pair was ovariectomized and only given the vehicle control treatment. None of these controls exhibited lordosis behavior. Then the microinjection into the VMH was followed by various

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survival times ranging from 2 hours to 2 days. The best results were at 4 hours and were positive in two very encouraging ways. Not only were there significantly more radioactively labeled proteins in the VMH tissue, but also some of these had been transported along axons to the midbrain central grey (MCG). One specific class of proteins in the central grey, probably a class of molecular weights based on the protein separation column we used, was more than ten times more abundant in the estrogen-treated animals than in the controls. A second approach to estrogen-stimulated protein synthesis in the hypothalamus was initiated by Charles Mobbs. Before joining us at Rockefel ler, Charles worked his way through MIT, and then got his doctorate in endocrine biochemistry at the University of Southern California. Charles tested our theory, based on some of the work summarized in Chapter 1, that systemic administration of estrogen would lead to novel proteins synthesized in the VMH. In doing so he brought the performance of two-dimensional gel analysis of radioactively labeled new proteins in the brain to a high art. This was time-consuming work, and while he was waiting for gels to run he would, in front of a huge mirror, practice his tap dancing in the marble-floored halls outside our laboratory doors. The basic technique of two- dimensional gel analysis relies on separation in one dimension according to the molecular weight of the protein, yielding gels in the form of tubes. Then those tube gels are separated again, using a carefully chosen pair of stacking gels and resolving gels; in this latter step, proteins are separated according to their isoelectric points. Thus we produce two-dimensional gel electrophoresis. In our work, ovariectomized female rats had been prepared either with constant estrogen treatment or with the vehicle control 7 days before further experimentation, at which time they were infused with radioactive amino acids. Our best results came 14 hours after the radioactivity infusion. Consequent radioactively labeled proteins were detected with a technique known as fluorography, in which a Kodak fi lm with high spatial resolution and high sensitivity to the S35 radioactive label is pressed against the gels produced as described. The high degree of strategic and methodological “art” that Charles Mobbs achieved (Mobbs et al. 1988) was based, at a minimum, on 1) the choice and timing of the first gel, 2) the choice and timing of the second gel,

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together with choice of electric field strength, 3) the choice of buffers, and 4) the exposure time of the Kodak film. As a result of this extensive work, we discovered an estrogen-induced protein that is synthesized in the VMH and transported to the MCG. In those days, our detecting more than 250 protein “spots” of black, radioactive proteins represented an achievement due to Charles’s scientific artistry. In addition, Charles’s gels had superb resolving power when subjecting the films to computerized densitometric analysis. The big story is that we had found a specific protein with an apparent molecular mass of 70 kilodaltons and an isoelectric point of 5.9 that was clearly present in almost all the estrogen-treated females’ VMH and in only two of sixteen of the control animals. The same significant result for that exact class of protein was obtained in the MCG. In fact, disrupting axoplasmic flow from the VMH to the MCG by colchicine treatment reduced the arrival of radioactivity at the central grey by approximately 85  percent (Mobbs et al. 1988). This finding logically sent us on a hunt for estrogen-induced gene expression in the hypothalamus. We had united molecular biology and behavioral biology (Mobbs, Fink, and Pfaff 1990; Mobbs et al. 1989). Robert Lustig, another MIT gradu ate, had trained as a medical endocrinologist; he worked with Charles to follow up his work. Subsequent to Robert’s laboratory work at Rockefel ler, he went on to a clinical endocrine practice and has become a nationally recognized expert, warning the public about the deleterious consequences of ingesting sugar at high levels. His methodology was similar to that described here, using two-dimensional gels, and his idea was to extend Charles’s observations to include quantitative mea surements of the synthesis of specific proteins in the VMH as a function of estrogen treatment (versus control) (Lustig, Pfaff, and Mobbs 1989). His results confirmed the induction of a protein whose apparent molecular mass is 70 kilodaltons with an isoelectric point of 5.9, but his method of analysis allowed us to go much farther. Instead of focusing exclusively on that one hormone-induced protein, Robert used a flatbed laser scanner to quantify the optical density of each of the two-hundred and forty spots on the gel. Three new protein-spots were subsequently identified as quantitatively induced (72 to 96  percent increases) by estrogen treatment. Further, slight increases in the isoelectric point of

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two other proteins suggested estrogen-induced posttranslational modification. Again, Robert’s results reinforced our efforts to discover estrogen-influenced gene expression in the hypothalamus as causal to lordosis behavior. Motivated by the foregoing proof that new protein synthesis is necessary for the estrogenic facilitation of lordosis behavior (Chapter  1), Rockefeller graduate student Bruce Parsons did work that justified Charles Mobbs’s and Robert Lustig’s studies (Parsons et al. 1982; Parsons, McEwen, and Pfaff 1982). Bruce used three dif ferent doses of the very effective protein synthesis inhibitor anisomycin (80  percent decrease in hypothalamus). Timing was crucial. If the anisomycin was administered just before an estrogen treatment, lordosis was abolished. If instead anisomycin was given well after estrogen treatments but hours before the behavioral assay, lordosis was at normal, control levels. This last control was important because it proved the anisomycin was not reducing lordosis simply because the animals were ill. Thus, the effect of protein synthesis reduction was specific to the estrogenic facilitation of lordosis. Again, this type of result made us want to mea sure levels of specific mRNAs in specific hypothalamic neurons. In situ hybridization. Moving on to the mRNA level, we wanted cellular resolution in our data and thus were the first to work out a technique for in situ hybridization in the brain (McCabe et al. 1986). Using a precise, quantitative approach to in situ hybridization, Andrea Lauber, as mentioned in Chapter 1, not only proved the expression of the mRNA for ER- α in VMH neurons but also demonstrated that 1) estrogen administration reliably led to a decrease in expression of mRNA for ER- α, and 2) this phenomenon is sexually differentiated, with the results of females being much stronger than those of males.

Specific Transcriptional Systems Proven to Be Involved Estrogen hormones bind to ER- α. Liganded, the receptor quickly recognizes and binds to specific DNA sequences—the consensus estrogen response element (ERE) on DNA is AGGTCAnnnTGACCT. The n can be any nucleotide base but there must be three of them. Some substitutions in the consensus ERE are allowed. These EREs for “turning on”

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Gene Turned On (in Hypothalamus) rRNA and Growth

Progesterone Receptor nNitricOxideSynthase ERa E

binds

ERb

Adrenergic a1 Receptor Muscarinic Receptors

Female Reproductive Behaviors

Enkephalin X Opioid Receptors Oxytocin X Oxytocin Receptor

(in Preoptic Area) GnRH X GnRH Receptor Figure 3.1. Several genes have two properties: 1) estrogens facilitate their expression, and 2) their products foster lordosis behavior. (Updated from Pfaff 1999.)

estrogen responsive genes in brain and other tissues can be found in the genes’ (proximal) promoters or their (distal) enhancers (see Klinge’s section in Kow et al. 2016). Here is the logic we will follow. Several transcriptional systems have the following two properties: 1) estrogen treatment raises their mRNA levels in VMH neurons, and 2) the final gene product facilitates lordosis behavior (Figure 3.1). Thus, because we know that new mRNA synthesis and new protein synthesis are required for estrogen effects on lordosis, we take these two properties and make a logical inference in the form of a syllogism. I like to use the idea of a syllogism, that is, reasoning in the form: “If (a) John is a scientist and (b) all scientists are good, then (c) John is good.” In the case of each transcriptional system here, the logical inference will be that revving up of that transcriptional step serves to facilitate estrogen-dependent lordosis behavior.

Progesterone Receptor Gary Romano entered the laboratory as a Rockefel ler gradu ate student and immediately dispensed with the stereotypes of what precise

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Figure 3.2. Black dots represent neurons in which the expression of the progesterone receptor gene has been turned on by estrogen administration. Strong estrogen effects were seen in the ventromedial nucleus (VM) and the arcuate nucleus (ARC) of the hypothalamus. Expression is bilateral, plotted here on one side. (Adapted from Romano, Krust, and Pfaff 1989.)

scientific operations require. Gary had hands with the size and muscularity of a professional football lineman, yet he operated at the laboratory bench with a degree of order and precision that could hardly be equaled. The fi rst breakthrough in his work was enabled by a long conversation with the great French molecular biologist Pierre Chambon. Pierre had cloned the progesterone receptor (PR) and generously gave us his reagents and advice. As a result, Gary learned our technique for in situ hybridization and compared PR mRNA levels in the VMH (with implications for lordosis) as a function of time after estrogen administration compared with control ovariectomized rats (Figure  3.2) (Romano, Krust, and Pfaff 1989). The PR mRNA levels already started up within 4 hours of estrogen administration and reached their peak at 24 hours. By 48 hours, they were headed back down. Andrea Lauber came to Rockefeller after she got her Ph.D. from the University of California, and she worked with Gary Romano to explore the specificity of the estrogenic induction of PR mRNA (Lauber et al.

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1991). She knew that the most important determinant of the ability of progesterone to facilitate reproductive behavior was the level of PR. First she replicated and extended Gary’s work by demonstrating a 3.5 times induction of PR in the VMH of the female. Then she showed that in the genetic male hardly any PR induction occurred after estrogen treatment. All these data will play into my “transcriptional formula” for behavioral regulation at the end of this chapter. Some of my earliest studies on lordosis behav ior had proven the efficacy of long-term estrogen exposure. In fact, I never considered molecular endocrine systems to be signaling systems with instantaneous time- constants because hormonal dynamics, as they affect various target tissues (including brain tissue), simply take time. Rockefeller graduate student Bruce Parsons took up the call. Crucially, Bruce demonstrated that long-term treatment with estrogens greatly facilitated the subsequent response of the brain to a later estrogen treatment as well as to a later estrogen plus progesterone treatment (Parsons et al. 1979). This was true whether that later estrogen treatment was followed by a lordosis behav ior assay or a PR assay. The practical importance of Bruce’s work was that it revealed the inappropriateness of a major National Institutes of Health (NIH) clinical trial of hormone replacement therapy in which the patients had been without their estrogens for years—those subjects were lacking the long-term estrogenic support revealed as crucial by the Parsons et al. study. Under the circumstances in which the trial was terminated, molecular endocrinologists summarized “wrong trial, wrong analysis, wrong decision.” Our case was much simpler. We knew that blocking the synthesis or action of PR would block the ability of progesterone to facilitate estrogen’s actions on lordosis behavior (Ogawa et al. 1994). To prove the parallelism between estrogenic effects on PR and estrogenic effects on lordosis behav ior, Bruce made clever use of discontinuous schedules of estrogen treatment (Parsons et al. 1982; Parsons, McEwen, and Pfaff 1982). He had already shown great parallelism between the molecular end point and behavior (Parsons et al. 1980). That is, as a function of time after estrogen administration both PR and behavior rose steadily, starting at 12 hours and peaking at about 48 hours. Likewise, after removal of estrogen from the body they both decayed, reaching levels indistinguishable from controls at 36

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hours after withdrawal. Then, regarding the discontinuous schedules of estrogen treatment, we analyzed the 24 hours of estrogen preceding the progesterone treatment that would finish the hormonal support for high levels of lordosis (Parsons et al. 1981, 1982). Brief estrogen exposures were accomplished by popping small silastic capsules in and out of the subcutaneous space. It turned out that two brief estrogen exposures during the 24 hours before progesterone were sufficient for lordosis if they were separated by at least 4 hours and not more than 13 hours. Well-timed use of the protein synthesis inhibitor anisomycin showed that each brief estrogen exposure needed to initiate new protein synthesis for the behav ior to occur. Most impor tant for this chapter: the same conditions that were adequate for lordosis behavior also were sufficient for PR synthesis. Beyond the parallels between the induction of PR and the initiation of lordosis, we were able to prove that PR expression is necessary for lordosis. Sonoko Ogawa in my laboratory (now a professor of neurobiology at Tsukuba University in Japan) pioneered our use of antisense DNA technology. In this approach, we synthesize a string of nucleotide bases that are complementary to and will bind to a specific messenger RNA (in this case the mRNA for PR), thus opening that mRNA to attack by nuclease enzymes and, as a consequence, to being put totally out of action. Sonoko microinjected antisense DNA among neurons of the VMH and massively reduced the amount of lordosis behav ior compared with control females given only the vehicle (Ogawa et  al. 1994). Sonoko’s work was replicated by Shaila Mani in Bert O’Malley’s laboratory at Baylor College of Medicine. Our results were also complemented by findings from other laboratories that used a PR blocker called RU486 [(8S,11R,13S,14S,17S)-11-[4-(dimethylamino)phenyl]-17-hydroxy13-methyl-17-prop-1-ynyl-1,2,6,7,8,11,12,14,15,16- decahydrocyclopent a[a]phenanthren-3- one]. Females given estrogen and progesterone but also RU486 showed only low levels of lordosis behav ior. As a side point, Anne Etgen, at Albert Einstein College of Medicine showed that RU486 also blocked the ability of the enkephalin / δ- opioid system to facilitate lordosis. The power ful causal role of PR transcription in fostering lordosis behavior was thus proven beyond a doubt. Thus, (a) estradiol, bound to the ligand- activated transcription factor ER- α, turns on the gene for the PR (another ligand-activated

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transcription factor). Chris Krebs in our laboratory followed up the progesterone-sensitive genes (Krebs et al. 2000). (b) In any case, the progesterone receptor is necessary for progesterone to be able to amplify estrogenic effects on lordosis. Therefore, (c) it follows that one molecular path by which estrogens facilitate lordosis is by ramping up the transcription of the PR.

Alpha Adrenergic Receptors Having worked with the massive amplification of estrogenic effects on behavior by progesterone through the PR, it now seemed logical—and well within the purview of late-twentieth- century neurobiology—to work with neurotransmitter receptors. We dealt effectively with two: α- adrenergic receptors and muscarinic receptors. It was clear from careful neuroanatomical studies using in situ hybridization that adrenergic- α1 mRNA is expressed in neurons of the VMH. The main question for us was what effect estrogens might have. The rate-limiting enzyme for the receptor’s ligand, norepinephrine, is tyrosine hydroxylase. Its gene’s transcription is reduced in the brain through deprivation of estrogen by ovariectomy and is increased by estrogen treatment. As reviewed authoritatively by Anne Etgen, the ligand’s release is increased by estrogens as well. Further, she showed that estrogens increase α1 binding sites in ovariectomized rats by four to six times in the hypothalamus, and this huge binding increase is accompanied by a significant rise in α1 mRNA levels. It follows that Lee-Ming Kow in our laboratory, using extracellular recordings of individual VMH neurons, was able to demonstrate that estrogen treatment heightens electrical responses to treatment (in the recording bath) with α1 receptor ligands (Kow and Pfaff 1985, 1995). What ion channels are involved in the excitation of VMH neurons by α1 receptor adrenergic agonists? Our work proceeded apace with that of Anne Etgen’s molecular work at Einstein. α1B -noradrenergic transmission can facilitate estrogen-dependent lordosis behavior (Kow, Weesner, and Pfaff 1992). Estradiol treatment in vivo increases levels of mRNA encoding the α1B receptor in the hypothalamus. In the VMH, estradiol treatment potentiates α1B - adrenergic signaling by increasing the proportion of neurons that respond to stimulation of α1B -adrenergic

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receptors (α1B -AR). Thus, estradiol may modulate α1B -adrenergic receptors in the VMN to promote neuronal excitability and ultimately lordosis. Members of the G-protein coupled receptor superfamily of membrane proteins, α1B -ARs are located on the cell surface. α1B -ARs can couple to a variety of G-proteins and second-messenger systems, but it is generally agreed that the primary pathway involves activation Gq/11, eliciting second messenger signals, including Ca 2+, diacylglycerol, and inositol triphosphate. Ion channels. Norepinephrine uses both L-type calcium channels and reduced conductance of potassium channels (A currents) to signal through Gq proteins, and thus to activate phospholipase C and protein kinase C. In contrast, glutamate, acting through NMDA (N-methyl-D aspartate) receptors coupled directly to ion channels, triggers an inward sodium current. Acetylcholine, acting through muscarinic receptors in the VMH, will excite VMH neurons through the inward flow of sodium ions. As will be noted here, histamine, acting through H1 receptors, works through the inhibition of a potassium leak current to depolarize VMH neurons. All these ion channel routes contribute to the VMH facilitation of lordosis. Calcium channels, especially the L-type channels, are targets of modulation by norepinephrine and α1-adrenergic agonists, but the majority of these studies have been carried out in cardiac myocytes. In VMH, both L- and N-type calcium channels contribute to the phenylephrine (PHE) response, although N-type calcium channels predominate. PHE also increases low-voltage-activated currents in the majority of VMH neurons. These results are important, as an increase in calcium entry can be expected to modulate second messenger systems and increase the release of neurotransmitters as well as modulate spike frequency by facilitating action potential firing. Although these effects may be important, in the VMH PHE-mediated membrane excitability is not solely dependent on an increase in intracellular calcium (Lee et al. 2008). The effect of adrenergic stimulation on VMN neurons is probably also due to effects on K+ conductances. It follows that the α-adrenergic agonist PHE depolarizes VMH neurons, in part by reducing membrane conductance for K+ via an A-type

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K+ channel (Lee et al. 2008). As shown by Anne Etgen, this pathway is potentiated by estradiol in two ways: by increasing levels of mRNA encoding the α1B -adrenergic receptor and by increasing protein kinase C. These results show a role for the noradrenergic system in modulating potassium channels, and this may provide a powerful way to increase the excitability of hormone-dependent VMH neurons that govern female sexual behavior. Thus, the intracellular signal transduction pathway for mediating the facilitation of lordosis can be outlined as follows: norepinephrine binds to α1B -adrenoceptor causing conformational change → activates α-subunit of Gαq/11 → stimulates phosphatidyl-inositol-specific phospholipase C- β1 in the plasma membrane → hydrolyzes membranebound phosphatidylinositol 4,5-biphosphate (PIP2) → generates second messengers inositol triphosphate and diacylglycerol → mobilizes intracellular Ca 2+ and activates protein kinase C, respectively → phosphorylates a variety of cellular substrates to achieve the fi nal functional results (Kow and Pfaff, 1998). As noted previously, we also have gathered some information about the ion channels involved. With respect to lordosis, Lee-Ming Kow showed clearly that α1adrenergic agents act on the VMH to increase female reproductive behav ior (Kow, Weesner, and Pfaff 1992). Lee microinjected into the VMH α1-agonists, methoxamine (MA) and phenylephrine (PhE), and various control agents. In ovariectomized rats treated with estrogen, infusion of MA, PhE, or a β-agonist isoproterenol into the lateral ventricle, or bilateral infusions of MA or PhE into the VMH significantly facilitated lordosis. Conversely, intra-VMH infusion of the α1-antagonist prazosin inhibited lordosis. The phenomenon was specific: intra-VMH infusion of isoproterenol or an α2-agonist clonidine had no effect. Neither was the intra-VMH infusion of MA effective if 1) the rats were not primed with estrogen, 2) the tips of the cannulae were outside the VMH, or 3) it was preceded by an intra-VMH infusion of the α1B-antagonist, chloroethyl clonidine. In parallel experiments with microelectrodes, he confirmed that, to go along with the lordosis-facilitating effect of α1-activation, the actions of MA and PhE on the electrical activity of single neurons are to increase firing rates. Our behavioral work extended the work of the late Robert Moss at Southwestern Medical School in Dallas. He showed that administering

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either adrenalin or noradrenaline into the VMH increased lordosis, an effect prevented by receptor blockers. Because (a) estrogens increase synthetic activity in adrenergic systems in the brain, and (b) activation of adrenergic receptors at the top of the lordosis circuit in VMH increase lordosis; it follows that (c) one way in which estrogens increase lordosis is through heightened adrenergic synapse activation in the VMH.

Histamine Histamine (HA) effects on VMH neurons (to promote lordosis) work differently. HA is well known to play an important role in electrophysiological actions of HA mediated by H1 that are predominantly excitatory, signaling through Gq linked to phospholipase C and then protein kinase C. Ion channels. In the mouse VMH, HA-induced depolarization was mediated by H1, but not H2, receptors and was not affected by the blockade of sodium or calcium channels, but was abolished by potassium channel blockade (Zhou et al. 2007). Further analyses indicated that HA depolarization was due to an inhibition of a potassium leakage current (Zhou et al. 2007). Both estrogens (Pfaff 1999, 2005) and HA are known to increase arousal (see Chapter 8). Estrogens potentiate the depolarizing actions in VMH due to HA administration by inhibiting outward K+ currents (probably the delayed rectifier) that would lead to depolarization.

Muscarinic Cholinergic Receptor As reviewed by the authoritative Anne Etgen, estrogen treatment of ovariectomized rats increases the activity of choline acetyltransferase, the rate-limiting enzyme in acetylcholine synthesis. Altogether, the estrogen effect can be detected at the mRNA, protein, and enzymatic activity levels of analysis. Release of acetylcholine in the hypothalamus is increased by estrogens as well. As far as muscarinic receptors are concerned, Kathie Olsen, working with the neuroendocrine pioneer Richard Whalen, demon-

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strated the estrogen-dependent enhancement of muscarinic agonist 3H-quinuclidinyl benzilate ([3H]QNB) binding in the preoptic area as compared with ovariectomized, estrogen-free controls. A significant sex difference was found in the ability of estrogen to induce [3H]QNB— estrogen was ineffective in altering [3H]QNB binding in either brain region of castrated males. This muscarinic binding goes along with our electrophysiology: Lee-Ming Kow and I used extracellular singleneuron recording in brain tissue slices through the VMH to show that estrogen treatment greatly increased the number of neurons responding to acetylcholine, and that effect depended on muscarinic receptors (Kow and Pfaff 1995; Kow et al. 1995). That is, we analyzed the effects of muscarinic agents on the singleneuron activity of VMH neurons recorded in brain tissue slices of estrogen-primed female rats. All the agonists tested, including acetylcholine (ACh), oxotremorine-M (OM), carbachol (CCh), and McN-A-343 (McN), evoked primarily excitation (80–100  percent), some inhibition (0–20  percent), or occasional biphasic responses (0–8  percent). By comparing the response magnitude and the effectiveness in evoking a response, the rank order for evoking excitation, the primary response, was found to be OM > CCh > ACh approximately McN, which is consistent with that (OM > CCh > McN) for facilitating lordosis reported by others. The consistency and frequency of occurrence suggest that the excitatory electric action of the muscarinic agonists is related to their facilitatory behavioral effect. Experiments with antagonists selective for M1 (pirenzepine), M2 (AF-DX 116 [otenzepad]), and M3 (4-DAMP [[3H]4-diphenylacetoxy-Nmethyl-piperidine methiodide] and p-F-HHSiD) indicate that muscarinic excitations are mediated by M1 and / or M3, but not M2. Because M1 receptors have been shown to be neither sufficient nor necessary to mediate the muscarinic facilitation, the M3 receptor may be crucially involved in this behavioral effect. Autoradiographic assays of binding to [3H]4-DAMP with or without pirenzepine and AF-DX 116 also indicate the presence of M3 receptors in the VMH. Altogether, the electrophysiology links VMH muscarinic synaptic transmission to lordosis. With respect to lordosis behavior, Gary Dohanich and his colleagues, as well as other laboratories, demonstrated facilitation of lordosis by  several receptor agonists that mimic acetylcholine: carbachol,

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bethanechol, eserine, oxotremorine, pilocarpine, and acetylcholine itself. We extended Dohanich’s work by showing that the antagonist N-methyl scopolamine inhibited lordosis (Kaufman, McEwen, and Pfaff 1988). In the matched the literature on other muscarinic receptor antagonists, atropine, hemicholinium, and scopolamine all blocked lordosis. Further, we used tritiated N-methyl scopolamine to show that when we put the muscarinic blocker into the VMH it did not diffuse beyond the VMH. Thus, in the VMH, stimulation through muscarinic cholinergic receptors is sufficient and necessary to facilitate lordosis. Given that (a) estrogens increase cholinergic excitation to VMH neurons, and (b) such cholinergic activity fosters lordosis, it follows that (c) one way in which estrogens facilitate lordosis is through ramping up synthetic activity to produce more VMH excitation through cholinergic receptors.

Gonadotropin-Releasing Hormone and Gonadotropin- Releasing Hormone Receptor Although we often think of neurotransmitters as achieving point-topoint neuronal signaling, neuropeptides, composed of several amino acids, are usually thought of in a more inclusive manner, comprising “systems” with par ticu lar, individual neurobiological themes. We dealt effectively with three of them, the most famous of which, gonadotropinreleasing hormone (GnRH), regulates all aspects of reproductive physiology. Gene expression for the ultimately integrative neuropeptide GnRH was crucial to our thinking about reproductive physiology. Joel Rothfeld entered the laboratory as one of the best distance runners we had ever seen and with a determination to learn in situ hybridization. His results showed that estrogen treatment raised the GnRH levels by an average of 63 percent (Rothfeld et al. 1989). By the mid-1990s we had been doing in situ hybridization in the brain for more than 10 years and wanted to put the technique to good use on a subject we expected to be difficult: the receptor for GnRH (Quiñones-Jenab, Jenab, et al. 1996). The GnRH system is crucial in regulating the reproductive system of female vertebrates. Thus it was

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compelling to analyze the estrogenic regulation of the GnRH receptor mRNA at the cellular level in female rats. Northern blot analysis detected three species (5.0, 4.5, and 1.4 kilobases) of GnRH receptor mRNA in pituitary tissues. The GnRH receptor mRNA levels of all three of these three species were increased by estrogen. Via in situ hybridization we observed a 3.5 times increase in GnRH receptor mRNA levels after 48 hours of estrogen treatment when compared with ovariectomized rats (12 hours of estrogen treatment did not change the GnRH receptor mRNA levels). Similar increases in GnRH receptor mRNA levels by estrogen were also found in female rat pituitary tissue. In situ hybridization analysis identified clusters of anterior pituitary cells that expressed the GnRH receptor mRNA. The estradiol effect depends on increased mRNA levels in these clusters, and a significant increase in the number of pituitary cells that expressed GnRH receptor was observed after 48 hours of estrogen treatment. GnRH neurons themselves do not express substantial levels of ER mRNA (Shivers et al. 1983). Some of the best information available tells us that the effect of estrogen on GnRH expression is mediated by ER- dependent GABA (γ-aminobutyric acid) neurons nearby. Several laboratories have confirmed and extended Joel Rothfeld’s results. One group concluded that enhanced GnRH receptor mRNA expression observed on the day of proestrus is largely due to the actions of estrogen, and other groups have agreed. Physiological concentrations of estradiol increase the steady-state levels of GnRH receptor mRNA in a dose-dependent manner. I note that estrogen-induced elevations of the ligand GnRH and of the mRNA for its receptor could produce a multiplicative hormone effect. What about lordosis? I got the idea for a direct GnRH effect on the behavior about 11:30 on a Wednesday night and had ordered the animals by 9:00 the next morning. The effect was that, clearly, GnRH injection elevated lordosis behavior responses (Pfaff, 1973), a phenomenon quickly replicated by the authoritative neuroendocrinologist Robert Moss in Dallas (for more detail, see Chapter 5). The conclusion: (a) estrogens increase GnRH indirectly and GnRH receptor transcription directly, and (b) GnRH increases lordosis

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behav ior; it follows that (c) one way in which estrogens increase lordosis behav ior is by signaling through the GnRH system.

Oxytocin and Oxytocin Receptor Sookja Kim Chung came to the laboratory after receiving her Ph.D. working with Rochelle Cohen at the University of Chicago, after a successful college career in South Korea that included participation on the national volleyball team. She was strong, as indicated by her work with oxytocin (OT) in addition to her ultrastructural work. She knew that oxytocinergic signaling was important throughout the reproductive axis (see Chapter 5) and that OT-containing synapses could be found in the VMH. In fact, after OT administration during in vitro electrophysiological recordings, it was clear that OT increased the firing of action potentials by VMH neurons (Kow et al. 1991). As a result of these data, Sookja decided to use in situ hybridization to study OT gene expression in preoptic and hypothalamic neurons as well as to determine estrogenic influences on OT expression (Chung, McCabe, and Pfaff 1991). The hybridization findings were confirmed by immunocytochemistry. Using these techniques, we documented OT expression in the medial preoptic area, the nucleus of the anterior commissure, the periventricular neurons, (importantly) the paraventricular and supraoptic nuclei, the perifornical nuclei, as well as the bed nucleus of the stria terminalis. In a new set of experiments, estrogen-free control ovariectomized rats were compared with short-term (2 days) or long-term (2 months) estrogen-treated rats. The most striking estrogen effects came from the preoptic area neurons, in which either short- or long-term estrogen treatment caused an approximately 75  percent increase in the number of grains per cell (i.e., OT mRNA levels per cell). Additional analyses used frequency distributions of the number of grains per cell. In both the supraoptic nucleus and in the nucleus of anterior commissure such graphs revealed subpopulations of neurons in the estrogen-treated groups (compared with controls) that “pushed the curves to the right”—revealed subpopulations with particularly high concentrations of estrogen-dependent OT mRNA expression. To pursue the OT system further, we analyzed the potential estrogen effects on the OT receptor mRNA levels (Quiñones-Jenab et al. 1997) in

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some areas integral to the limbic-hypothalamic system discovered (as described in Chapter 1), namely, the VMH, posterior medial nucleus of amygdala, arcuate nucleus, caudate putamen, CA1 region of the hippocampus, anterior pituitary, and uterine tissue of ovariectomized female rats. Via in situ hybridization we observed a 4.4 times increase in OT receptor mRNA levels in the VMH after 48 hours of estrogen treatment when compared with ovariectomized rats. No other place in the brain had such a large estrogen effect. Later I claim the potential multiplicative actions of estrogens acting at both the levels of ligand transcription and corresponding receptor transcription. Here, the estrogenic effects on OT expression could be multiplied in their effectiveness by estrogenic effects on OT receptor expression. Because pioneers such as Charles Pedersen at the University of North Carolina had shown OT administration to the hypothalamus to be strongly facilitatory and in some cases essential for lordosis, it remained for us to replicate and extend his work. Michael Schumacher at Rockefel ler (now an INSERM leader in Paris), for example, specified the relations of progesterone effects and OT effects on female reproductive behav ior (Schumacher et al. 1990). So we thought that OT receptor activation would be required for high levels of lordosis behavior. To prove this, we used infusions of antisense oligodeoxynucleotides directed against OT receptor mRNA, microinjected directly into the VMH (McCarthy et al. 1994). Control infusions consisted of a scrambled-sequence oligo that had little or no homology to known mRNAs. The OT receptor antisense oligo infusion significantly reduced lordosis frequency and intensity in females primed with estrogen. There were also a significantly greater number of male-rejection behaviors exhibited by antisense-oligo-infused estrogen-treated females versus controls. These behavioral results are supported by our electrophysiological findings. Lee-Ming Kow recorded single-neuron activity from the lordosisrelevant VMH in hypothalamic slices to characterize the electrophysiological actions of OT (Kow et al. 1991). To examine the effects of ovarian steroids on OT actions, we used brain slices prepared from ovariectomized rats either treated with estrogen or not, and some slices were treated with progesterone in vitro. OT affected the activity of a large

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number of VMH—and of those neurons affected, 94 percent responded with excitation. This predominant stimulatory action of OT is consistent with its lordosis-facilitating effect, because increases in the activity of VMH neurons are generally associated with the facilitation of lordosis. Pharmacological analyses with selective OT agonists and antagonists as well as structurally related peptides showed that the excitatory action of OT is, as expected, mediated by OT receptors. Estradiol modulated several aspects of OT transmission. First, it increased neuronal responsiveness to OT, especially at the lowest concentration used (0.2 nM). In addition, it caused neuronal responses to OT to correlate significantly with responses to acetylcholine and norepinephrine, which also can act on the ventromedial hypothalamus to facilitate lordosis. In summary, electrophysiology reinforces our applied genomic results. Thus, knowing that (a) estrogens increase transcription rates in both OT and OT receptor systems; and (b) OT working through its OT receptor facilitates lordosis; it follows that (c) one way in which estrogenic hormones increase lordosis behavior is by revving up the transcription of OT and the OT receptor genes.

Enkephalin and the Delta Opioid Receptor Gary Romano, whose laboratory skills I lauded earlier, not only wanted to study estrogen effects on the opioid peptide gene for preproenkephalin (PPE) but also to determine whether there is a sex difference in steroid hormone regulation of PPE gene expression. Slot blot hybridization analyses of RNA isolated from the VMH indicated that estrogen treatment increased the PPE mRNA levels in the ventrolateral portion of the ventromedial hypothalamic nucleus (VL-VM) of ovariectomized female rats (2.2-fold) but had no mea surable effect on the PPE mRNA levels in gonadectomized males (Romano et al. 1990). Gary’s work was replicated and extended a few years later in my laboratory by Vanya Quiñones-Jenab (Quiñones-Jenab, Ogawa, et al. 1996). In these experiments we studied the effects of estrogen treatment on PPE mRNA expression in female ovariectomized Swiss Webster mice after 0, 1, 6, 12, 24, or 48 hours using the in situ hybridization technique. The surprising aspect of the results was the amount of time

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required for a maximal effect; VMH neurons showed a three times increase in PPE mRNA levels after 48 hours of estrogen treatment when compared with ovariectomized control females. Andrea Lauber, working with Gary, performed what might be considered some of the most impor tant experiments of all for the purposes of this book (Lauber et al. 1990a,b). That is, the hormone effects on PPE mRNA allowed us an opportunity to compare a brain region-specific molecular change with a quantifiable behavior in an animal by individual animal basis. Slot blots were used to mea sure PPE mRNA levels in the VMH as a function of the dose of estrogen administered to ovariectomized rats. Every rat used had been characterized for the ability to display lordosis behavior. Estradiol treatment led to a monotonic dose-dependent increase in PPE mRNA level in the VMH. Lordosis behavior, assessed by two dif ferent types of quantitative assays, also increased monotonically with estradiol dose. The data indicated that an apparent threshold level of PPE mRNA in VMH coincided with the display of reproductive behavior. One potential reason for this came from the ultrastructural work done by Katie Commons when she joined the laboratory years later (Commons and Pfaff 2001). Because enkephalin is thought to act in several brain areas to modulate the activity of GABAergic neurons, we studied the ultrastructural morphology and relationship between neurons containing these neurochemicals using dual-labeling immunocytochemistry in ovariectomized rats, half of which received estrogen replacement. Immunolabeling for enkephalin was almost always detected within axon terminals, while GABA immunoreactivity was more often localized to cell bodies and dendrites. Crucially, axon terminals containing enkephalin immunolabeling provided a major innervation to soma or dendrites containing GABA. That is, over one-third of the axon terminals in contact with GABAimmunoreactive dendrites contained enkephalin. Furthermore, these GABA-immunoreactive dendrites accounted for a fifth of the somatodendritic processes associated with enkephalin-containing axon terminals. These findings support the hypothesis that enkephalin may act in the VMH by inhibiting GABAergic neurons, which could result in the disinhibition of neural circuits relevant for lordosis. Thus, we think that the estrogenic induction of PPE has one of its major sources of

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behavioral import in the disinhibition of VMH neurons as they signal to the midbrain as shown in Chapter 2. There are fewer data on estrogens and δ-opioid receptors (primarily responsible for enkephalin signaling). The most important come from the laboratory of Theresa Milner at Cornell Medical College. Her earliest observations suggested increased δ-opioid receptor internalization and trafficking from dendrites toward cell bodies of hippocampal neurons in females at a time in their cycle when estrogen levels are high. In such females there was also increased colocalization with corticotropinreleasing hormone receptors, better capacity for long-term potentiation of electrical excitability, and more δ-opioid receptor (immunoreactivity) in dendritic spines. Whether these data would hold up for hypothalamic neurons remains to be determined. What about lordosis behaviors? When Jim Pfaus joined the laboratory we knew that previous studies suggested that opioid receptor agonists infused into the lateral ventricles can facilitate (through δ-receptors) the lordosis behavior of ovariectomized rats treated with estrogen and a low dose of progesterone. In an elaborate experiment, we found that hypothalamic application of either δ-receptor agonist [D-Pen2,5]-enkephalin hydrate (DPDPE) or U-50488H [2-(3,4- dichlorophenyl)-N-methyl-N[(1R,2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide;methanesulfonic acid] increased lordosis quotients and lordosis magnitudes, and that the facilitation of lordosis behav ior by δ-receptor agonists is independent of progesterone treatment (Pfaus and Pfaff, 1992). Anne Etgen’s laboratory got similar results and further showed that pretreatment with the selective δ- opioid receptor antagonist naltrindole (NTDL) blocked DPDPE effects on lordosis be hav ior. As expected, Anne’s results showed the specific importance of the VMH. As an additional point she showed that the PR antagonist RU486 blocked receptive (lordosis) and proceptive (“courtship”) behav iors induced by DPDPE. Laboratories in Japan replicated these fi ndings and further showed differences in efficacy among dif ferent chemical forms of enkephalin. The necessity of the enkephalin stimulation in the VMH for high levels of lordosis behavior was proved when Arnaud Nicot joined my laboratory. That is, to assess the physiological role of hypothalamic opioid expression in lordosis behavior, we synthesized a 16-mer oligodeoxynucleotide directed toward PPE mRNA and microinjected it the

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VMH of estradiol-primed ovariectomized rats (Nicot et  al. 1997). Estradiol-induced lordosis behavior was observed in response to a stud male 2 days thereafter. Antisense injections near the ventrolateral portion of the VMH resulted in a significant reduction in lordosis quotient compared with the control treatments. The results were specific to the VMH. We also validated the effectiveness of our antisense VMH treatment both by checking enkephalin immunoreactive levels determined by radioimmunoassay and by using in situ hybridization. Thus, we could say with confidence that enkephalin gene expression in the VMH contributes causally to lordosis behavior. In summary, because (a) estrogens heighten PPE gene expression, and (b) enkephalin helps to cause lordosis behav ior; it follows that (c) one way in which estrogens facilitate lordosis behav ior is through their activation of PPE gene transcription.

Neuronal Nitric Oxide Synthase Solomon Snyder, chief of neuroscience at the Johns Hopkins School of Medicine, had already had a sparkling career marked by several highprofi le discoveries when he found out that nitric oxide (NO) can act as a gaseous transmitter. This was unheard of. As a gaseous neurotransmitter NO, in part, works in tandem with glutamatergic neurons to transmit neuronal excitation. Of course, we wanted to investigate this novel transcriptional system with respect to estrogen action in the brain. Neuronal nitric oxide synthase (nNOS) is an isoform of the enzyme responsible for the synthesis of the gaseous NO. First, Sandra Ceccatelli, now a professor at the Karolinska Institutet, studied expression and estrogen regulation of the genes for NO- synthesizing enzymes (NO synthase, NOS) using in situ hybridization (Ceccatelli et al. 1996). Brains were sectioned and hybridized with antisense riboprobes for neuronal NOS, macrophage NOS, and endothelial NOS. In the hypothalamus, mRNA was clearly detectable only for the neuronal NOS with the probes used. A strong hybridization signal was observed in the hypothalamic cell group of greatest interest—at the top of our lordosis circuit. Quantitative analysis showed an increase in neuronal NOS mRNA in the VMH of the ovariectomized rats treated

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with estradiol benzoate. The increase was mainly in the ventrolateral aspect of the VMH, exactly where the most ER- expressing cells are located. Ilya Rachman working with my longtime collaborator cell biologist Rochelle Cohen replicated and extended Ceccatelli’s results (Rachman et al. 1998). Short-term estrogenic regulation specifically of neuronal nNOS mRNA in the ventrolateral subdivision of the VMH was demonstrated using in situ hybridization. Estrogen-treated animals showed a significantly greater signal in the same portion of the VMH as Sandra Ceccatelli had reported. As a retrograde messenger, NO may mediate some of estrogen’s actions on various inputs to VMH neurons. In parallel work, Ilya Rachman studied the distribution of the enzymes NADPH diaphorase (an NO marker) and NOS in the VMH (Rachman, Pfaff, and Cohen 1996). Some, but not all, neurons in the ventrolateral subdivision of the VMH contained both NADPH diaphorase and brain NOS, as demonstrated by colocalization of these two enzymes in individual cells of this area. That NADPH diaphorase and brain NOS were found in estrogen-binding cells was shown by colocalization of NADPH diaphorase and ER and brain NOS and ER at the light and ultrastructural levels, respectively. Thus, both of these enzymes are in the right place in the hypothalamus to be subject to estrogenic influence (as reported earlier) and impor tant for lordosis (as covered later). nNOS re lordosis: Samuel McDonald McCann was one of the most effervescent figures in American endocrinology. At international meeting after meeting, he was the loud, happy, hard-drinking Yank who could entertain every scientist present late into the evening. With respect to nNOS, as he planned his work he already knew the importance of NO in the neuroendocrine control of reproduction. Indeed, building on the pioneering work of Solomon Snyder of the Johns Hopkins School of Medicine, who had proved NO as a transmitter, McCann provided the evidence that the excitatory transmitter glutamic acid works together with NO to stimulate the release of luteinizing hormone-releasing hormone (now known as GnRH; see Chapter 5) for the ovulation-causing pituitary release of luteinizing hormone (LH). Because it makes sense that female reproductive behavior, exposing the female to predation,

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should be coupled with ovulation, McCann and his colleagues explored the role of NO in lordosis and demonstrated that third ventricle injection of the NO donor sodium nitroprusside, a treatment what would bathe the VMH in NO, increased female reproductive behavior; this in turn could be blocked by inhibitors of NOS. The leading molecular endocrine laboratory of Bert O’Malley at Baylor College of Medicine followed up McCann’s work by studying the mating behav iors of female rats after administration of an inhibitor of NOS, NG-monomethyl- L -arginine, into the cerebral ventricle adjacent to the hypothalamus. This NOS blocker prevented estrogen and progesterone-facilitated lordosis, while a control injection of NGmonomethyl-D -arginine, which does not inhibit NOS, did not inhibit lordosis under the same experimental conditions. Further, microinjection into the 3V of sodium nitroprusside, which spontaneously releases NO, facilitated lordosis. O’Malley’s team concluded, in agreement with McCann, that the NOS / NO system, in addition to fostering ovulation, boosts female mating behavior. Thus, (a) estrogens increase transcription from the NOS gene; and (b) NO activity is both sufficient and necessary to increase lordosis; so it follows that (c) one of the transcriptional systems through which estrogens act to increase female reproductive behav ior is the NOS pathway.

Estrogens Also Trigger Growth Processes in Hypothalamic Neurons Consonant with estrogen- stimulated mRNA and protein synthesis introduced at the beginning of this chapter (and consistent with the requirements for lordosis behav ior), estrogens increase synthesis of ribosomal RNA (rRNA), and the resulting synthetic processes lead to  cell biological signs of neuronal growth. An amplified hormonedependent signal likely results. Katherine  J. Jones entered my laboratory shouting. The younger sister of a bunch of loud boys, she had learned that she had to shout in order to be heard at all. My office in Smith Hall at Rockefel ler was then about 50 meters from the elevator, but I nevertheless could hear Kathy clearly as soon as she got off the elevator at our floor.

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Kathy began by collaborating with molecular biologist Dona Chikaraishi of Duke University to use Dona’s ribosomal RNA probes with in situ hybridization to demonstrate estrogenic stimulation of rRNA synthesis in VMH neurons ( Jones et al. 1986). The background of our thinking was that in tissues throughout the body important for reproduction, sex steroids make those tissues grow. And in many nonneural steroid-sensitive tissues, hormonal regulation of the polymerase I system has been shown to be a major aspect of the mechanisms by which those steroids alter cellular functions. At that time, no effect of steroids on nucleolar gene expression had been reported for any neuronal regions. In our case we used estrogen-free ovariectomized female rats to quantify effects of 6 hours, 24 hours, or 15 days of estrogen treatments on rRNA synthesis in the brain. At 6 hours a highly significant 70 percent increase due to estrogen was observed; at 24 hours a significant 60  percent increase was seen. Surprisingly, at 15 days we saw no effect. I note the size of these hormone-induced inductions because, typically, in the brain changes in transcription rates are not as large as in peripheral tissues. The results were robust whether we quantified grains per neuron or grains per unit area ( Jones et al. 1986). Kathy was dealing with the “polymerase I” system, one of three RNA polymerase systems discovered by my Rockefel ler colleague Robert Roeder. This system is extremely important in cells with high protein synthetic rates and metabolic rates such as neurons. Given the importance of the rRNA system and the strength of her initial fi ndings (as mentioned), Kathy wanted to follow up by determining “precursor / product” relations in transcription from the DNA that encodes rRNA ( Jones et  al. 1990). That is, the initial transcript (“precursor”) is relatively short-lived compared with mature, stable (“product”) rRNA. Thus, two types of ribosomal DNA probes were used for in situ hybridization of the estrogen effect: rDNA with the short-lived external transcribed spacer region (precursor) and rDNA of stable 18S RNA coding region (product). Already at 30 minutes of estrogen exposure, the estrogentreated females had significantly greater precursor rRNA synthesized than control ( Jones et al. 1990). The result was specific in that we did not see results in other parts of the hypothalamus. Likewise, at the protein synthesis level, only three proteins out of 39 whose synthesis was

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elevated by estrogens in the VMN were also among the proteins affected in the preoptic area ( Jones et al. 1988). The VMH result at 30 minutes comprises the fastest genomic effect of hormone actions in the brain. Thinking back now to all of Kathy Jones’s work in the laboratory, it turned out, in a most logical fashion, that at very early time points, the estrogen effect could be seen on the amount of precursor rRNA per VMH neuron. At much later times, after the onset of estrogen treatment, the major effect is on mature, stable product rRNA.

Ultrastructure I had the courage to begin electron microscopic work because of a collaboration I struck up with cell biologist Dr. Rochelle Cohen, a postdoctoral researcher in the Rockefel ler University laboratory of Phillip Siekevitz, a member of the Nobel Prize–winning cell biology team headed by George Palade. With Rochelle’s extremely discriminating methodology we were able to show massive elaboration of the rough endoplasmic reticulum—the “protein synthetic machinery” of individual VMH neurons caused by estrogen treatment (Cohen and Pfaff 1981). When Bob Meisel came to the laboratory he replicated and extended Rochelle’s work (Meisel and Pfaff, 1985). The percentage of VMH neurons with what we called “stacked” endoplasmic reticulum went from 21 percent in the ovariectomized control females to 51 percent in females that had received estrogen treatment for 15 days, a more than two times increase. Further, Bob’s results were specific to the VMH. And the ultrastructural results in the VMH were tightly correlated, on an animal-byanimal basis, with the amount of lordosis behavior. Most relevant to these rRNA results is the function of the nucleolus, the primary site of rRNA synthesis. From studies of several cell types it was known that separation or segregation of nucleolar components can occur when the demand for rRNA by a cell is greater than its synthesis. Thus, we were alert to the appearance of the nucleolus when we compared VMH neurons of ovariectomized control animals with those from animals exposed to estrogens for 15 days. We began with light microscopy at the highest magnifications possible and then moved on to study ultrathin sections stained by sodium tungstate (Cohen, Chung, and Pfaff 1984). We concentrated on the portion of the VMH

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Figure 3.3. Top: Effect of very short-term estrogen treatment (2 hours). (A) A representative ventromedial hypothalamus (VMH) neuronal nucleus, estrogen free. (B) After only 2 hours of estrogen exposure, the nucleus (N) and nucleolus (Nu) are larger and the

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that I had earlier determined has the highest concentration of ER- α expressing neurons. Already at the light microscope level we noticed protuberances on the surfaces of some neuronal nucleoli. In fact, these protuberances were more than two times more frequent in the VMH of estrogen-treated animals than in controls. In every matched pair of animals, the estrogen-treated animal had more nucleolar protuberances than the control in that pair. Then we moved on to the ultrastructural examination. As expected, we found cell nuclei whose nucleoli had aggregations of electron-dense material corresponding to the surface features of nucleoli quantified at the light microscopic level. At high magnification, this material could be seen to be separated from the main part of the nucleolus by a narrow gap, penetrated by stands of this electron-dense material that connected it to the main portion of the nucleolus. We became very interested in this phenomenon, so we then used the sodium tungstate staining method, which differentiates between RNA- and DNA-containing structures in ultrathin sections. The protuberant surface feature was shown to be stained more densely than the nucleolus proper, supporting the interpretation that the protuberance contains DNA and thus could be considered to be nucleolus-associated chromatin, obviously there as a mechanisms for increased rates of synthesis of rRNA (consistent with our VMH data mentioned). Based on the cell biological literature, the obvious interpretation was that VMH neurons exposed adequately to estrogen face a demand for rRNA to support the increased rates of protein synthesis necessary in turn for lordosis behavior. Elsewhere in the laboratory, Kathy Jones, who was cognizant of Rochelle’s and Sookja’s work, undertook a more comprehensive ultrastructural study of VMH neuronal nuclei in the presence or absence of estrogens (Figure 3.3) ( Jones, Pfaff, and McEwen 1985). Several major

nucleus more spherical. Fewer clumps of heterochromatin are scattered throughout the nucleus. In the lower right, a truncated view of a major change: an increase in stacked endoplasmic reticulum within an enlarged cytoplasm. Bottom: Effect of a longer estrogen treatment, behaviorally effective. (C) A representative neuronal nucleus from VMH, estrogen-free control. (D) After a long and discontinuous behaviorally effective estrogen treatment, not only is the nucleus more spherical with fewer dark heterochromatin clumps scattered within the nucleoplasm, but also there is an obvious mass of nucleolus-associated chromatin. (Adapted from Jones, Pfaff, and McEwen 1985.)

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findings emerged: a replication of the discovery of estrogen-associated protuberances on the surfaces of VMH nucleoli, significant alterations in nuclear size and appearance, and another replication of the appearance of massive “stacked” rough endoplasmic reticulum in the VMH neurons after estrogen treatment (increased more than three times by estrogen). Regarding nuclear structure, under the influence of estrogens, VMH neuronal nuclei 1) increased in size, 2) changed toward a spherical shape, 3) reduced the appearance of chromatin clumps adjacent to the nuclear envelope, and 4) the appearance of a homogeneous nucleoplasm. Most amazing, we discovered marked invaginations of the nuclear envelope in the ovariectomized controls that simply disappeared as a result of estrogen treatment. Overall, this ultrastructural study gave evidence of a cascade of nuclear changes that support higher rates of VMH synthetic activity after estrogen treatment, consistent with estrogenic effects on nucleolar ultrastructure, rRNA, increases in several transcriptional systems, and estrogen-induced protein synthesis. We were excited that the ultrastructural results of estrogen treatment extended back to the MCG, consonant with ER and estrogen nuclear binding there, at the next lower step of our lordosis behav ior circuit. We had already reported transsynaptic degeneration in the MCG after VMH lesions, thus revealing that link in the lordosis circuit (Chung, Pfaff, and Cohen 1990b). Now we wanted to look at estrogen effects on synaptic morphology in the central grey (Chung, Pfaff, and Cohen 1988). Estrogen treatment for 20 days (compared with estrogenfree ovariectomized control animals) revealed 1) an estrogen-dependent increase in the number of dense- cored vesicles and a corresponding increase in the number of terminals containing dense- cored vesicles, 2) an increase in the lengths of postsynaptic densities, 3) an increase in the number of densities showing perforations, 4) an increase in the absolute number of synapses per unit area, and 5) an increase in the number of synapses with positive synaptic curvature. It seems likely that the dense cored vesicles contain neurotransmitters or neuromodulators impor tant for central grey neuronal excitation. Likewise, cell biologists had shown the dynamism of postsynaptic densities during the imposition of various physiological changes, even including the suggestion that their perforations could lead to an increase in the number of functional synapses.

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Summarizing these several demonstrations in logical order, in the ser vice of transcriptional systems important for lordosis behavior, estrogens acting in the VMH 1) increase nucleolus-associated chromatin, and, as a result, 2) increase rRNA synthesis, associated with 3) marked enlargement and structural changes in the cell nucleus and nucleoplasm. Assuming that all of these are associated with higher mRNA and protein synthetic rates, it fits that estrogens in the VMH also 4) increase the amount and stacking of the rough endoplasmic reticulum. Thus, in addition to increases in specific transcriptional systems, an overall greater synthetic capacity is part of the program after hormonal treatment. It seems likely that because estrogens heighten rRNA synthesis and VMH neuronal growth, and new mRNA and protein synthesis in the hypothalamus are required for lordosis, these hormone-dependent neuronal growth processes play a role in estrogen-facilitated lordosis behavior. A stronger hormone-dependent signal from the VMH to the central grey in the lordosis circuit (Chapter 2) should result.

Transcription Factor Competition When she was in the laboratory, Maria Morgan, a skilled behavioral neuroscientist, had shown that rendering female mice hyperthyroid significantly reduced lordosis behav ior in estrogen-primed female mice (Morgan, Dellovade, and Pfaff 2000). Neuroanatomist Tammy Dellovade followed that and showed the same result for female rats (Dellovade et  al. 1996): high thyroid hormone results inhibit lordosis. We would use the relative simplicity of nuclear receptor signaling to make a point, as I tell here. My first thought about how to explain the ability of thyroxine to down-regulate female reproductive behavior centered on the overlapping nature of the DNA response elements for T3 and estradiol. As previously mentioned, a consensus ERE, beginning from the 5ʹ end, is AGGTCAnnnTGACCT. The n can be any nucleotide base, but there must be three of them in order to get the spacing right for ERs to bind as dimers. A consensus thyroid hormone response element, starting from the 5ʹ end, is AGGTCA, which essentially is one-half of an ERE. So I thought that high levels of thyroxine would bind to TR and compete

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with ER for DNA binding and thus hormone-regulated transcription. This thinking, plus the collaboration with a TR expert, the Harvard molecular endocrinologist William Chin, led to the three experiments summarized here, and subsequently to the epigenetic (histone modification) work covered in the next section. Tammy Dellovade followed up her previously discussed behavioral work by considering that expression of the enkephalin gene in the VMH of the female rat had been correlated with the per for mance of lordosis behav ior (Dellovade et al. 1999b). By antisense DNA evidence, it has been drawn into a causal role as well. So we explored whether, parallel to these earlier molecular and behavioral results, thyroid hormone coadministration could disrupt the estrogenic induction of PPE mRNA. As expected, estradiol benzoate treatment to ovariectomized rats led to a large and significant increase in PPE gene expression in the VMH. The main point was that this increase was inhibited by coadministration of thyroid hormone. The thyroid hormone interference in PPE gene expression was specific to the VMH, as there were no significant effects in the central nucleus of the amygdala or in the caudate / putamen. These in situ hybridization histochemical results formed a direct parallel both to previous transcriptional mea surements and to the reproductive behavior assays in which thyroid hormones were able to oppose estrogenic facilitation. Further, Tammy Dellovade showed that treatment with exogenous thyroid hormones significantly reduced estrogen effects on the expression of the OT gene (Dellovade et al. 1999a). Previous evidence supported the notion of competitive DNA binding and protein–protein interactions, providing mechanisms for nuclear TR to affect ER function; but whatever the mechanisms, these results with PPE mRNA and OT mRNA reinforced the parallelisms between estrogen-induced gene expression and lordosis behavior. Nandini Vasudevan, fresh in New York from a molecular endocrinology laboratory in a national Indian research center in Bangalore, replicated and extended Tammy’s work. She examined the effect of multiple ligand-binding TR isoforms on the ER-mediated induction of the PPE gene in transient transfection assays in CV-1 cells (Vasudevan, Zhu, et al. 2001). On a natural PPE gene promoter fragment containing two putative EREs, both ER- α and ER-β isoforms mediate a four to five

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times greater induction by estrogen. Cotransfection of TR- α1 along with ER- α inhibited the ER- α transactivation of PPE by approximately 50 percent. However, cotransfection with either TR- β1 or TR-β2 expression plasmids produced no effect on the induction of PPE mediated by ER- α or ER- β. Therefore, under these experimental conditions, interactions with a single ER isoform are specific to an individual TR isoform. By the way, transfection with a TR- α1 DNA-binding mutant could also inhibit ER- α transactivation, suggesting that my idea of competition for binding on the ERE may not be the exclusive mechanism for inhibition. In fact, data with the coactivator SRC-1 suggested that coactivator squelching may participate in the inhibition. In dramatic contrast, when ER-β is cotransfected, TR- α1 stimulated ER-β–mediated transactivation of PPE by approximately eight times over control levels. While not directly related to lordosis behavior, this was the first study revealing specific interactions among nuclear receptor isoforms on a neuroendocrine promoter. These data also suggested that the combinatorics of ER and TR isoforms allow multiple forms of flexible gene regulations in the ser vice of neuroendocrine integration (Vasudevan, Davidkova, et al. 2001; Vasudevan, Koibuchi, et al. 2001; reviewed in Vasudevan, Ogawa, and Pfaff 2002). Y. S. Zhu, a medical doctor with expertise in molecular biology, took up this phenomenon of TR / ER interactions for the purpose of a more thorough molecular analysis (Zhu et al. 2001). Knowing that ER and TR are ligand-dependent nuclear transcription factors and that estrogeninduced PPE gene expression in the hypothalamus is directly related to estrogen-induced lordosis behav ior in the rat, Zhu wanted more mechanistic detail. Using transient transfection and electrophoretic mobility shift assays (EMSA), functional ERE were identified between −437 and −145 base pairs of the rat PPE gene promoter region. Two ERElike elements are present between −405 and −364 of the rat PPE gene promoter, which bind ER- α as demonstrated by EMSA. Estrogen produced a dose- dependent increase in chloramphenicol acetyl transferase (CAT) activity in cotransfection assays with ER-α expression vector and a 437PPE-CAT reporter construct containing 437 base pairs of the rat PPE gene promoter and the CAT reporter gene. Crucially, this estrogeninduced PPE promoter activity was inhibited by liganded TR in transient

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cotransfection assays. The analysis of DNA–protein interactions by EMSA revealed that both ER-α and TR could bind to the ERE in the rat PPE gene promoter. Furthermore, we replicated that estrogen induction of PPE mRNA in the VMH of the ovariectomized female rat was significantly attenuated by concomitant administration of triiodothyronine. Thus, we showed that estrogen regulation of the hypothalamic PPE gene expression is mediated through an ER complex directly interacting with the functional ERE in its promoter region and that this estrogen effect can be modified by thyroid hormones. How these TR / ER interactions come to occur is explored by histone chemical work from Larissa Faustino in the next section. In summary, it is easy to see how the relative simplicity of nuclear hormone receptor signaling to transcription regulating mechanisms could be used to advantage to push forward the field of molecular neuroendocrinology. How do these phenomena come about?

How Transcription Is Altered— Epigenetic Methodology At this point we had proven that several transcriptional systems operating in specific sets of hypothalamic neurons work to produce lordosis behavior. Luckily we were just entering an era in which the regulation of transcription was benefiting from new insights based on protein chemistry. We took advantage of those insights as follows. How do those transcriptional events I documented above come about? How are they regulated? A few years ago, molecular chemical methodology presented me with three choices of paths to follow: DNA methylation, histone N-terminus modifications, and noncoding RNAs. DNA methylation seemed altogether too simple to participate in dynamic, subtle epigenetic alterations during the female’s estrous cycle. At the other extreme, noncoding RNAs were being reported in such large numbers and were so obscure that at that time I could not handle the complexity. Thus, I chose histone modifications. In sum, there were two reasons for me to encourage biochemist Khatuna Gagnidze in my laboratory to begin to study histone protein chemistry as a function of estrogen treatment. First, the chemistry did not seem overwhelmingly complex, yet it has the variety and subtlety that you might expect for

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dynamic adjustments in the central ner vous system regulation of behav ior. Second, and just as impor tant, my laboratory was blessed with the proximity of the Rockefeller University laboratory of C. David Allis, a histone protein chemist of brilliant clarity, who is generous besides. I briefly noted earlier that if and only if ER proteins are liganded by an estrogen molecule will they join together as dimers (two ERs joined) and bind to a consensus ERE (AGGTCAnnnTGACCT) or certain permitted variants of the consensus ERE. But remember that in the usual case there is no such thing as naked DNA. DNA is covered with proteins collectively called chromatin ( because of their classically recognized staining properties). The most important elements of chromatin are basic proteins called histones, the chemistry of which has been explicated in yeast cells. Segments of approximately 146 nucleotide bases of inactive DNA are wound around histone protein assemblies in structures called nucleosomes. The saving grace, for the purpose of freeing up DNA to initiate gene transcription is that one end of some of those histone proteins in nucleosomes (the N-terminus) sticks out of the nucleosome and is susceptible to chemical modification. One kind of modification, acetylation, fosters transcription. Another kind of modification, methylation, will either foster transcription or repress transcription, depending on exactly where—on exactly what— amino acid the methyl group is added. As Dave Allis might say, every amino acid is important in this game. There are other types of modifications, too, but I will not mention them because we have not studied them. So for us, the question was whether we could discover estrogendependent modifications in the VMH that would help to shed light on exactly how estrogens facilitate the transcriptional increases I documented earlier in this chapter. The answer was yes. Biochemist Khatuna Gagnidze, whose calm and unfailingly pleasant demeanor conceals her scientific zeal, entered the laboratory and began the study of estrogenic effects on histone chemistry in the VMH. The minimal latency from estradiol administration to lordosis is 18 hours. During that time, ligand-bound ER, members of a nuclear receptor superfamily, recruit transcriptional coregulators, which induce covalent modifications of histone proteins, thus leading to transcriptional activation or repression of target genes. Khatuna’s study investigated the early molecular epigenetic

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events underlying estrogen-regulated transcriptional activation of the Pgr gene in the VMH of female mice (Gagnidze et al. 2013). Estrogen administration induced rapid and transient global histone modifications in the VMH of ovariectomized female mice. Histone H3 N-terminus phosphorylation (H3S10phK14Ac), acetylation (H3Ac), and methylation (H3K4me3) exhibited distinct temporal patterns facilitative to the induction of transcription. These particu lar histone modifications create a permissive environment for the transcriptional activity necessary for lordosis, within 3 to 6 hours after estradiol treatment. In the VMH, transcription-promoting changes in the H3Ac and H3K4me3 levels of histone H3 were also detected at the promoter region of the PR gene within the same time window. Moreover, examination of histone modifications associated with the promoter of another ER-target gene, whose product facilitates lordosis, the OT receptor (Oxtr), revealed gene- and brain-region specific effects of estrogens. In sum, Khatuna showed that, early responses to estradiol treatment involve highly specific changes in chromatin structure and clearly show how histone modifications in the VMH can promote estrogen- dependent transcription. In fact, raising VMH acetylation levels by inhibiting the enzyme that would break it down (histone deacetylase) can increase lordosis behavior (as illustrated in the next chapter; see Figure 4.3). When Brazilian molecular biology student Larissa Faustino came to the laboratory, Khatuna and I wanted to continue studies of histone modifications in the VMH (Faustino et al. 2015). I wanted to make the experiments simpler, but with Larissa’s insistence we made them more complicated. That is, Larissa referred to Tammy Dellovade’s and Maria Morgan’s data, discussed earlier, that showed inhibiting effects of high thyroid hormone levels on lordosis behavior. And Y.-S. Zhu, also discussed earlier, had shown the binding of TR to a consensus ERE, as I had predicted. Therefore, Larissa hypothesized that high levels of thyroid hormones would inhibit protranscriptional estrogen-dependent histone modifications in the VMH and the preoptic area. Larissa started by replicating our earlier results: as expected, estrogen treatment increased the PR mRNA levels by more than two times and the OT receptor mRNA levels by about two times.

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Indeed, as we hypothesized, making the animals hyperthyroid abolished these transcriptional increases ( just as hyperthyroidism reduces lordosis). But we were surprised to find that the opposite thyroid condition, hypothyroidism, could also reduce the estrogen effect. Epigenetic studies yield the same mixture of predicted and unpredicted results. Two protranscriptional histone modifications were studied on the promoters of the PR and OT receptor genes: histone H3 acetylation and histone H3 lysine 4 trimethylation. First, Larissa replicated Khatuna’s results: for instance, in the VMH, estrogen treatment could increase acetylation about four times (Faustino et  al. 2015). Yes, rendering animals hyperthyroid could interfere with estrogen effects; but, against prediction, rendering animals hypothyroid could as well. And when we went farther, to study gene expression levels for an ER coactivator— SRC-1, a nuclear protein that enhances ER effects on estrogen- dependent genes— again in the VMH estrogen treatment increased SRC-1 mRNA levels by almost two times. Yes, parallel to lordosis, high thyroxine levels blocked the estrogen effect; but a hypothyroid condition did as well. In summary, in this complicated study we got many parallelisms between lordosis behav ior and epigenetic changes on the PR and OT receptor promoters, but the hypothyroid results continue to puzzle me. Thinking of our competitive ER / TR binding theory, Rod Scott, a molecular endocrinologist trained in Glasgow, investigated the activity of an ERE identified in the PR proximal promoter and its interactions with the ER and TR (Scott et al. 1997). In addition, we compared ER and TR interactions on the PR ERE construct with that of the vitellogenin A2 consensus ERE. Electrophoretic mobility shift assays demonstrated that TR does indeed bind to the PR ERE as well as to the consensus ERE sequence in vitro. Further, these two EREs were differentially regulated by T3 in the presence of TR. T3 in the presence of TR- α increased transcription from a PR ERE construct approximately fivefold and had no inhibitory effect on estrogen induction. Similarly, T3 also activated a β-galactosidase reporter construct containing PR promoter sequences spanning −1400 to +700. In addition, the TR isoforms β1 and β2 also stimulated transcription from the PR ERE construct by fivefold to sixfold. A TR α mutant lacking the ability to bind AGGTCA sequences in vitro failed to activate transcription from the PR ERE construct,

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demonstrating dependence on DNA binding. In contrast to its actions on the PR ERE construct, TR- α did not activate transcription from the vitellogenin A2 consensus ERE but rather attenuated estrogenmediated transcriptional activation. Surprisingly, attenuation from the vitellogenin A2 consensus ERE is not necessarily dependent on DNA binding, as the TR- α DNA binding mutant was still able to inhibit estrogen- dependent transactivation. As with Larissa Faustino’s results, our competitive DNA binding theory did not account for all of our data. Instead, divergent pathways must exist for activation and inhibition by TR. Because ER, PR, and TR are all present in VMH neurons, these findings must indicate neuronal integration (regarding TR, probably relevant to the seasonality of reproduction), impor tant for the regulation of female reproductive behav ior. When Mitsuhiro Kawata came to the laboratory, I was re-exposed to traditional Japa nese loyalty. Mitsuhiro (“Mike”) visited the graves of famous Japa nese scientists who had worked in the United States, continued to practice kendo (a Japa nese sword-fighting discipline), and showed me how to make Japa nese tea in the correct manner (using traditional Japa nese pottery). After he returned to Kyoto he became professor of neuroanatomy and, as head of his department there, led quite an ambitious study of sexual development (Matsuda et al. 2011). He knew that epigenetic histone modifications are emerging as important mechanisms for conveyance of and maintenance of the effects of the hormonal milieu on the developing brain. Thus, he hypothesized that alteration of histone acetylation status early in development by sex steroid hormones would be important for sexual differentiation of the brain. His team in Kyoto found that during the critical period for sexual differentiation, histones associated with promoters of essential genes in masculinization of the brain (ER- α and aromatase enzymes) in the medial preoptic area, an area necessary for male sexual behavior, were differentially acetylated between the sexes. Consistent with these fi ndings, binding of histone deacetylase (HDAC) 2 and 4 to the promoters was higher in males than in females. Most importantly, to examine the involvement of histone deacetylation on masculinization of the brain at the behavioral level, his team inhibited HDAC in vivo by intracerebroventricular infusion of the HDAC inhibitor trichostatin A or anti-

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sense oligodeoxynucleotide directed against the mRNA for HDAC2 and HDAC4 in newborn male rats. Aspects of male sexual behav ior in adulthood were significantly reduced by administration of either trichostatin A or antisense oligodeoxynucleotide. These results demonstrated that histone modifications in the brain play a role in the sexual differentiation of behavior. Kawata’s result represented a specific example of what Cambridge University biologist Barry Keverne and I mean when we talk about epigenetic effects in the developing brain (reviewed in Keverne, Tabansky, and Pfaff 2015). Now, in 2016, we have convincing examples of DNA methylation effects and noncoding RNA effects as well as important histone N-terminus modifications. One of the consequences of neonatal androgen exposure during the neonatal critical period for sexual differentiation is the absence of lordosis in the male. Rod Scott, mentioned earlier, came to the laboratory and investigated sex differences with respect to the PR promoter (Scott, Wu-Peng, and Pfaff 2002). He mea sured protein binding on the PR ERE and mRNA levels for PR-A (the shorter transcript) and PR-B (the longer transcript) and compared the results between female and male rats. In both sexes, protein extracts demonstrated an increase in nuclear protein binding activity to a PR ERE after estradiol treatment. However, females’ ERE protein binding was greater than males’. In both cases, reflecting the binding data, estradiol pretreatment led to an increase in PR-B messenger RNA, although this increase was significantly larger in females than in males. Estradiol treatment also led to a significant increase in specific binding of hypothalamic nuclear proteins to the PR ERE from both female and male hypothalamic extracts. The predominance of PR-B over PR-A mRNA in the rat hypothalamus and pituitary, and the quantitative differences between female and male rats could both contribute to the greater responsiveness of female rats to progesterone with respect to control over luteinizing hormone release from the pituitary and lordosis behavior. Rod Scott further analyzed the PR promoter using a construct made from a herpes simplex viral vector (Scott et al. 2003). That is, to demonstrate that estradiol (E) induces transcription via the PR promoter, and to identify sequences within the PR promoter responsible for tissuespecific and hormonal regulation, he used a defective herpes simplex

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virus vector for direct gene transfer into the rat pituitary and brain. A viral amplicon expressing the β-galactosidase gene (for a reporter, lacZ) under the regulation of a 2.1-kilobase PR promoter fragment to create a defective viral vector for gene transfer was microinjected into the brain. In the pituitary, lacZ activity was observed in cells of the anterior lobe. However, no activity was seen in the neurointermediate lobe, demonstrating tissue-specific transcriptional regulation. An approximately six times increase in cells demonstrating β-galactosidase activity was observed in the anterior lobe after treatment with estrogen. Likewise, injection of defective viral vector into the hypothalamus followed by treatment with estrogen resulted in an approximately eight times increase in cells demonstrating β-galactosidase activity including the VMH, the very nerve cell group responsible for estrogen-dependent reproductive behavior. Finally, a more complex point about relations among hormonesignaling neuroendocrine systems: female sexual behaviors are inhibited by stress (reviewed in Magariños and Pfaff 2016). Ironically, it was the stress effect on histone modifications that led us into the cooperation with Dave Allis’s laboratory to begin with. Richard Hunter showed the effect of acute restraint stress on transcriptional-repressive histone methylation in hippocampal neurons (Hunter et al. 2009). He later conceived hippocampal histone H3 lysine 9 trimethylation as suppressing transcription of potentially disruptive transposons (Hunter, McEwen, and Pfaff 2013; Hunter et al. 2012, 2015)—these are the “jumping genes” of Nobel laureate Barbara McClintock.

How Multiple Transcriptional Systems Overdetermine Reproductive Behavior and Synchronize It with Ovulation First of all, we have several interesting examples of potential multiplicative effects of estrogens on transcriptional systems in VMH neurons: 1) both GnRH and the GnRH receptor, 2) both OT and the OT receptor, 3) both tyrosine hydroxylase for the production of norepinephrine and the α1-receptor, and 4) both enkephalin and the δ-opioid receptor showed estrogen sensitivity. Obviously, when both the ligand and the receptor are increased after estrogen treatment, the two hormone effects could multiply. On top of that, the overall trophic effects of estro-

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gens in VMH neurons, as documented earlier, have the potential to multiply all VMH outgoing signals. Further, we have the concept of separate but converging contributions of individual transcriptional systems to regulate lordosis behavior and thus all of the female’s contributions to reproduction, because lordosis is required for fertilization. Consider the eight transcriptional systems I reviewed early in this chapter (Figure 3.1). 1) The PR induction amplifies the estrogen effects because of downstream genes regulated by PR. Additionally, PR induction brings into play the same hormone, progesterone, used to control the ovulatory surge of LH from the pituitary, thus helping to synchronize ovulation and lordosis. The female should not be exposed to predation, as required by mating, if that mating would not be productive of a litter. 2) Likewise, GnRH working through the GnRH receptor promotes mating behavior and is also required for the ovulatory surge of LH, thus synchronizing mating and ovulation. 3) Estrogens enhance the transcription of neuronal nNOS. Release of NO from these neurons stimulates the pulsatile release of GnRH, which not only induces LH release to induce ovulation but also fosters lordosis. Again, there is a synchrony between the endocrine and the behavioral requirements for reproduction. In addition, NO coordinates with glutamatergic neurons to heighten excitability and thus amplify the VMH effect. 4–5) Both adrenergic inputs and cholinergic inputs are neurotransmitters used by ascending brain arousing systems, serving an arousal state necessary for the initiation of mating behavior. Estrogen in the absence of arousal does not work to facilitate lordosis (see Chapter 8). 6) OT working through the OT receptor (Chapter 6) reduces the anxiety-provoking effects of environmental stress: anxiolysis. The female must leave the burrow in order to mate, thus exposing herself to predation. Anxiolysis reduces the behaviordisrupting effects of this stress. 7) Transcription for the neuropeptide enkephalin working through its δ- opioid receptor, both increased by estrogens, can be seen as impor tant for the induction of analgesia. Not only must the female be able to mate whether or not in some degree in pain, but also small females accosted by large males must allow mating to go forward. 8) And all these effects are amplified by the trophic actions of estrogens, as previously detailed, on VMH neurons.

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Putting these findings in historical perspective, I note that decades ago George Beadle and Edward Tatum worked with Neurospora and won the Nobel Prize for their “one gene, one enzyme” concept. Now, working with mammalian reproductive behaviors, I can see clearly that we have patterns of genes governing the expression of patterns of behavior. From this overwhelming list of molecular links between estrogen administration, transcription of specific mRNAs in hypothalamic neurons, and lordosis behavior, it appears clearly that in transcriptional terms lordosis is “overdetermined” by estrogenic actions in VMH neurons. No chances of failure have been risked. Principle inferred: Several transcriptional systems participate to overdetermine the per formance of female reproductive behavior at the time of ovulation. I have conceived how their separate contributions dovetail to contribute to the behavior’s regulation. One step deeper, we use epigenetic methodologies to investigate how those transcriptional mechanisms are themselves controlled. In the next chapter I will show how this field of work was the first to establish causal gene / behavior relations in the vertebrate brain.

Further Reading Ceccatelli, S., L. Grandison, R. E. M. Scott, D. W. Pfaff, and L.-K. Kow. 1996. “Estradiol Regulation of Nitric Oxide Synthase mRNAs in Rat Hypothalamus.” Neuroendocrinology 64: 357–363. Chung, S.  R., J.  T. McCabe, and D.  W. Pfaff. 1991. “Estrogen Influences on Oxytocin mRNA Expression in Preoptic and Anterior Hypothalamic Regions Studied by in Situ Hybridization.” Journal of Comparative Neurolog y 307: 281–295. Chung, S. R., D. W. Pfaff, and R. S. Cohen. 1988. “Estrogen-Inducted Alterations in Synaptic Morphology in the Midbrain Central Gray.” Experimental Brain Research 69: 522–530. ———. 1990a. “Projections of Ventromedial Hypothalamic Neurons to the Midbrain Central Gray: An Ultrastructural Study.” Neuroscience 38: 395–407. ———. 1990b. “Transneuronal Degeneration in the Midbrain Central Gray following Chemical Lesions in the Ventromedial Nucleus: A Qualitative and Quantitative Analysis.” Neuroscience 38 (2): 409–426. Cohen, R. S., S. R. Chung, and D. W. Pfaff. 1984. “Alteration by Estrogen of the Nucleoli in Nerve Cells of the Rat Hypothalamus.” Cell and Tissue Research 235: 485–489. 120

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Cohen, R. S., and D. W. Pfaff. 1981. “Ultrastructure of Neurons in the Ventromedial Nucleus of the Hypothalamus in Ovariectomized Rats with or without Estrogen Treatment.” Cell and Tissue Research 217: 451–470. Commons, K. G., and D. W. Pfaff. 2001. “Ultrastructural Evidence for Enkephalin Mediated Disinhibition in the Ventromedial Nucleus of the Hypothalamus.” Journal of Chemical Neuroanatomy 21: 53–62. Davis, P. G., B. McEwen, and D. W. Pfaff. 1979. “Localized Behavioral Effects of Tritiated Estradiol Implants in the Ventromedial Hypothalamus of Female Rats.” Endocrinology 104: 898–903. Dellovade, T.  L., H.  K. Kia, Y.-S. Zhu, and D.  W. Pfaff. 1999a. “Thyroid Hormones and Estrogen Affect Oxytocin Gene Expression in Hypothalamic Neurons.” Journal of Neuroendocrinology 11: 1–10. ———. 1999b. “Thyroid Hormone Coadministration Inhibits the EstrogenStimulated Elevation of Preproenkephalin mRNA in Female Rat Hypothalamic Neurons.” Neuroendocrinology 70: 168–174. Dellovade, T. L., Y.-S. Zhu, L. Krey, and D. W. Pfaff. 1996. “Thyroid Hormone and Estrogen Interact to Regulate Behav ior.” Proceedings of the National Academy of Sciences of the United States of Amer ica 93: 12581–12586. Devidze, N., J. A. Mong, A. M. Jasnow, L.-M. Kow, and D. W. Pfaff. 2005. “Sex and Estrogenic Effects on Co- expression of mRNAs in Single Ventromedial Hypothalamic Neurons.” Proceedings of the National Academy of Sciences of the United States of Amer ica 102 (40): 14446–14451. Faustino, L. C., K. Gagnidze, T. Ortiga- Carvalho, and D. W. Pfaff. 2015. “Impact of Thyroid Hormones on Estrogen Receptor-Alpha-Dependent Transcriptional Mechanisms in Ventromedial Hypothalamus and Preoptic Area.” Neuroendocrinology 101: 331–346. Gagnidze, K., and D. W. Pfaff. 2016. “Epigenetic Mechanisms: DNA Methylation and Histone Protein Modification.” In Neuroscience in the 21st  Century: From Basic to Clinical. 2nd edition. Edited by D. W. Pfaff and N. D. Volkow. Berlin: Springer, 1939–1978. Gagnidze, K., D. W. Pfaff, and J. A. Mong. 2010. “Gene Expression in Neuroendocrine Cells during the Critical Period for Sexual Differentiation of the Brain.” Progress in Brain Research 186: 97–111. Gagnidze, K., Z.  M. Weil, L.  C. Faustino, S.  M. Schaafsma, and D.  W. Pfaff. 2013. “Early Histone Modifications in the Ventromedial Hypothalamus and Preoptic Area following Oestradiol Administration.” Journal of Neuroendocrinology 25: 939–955. Gagnidze, K., Z. M. Weil, and D. W. Pfaff. 2010. “Histone Modifications Proposed to Regulate Sexual Differentiation of Brain and Behavior.” Bioessays 32 (11): 932–939. Hunter, R. G., K. Gagnidze, B. S. McEwen, and D. W. Pfaff. 2015. “Stress and the Dynamic Genome: Steroids, Epigenetics and the Transposome.” Proceedings of the National Academy of Sciences of the United States of Amer ica 112 (22): 6828–6833. HORMONAL REGULATION OF GENE EXPRESSION IN THE BRAIN

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Hunter, R. G., K. J. McCarthy, T. A. Milne, D. W. Pfaff, and B. S. McEwen. 2009. “Regulation of Hippocampal H3 Histone Methylation by Acute and Chronic Stress.” Proceedings of the National Academy of Sciences of the United States of Amer ica 109: 17657–17662. Hunter, R. G., B. S. McEwen, and D. W. Pfaff. 2013. “Environmental Stress and Transposon Transcription in the Mammalian Brain.” Mobile Genetic Elements 3 (2): e24555. Hunter, R. G., G. Murakami, S. Dewell, M. Seligsohn, M. E. Baker, N. A. Datson, B.  S. McEwen, and D.  W. Pfaff. 2012. “Acute Stress and Hippocampal Histone H3 Lysine 9 Trimethylation, a Retrotransposon Silencing Response.” Proceedings of the National Academy of Sciences of the United States of Amer ica 109 (43): 17657–17662. Hunter, R. G., D. W. Pfaff, and B. S. McEwen. 2016. “Epigenetic Effects of Stress on the Mitochondrial Genome.” Proceedings of the National Academy of Sciences of the United States of Amer ica (in press). Jones, K. J., D. M. Chikaraishi, C. A. Harrington, B. S. McEwen, and D. W. Pfaff. 1986. “In Situ Hybridization Detection of Estradiol-Induced Changes in Ribosomal RNA Levels in Rat Brain.” Brain Research: Molecular Brain Research 1: 145–152. Jones, K. J., C. A. Harrington, D. M. Chikaraishi, and D. W. Pfaff. 1990. “Steroid Hormone Regulation of Ribosomal RNA in Rat Hypothalamus: Early Detection Using in Situ Hybridization and Precursor-Product Ribosomal DNA Probes.” Journal of Neuroscience 10 (5): 1513–1521. Jones, K. J., B. S. McEwen, and D. W. Pfaff. 1988. “Quantitative Assessment of Early and Discontinuous Estradiol-Induced Effects on Ventromedial Hypothalamic and Preoptic Area Proteins in Female Rat Brain.” Neuroendocrinology 48: 561–568. Jones, R.  J., D.  W. Pfaff, and B.  S. McEwen. 1985. “Early Estrogen-Induced Nuclear Changes in Rat Hypothalamic Ventromedial Neurons: An Ultrastructural and Morphometric Analysis.” Journal of Comparative Neurology 239: 255–266. Kaufman, L. S., B. S. McEwen, and D. W. Pfaff. 1988. “Cholinergic Mechanisms of Lordotic Behav ior in Rats.” Physiology and Behavior 43: 507–514. Keverne, E. B., D. W. Pfaff, and I. Tabansky. 2015. “Epigenetic Changes in the Developing Brain: Effects on Behav ior.” Proceedings of the National Academy of Sciences of the United States of Amer ica 112: 6789–6795. Kow, L.-M., A. E. Johnson, S. Ogawa, and D. W. Pfaff. 1991. “Electrophysiological Actions of Oxytocin on Hypothalamic Neurons in Vitro: Neuropharmacological Characterization and Effects of Ovarian Steroids.” Neuroendocrinology 54: 526–535. Kow, L.-M., A. W. Lee, C. Klinge, J.-A. Gustafsson, and D. W. Pfaff. 2016. “Molecular and Cellular Mechanisms for Estrogenic Effects on Brain and Behav ior.” In Hormones, Brain and Behavior. 3rd edition. Edited by D. W. Pfaff and M. Joels. Cambridge: Elsevier, in press. 122

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Kow, L.-M., and D. W. Pfaff. 1985. “Estrogen Effects on Neuronal Responsiveness to Electrical and Neurotransmitter Stimulation: An in Vitro Study on the Ventromedial Nucleus of the Hypothalamus.” Brain Research 347: 1–10. ———. 1995. “Functional Analyses of α1-Adrenoceptor Subtypes in Rat Hypothalamic Ventromedial Nucleus Neurons.” European Journal of Pharmacology 282: 199–206. ———. 1998. “Mapping of Neural and Signal Transduction Pathways for Lordosis in the Search for Estrogen Actions on the Central Ner vous System.” Behavioral Brain Research 92: 169–180. Kow, L.-M., Y. F. Tsai, N. G. Weiland, B. S. McEwen, and D. W. Pfaff. 1995. “In Vitro Electro-Pharmacological and Autoradiographic Analyses of Muscarinic Receptor Subtypes in Rat Hypothalamic Ventromedial Nucleus: Implications for Cholinergic Regulation of Lordosis.” Brain Research 694: 29–39. Kow, L.-M., G. D. Weesner, and D. W. Pfaff. 1992. “Alpha 1-Adrenergic Agonists Act on the Ventromedial Hypothalamus to Cause Neuronal Excitation and Lordosis Facilitation: Electrophysiological and Behavioral Evidence.” Brain Research 588: 237–245. Krebs, C., E. Jarvis, J. Chan, J. Lydon, S. Ogawa, and D.  W. Pfaff. 2000. “A Membrane-Associated Progesterone Binding Protein, 25-Dx, Is Regulated by Progesterone in Brain Regions Involved in Female Reproductive Behav iors.” Proceedings of the National Academy of Sciences of the United States of Amer ica 97: 12816–12821. Lauber, A. H., C. V. Mobbs, M. Muramatsu, and D. W. Pfaff. 1991. “Estrogen Receptor mRNA Expression in Rat Hypothalamus as a Function of Genetic Sex and Estrogen Dose.” Endocrinology 129 (6): 3180–3186. Lauber, A. H., G. J. Romano, C. V. Mobbs, R. D. Howells, and D. W. Pfaff. 1990a. “Estradiol Induction of Proenkephalin Messenger RNA in Hypothalamus: Dose-Response and Relation to Reproductive Behavior in the Female Rat.” Brain Research: Molecular Brain Research 8: 47–54. ———. 1990b. “Estradiol Regulation of Estrogen Receptor Messenger Ribonucleic Acid in Rat Mediobasal Hypothalamus: An in Situ Hybridization Study.” Journal of Neuroendocrinology 2 (5): 605–611. Lauber, A. H., G. J. Romano, and D. W. Pfaff. 1991. “Sex Difference in Estradiol Regulation of Progestin Receptor mRNA in Rat Mediobasal Hypothalamus as Demonstrated by in Situ Hybridization.” Neuroendocrinology 53: 608–613. Lee, A. W., A. Kyrozis, V. Chevaleyre, L.-M. Kow, N. Devidze, Q. Zhang, A. M. Etgen, and D. W. Pfaff. 2008. “Estradiol Modulation of PhenylephrineInduced Excitatory Responses in Ventromedial Hypothalamic Neurons of Female Rats.” Proceedings of the National Academy of Sciences of the United States of Amer ica 105 (20): 7333–7338. Lustig, R. H., D. W. Pfaff, and C. V. Mobbs. 1989. “Two-Dimensional Gel Autoradiographic Analysis of the Acute Effects of Estradiol on Protein HORMONAL REGULATION OF GENE EXPRESSION IN THE BRAIN

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Receptor Messenger RNA in Female Rat Pituitary Tissue.” Brain Research: Molecular Brain Research 38: 243–250. Quiñones-Jenab, V., S. Ogawa, S. Jenab, and D. W. Pfaff. 1996. “Estrogen Regulation of Preproenkephalin Messenger RNA in the Forebrain of Female Mice.” Journal of Chemical Neuroanatomy 12: 29–36. Rachman, I. M., D. W. Pfaff, and R. S. Cohen. 1996. “NADPH Diaphorase Activity and Nitric Oxide Synthase Immunoreactivity in Lordosis-Relevant Neurons of the Ventromedial Hypothalamus.” Neuroendocrinology 64: 357–363. Rachman, I. M., J. R. Unnerstall, D. W. Pfaff, and R. S. Cohen. 1998. “Regulation of Neuronal Nitric Oxide Synthase mRNA in Lordosis-Relevant Neurons of the Ventromedial Hypothalamus following Short-Term Estrogen Treatment.” Brain Research: Molecular Brain Research 59: 105–108. Romano, G. J., A. Krust, and D. W. Pfaff. 1989. “Expression and Estrogen Regulation of Progesterone Receptor mRNA in Neurons of the Mediobasal Hypothalamus: An in Situ Hybridization Study.” Molecular Endocrinology 3: 1295–1300. Romano, G.  J., C.  V. Mobbs, R.  D. Howells, and D.  W. Pfaff. 1989. “Estrogen Regulation of Proenkephalin Gene Expression in the Ventromedial Hypothalamus of the Rat: Temporal Qualities and Synergism with Progesterone.” Brain Research: Molecular Brain Research 5: 51–58. Romano, G. J., C. V. Mobbs, A. Lauber, R. D. Howells, and D. W. Pfaff. 1990. “Differential Regulation of Proenkephalin Gene Expression by Estrogen in the Ventromedial Hypothalamus of Male and Female Rats: Implications for the Molecular Basis of a Sexually Differentiated Behav ior.” Brain Research 536: 63–68. Rothfeld, J.  M., J.  F. Hejtmancik, P.  M. Conn, and D.  W. Pfaff. 1987. “LHRH Messenger RNA in Neurons in the Intact and Castrate Male Rat Forebrain Studied by in Situ Hybridization.” Experimental Brain Research 67: 113–118. ———. 1989. “In Situ Hybridization for LHRH mRNA following Estrogen Treatment.” Brain Research: Molecular Brain Research 6: 121–125. Schumacher, M., H. Coirini, D. W. Pfaff, and B. S. McEwen. 1990. “Behavioral Effects of Progesterone Associated with Rapid Modulation of Oxytocin Receptors.” Science 250: 691–694. Scott, R. E., X. S. Wu-Peng, M. G. Kaplitt, and D. W. Pfaff. 2003. “Gene Transfer and in Vivo Promoter Analysis of the Rat Progesterone Receptor Using a Herpes Simplex Virus Viral Vector.” Brain Research: Molecular Brain Research 114: 91–100. Scott, R. E. M., S. Wu-Peng, and D. W. Pfaff. 2002. “Regulation and Expression of Progesterone Receptor mRNA Isoforms A and B in the Male and Female Rat Hypothalamus and Pituitary following Oestrogen Treatment.” Journal of Neuroendocrinology 14: 175–183.

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Scott, R. E. M., S. Wu-Peng, P. M. Yen, W. W. Chin, and D. W. Pfaff. 1997. “Interactions of Estrogen- and Thyroid Hormone Receptors on a Progesterone Receptor Estrogen Response Element (ERE) Sequence: A Comparison with the Vitellogenin A2 Consensus ERE.” Molecular Endocrinology 11: 1581–1592. Shivers, B., R. Harlan, J. Morrell, and D. W. Pfaff. 1983. “Absence of Oestradiol Concentration in Cell Nuclei of LHRH-Immunoreactive Neurones.” Nature 304: 345–347. Vasudevan, N., G. Davidkova, Y. S. Zhu, N. Koibuchi, W. W. Chin, and D. W. Pfaff. 2001. “Differential Interactions of Estrogen Receptor and Thyroid Hormone Receptor Isoforms on the Rat Oxytocin Receptor Promoter Leads to Differences in Transcriptional Regulation.” Neuroendocrinology 74: 309–324. Vasudevan, N., N. Koibuchi, W. W. Chin, and D. W. Pfaff. 2001. “Differential Crosstalk between Estrogen Receptors (ER- α) and (ER-β) and Thyroid Hormone Receptor Isoforms Results in Flexible Regulation of the Consensus ERE.” Brain Research: Molecular Brain Research 95: 9–17. Vasudevan, N., S. Ogawa, and D. W. Pfaff. 2002. “Estrogen and Thyroid Hormone Receptor Interactions: Physiological Flexibility by Molecular Specificity.” Physiological Reviews 82: 923–944. Vasudevan, N., Y. S. Zhu, S. Daniel, N. Koibuchi, W. W. Chin, and D. W. Pfaff. 2001. “Crosstalk between Oestrogen Receptors and Thyroid Hormone Receptor Isoforms Results in Differential Regulation of the Preproenkephalin Gene.” Journal of Neuroendocrinology 13: 779–790. Zhao, X., H. Lorenc, H., H. Stephenson, Y. J. Wang, D. Witherspoon, B. Katzenellenbogen, D. W. Pfaff, and N. Vasudevan. 2005. “Thyroid Hormone Can Regulate Estrogen-Mediated Transcription from a Consensus Estrogen Response Element In Neuroblastoma Cells.” Proceedings of the National Academy of Sciences of the United States of America 102 (13): 4890–4895. Zhou, J., A. W. Lee, Q. Zhang, L.-M. Kow, and D. W. Pfaff. 2007. “HistamineInduced Excitatory Responses in Mouse Ventromedial Hypothalamic Neurons: Ionic Mechanisms and Estrogenic Regulation.” Journal of Neurophysiology 98 (6): 3143–3152. Zhu, Y. S., L. Q. Cai, X. You, Y. Duan, J. Imperato-McGinley, W. W. Chin, and D. W. Pfaff. 2001. “Molecular Analysis of Estrogen Induction of Preproenkephalin Gene Expression and Its Modulation by Thyroid Hormones.” Brain Research: Molecular Brain Research 91: 23–33.

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4 GENE S REGU L ATING BE H AV IOR

Problem: My field was faced with questions about the reliability of studies using gene manipulation. Could we come up with a novel, reliable set of findings for a complete, normal mammalian behavior? How do genes regulate the reproductive behavior that we analyzed at the neuroanatomical, neurophysiological, and transcriptional levels in Chapters 1, 2, and 3? From genetics to physiology to behavior.

Up to this point in our effort to show how a mammalian behav ior is produced, I had spelled out the neuroanatomy of estrogen-binding neurons and the neurophysiology of the neural circuit that produces lordosis behav ior. Because these estrogen-binding neurons serve estrogen-regulated transcription (Chapter  3), I now could use that family of facts to explore directly the impact of specific genes on reproductive behavior. That is, my field developed into applied genomics: a crescendo of neurobiological accomplishment with respect to reproductive (social) behavior as it subsumed genetic and epigenetic techniques. Thus, here I will relate how I used the chance to add up-to-date genetic and genomic technologies further to demonstrate exactly how estrogen-responsive neurons work in the lordosis neural circuitry to manage the behavioral side of reproduction. The fi ndings provided the

first and clearest gene / behavior causal relations available. I show how genetic alterations regulate reproductive behavior. Putting it another way, showing the estrogen-sensitive neurons that regulate our neural circuit for lordosis behavior and now showing how estrogen-sensitive genes regulate that behavior, we demonstrated for the first time how specific signaling chemicals from the body (hormones) acting on specific nerve cell groups regulate an entire instinctive mammalian behavior—a social behavior at that.

Initial Uncertainties Keeping in mind Sir Peter Medawar’s phrase about “science being the art of the soluble,” I had focused our studies on a relatively simple, biologically crucial mammalian social behavior, calculating that it would provide a reasonable opportunity for a defi nitive explanation, an explanation with the kind of certainty approaching that of the physical sciences. That is, the intellectual paths that some scientists, including myself, have followed toward behavioral experimentation have traversed into physics as well as biology and other academic territories. Those who have studied physics may well be disposed to strive for demonstrations of universal laws, expressed quantitatively, in the proof of reliable stimulus–response connections. This endeavor can be hard because of the complexity of some stimuli and certain behavioral responses and because of the need to recognize and control the relevant environmental variables. The delineation of causal relations between particular genes and specific behaviors (Pfaff 2001) is even harder than purely behavioral research. The pleiotropy of individual genes and overlapping functions between genes makes for trouble—difficulties of analysis—to begin with. Our lack of understanding of the mechanisms for penetrance of dominant alleles makes it difficult to construct meaningful gene dose–response relationships. Interpretations of gene knockout data can stumble over unexpected compensations, effects on other gene products, and altered endocrine and neuronal feedback loops, as well as a lack of control over the temporal and spatial impact of the genetic manipulation. Nevertheless, as I will show, a significant body of mouse behavior genetics work that depends on modern molecular manipulations has

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emerged. When the experimenter chooses well-controlled stimuli and biologically important responses of enough simplicity, and their connections are driven by identified neuroendocrine or neurochemical influences of sufficient power, he or she can gain reliable, important knowledge. Therefore, it was surprising when John Crabbe at Oregon Health & Science University reported that he and his colleagues got somewhat dif ferent results on dif ferent campuses during analyses of mouse strain differences in behavior. More disturbing were the echoes in secondary sources that seemed to reinforce the old stereotype that many behavioral results are unreliable. Brutally clear are the effects of genes whose alterations lead to developmental pathologies in the cerebellum causing simple sensory-motor control abnormalities. A deletion within the RAR-related orphan receptor- α gene produces the staggerer mouse, showing abnormalities in cerebellar Purkinje cells associated with severe motor ataxia. This phenotype can be contrasted with the whole-body action tremors characteristic of the vibrator mouse. Here the pathology is due to a retroposon insertion in an intron of the phosphatidylinositol transfer protein α gene, preventing a normal accumulation of its RNA. Mutation of the subunit of a voltage-gated calcium channel could produce the tottering phenotype, which includes both ataxia and paroxysmal dyskinesis. So those motor abnormalities were clearly replicable, and that literature gave me the confidence to go forward to explain entire, biologically crucial, instinctive behaviors. I linked genes and consequent neurochemistry to physiology to behavior, and we can see exactly how gene / behavior causal relations can work out.

The Gene for Estrogen Receptor α We investigated the role of gene expression of the estrogen receptor α form (ER- α) in the regulation of female reproductive behavior by using ER- α knockout (αERKO) mice, females deficient specifically for the ER- α but not the ER-β gene (Ogawa, Eng, et al. 1998). Estrogen-treated, or estrogen plus progesterone–treated αERKO mice did not show any lordosis response under any experimental conditions (Figure 4.1). That is, even with maximal hormonal facilitation, αERKO females could not show lordosis behavior.

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Proceptive posture (percent occurrence)

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Experiment 2 AAV.H1.ER1

Figure 4.1. Microinjection of a viral vector encoding a small-interfering RNA that blocks expression of the estrogen receptor- α gene (AAV.H1.ER1) into ventromedial hypothalamus (A) abolishes courtship behaviors, (B) abolishes lordosis behavior, and (C) increases rejection of the male. (Adapted from Ogawa, Eng, et al. 1998.)

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Detailed behavioral analysis revealed that αERKO females were also deficient in the sexual behavioral interactions that usually precede the lordosis response. They were extremely rejective toward attempted mounts by stud male mice, which could not show any intromissions. During resident–intruder aggression tests, gonadally intact αERKO females were more aggressive toward female intruder mice than were the wild-type (WT) mice. Taken together, a long set of experiments showed that ER- α gene expression plays a key role in female mice, most prominently for sexual behavior but also for other interrelated behaviors such as parental and aggressive behaviors. Emilie Rissman’s laboratory at the University of Virginia replicated our results. She and others have worked for years to turn this set of findings into an entire field of neuroendocrinology. In the most striking experiment, Sonoko Ogawa (1996) put αERKO females in cages with resident stud males; when the females were mounted by the males, the lordosis behavior was much reduced for at least two reasons: less responsiveness to somatosensory stimuli on the hindquarters of the female (which ordinarily would lead to lordosis behavior), and the αERKO females being treated as intruder males by the resident stud males and thus being attacked. When mounted, the αERKO females showed strong rejection behavior, including kicking the male with the hind legs and rapid fl ight. In addition, aggression by αERKO females toward other females was significantly increased, and maternal-like retrieving was reduced. Thus, disruption of the ER- α gene led these females to lose their normal female-typical behav ior and to behave and to be treated more like males. That is, imposing a knockout of the ER- α gene led to a reversal of sex roles. Our approaches to genetic manipulations in the brain were vastly expanded when Michael Kaplitt came to the laboratory in 1988. A virologist, Michael had training in Tom Schenck’s laboratory at Princeton; being a highly articulate and enthusiastic student, he quickly convinced me that we would be able to use viruses to modify gene expression in the brain. Using a replication-deficient herpes simplex virus, he quickly achieved the first use of a virus to express a foreign gene in a mammalian brain (Kaplitt et al. 1991). Then, we published on the first use of adeno-associated virus (AAV) as a gene delivery vehicle for the brain (Kaplitt, Leone, et al. 1994; Kaplitt, Kwong, et al. 1994).

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Just as important, he made us comfortable with the design and use of viral vectors, which Sonoko Ogawa and I would use for years. For example, short hairpin RNA (shRNA) are artificial RNA molecules with a tight hairpin turn that can be used to silence target gene expression via RNA interference, with a relatively low rate of degradation and turnover. Both vectors (the control AAV-luciferase and the experimental AAV-ER- α) contain, in addition, an independent enhanced green fluorescent protein (EGFP) expression cassette under the control of a hybrid cytomegalovirus / chicken β-actin promotor. The control AAV-luciferase was used to control for any potential nonspecific adverse effects of surgery or toxicity of encoded products, and the EGFP was used as a reporter to visualize transduced neurons. Michael’s assistant, Dr. Sergei Musatov, would provide these reagents that would allow us to discover the necessity of patterns of gene expression in specific nerve cell groups such as the ventromedial nucleus of the hypothalamus (VMH) for the proper regulation of instinctive behaviors. Thus, Sergei Musatov started his work with the certainty that ER- α plays a major role in the regulation of neuroendocrine functions and behaviors by estrogens (Musatov et al. 2006). Although the generation of ER- α knockout mice had advanced our knowledge of ER- α functions, gene deletion using this method is global and potentially confounded by developmental consequences. To achieve a site-specific knockdown of ER- α in the normally developed adult brain, we generated an AAV vector expressing a shRNA targeting ER- α. After bilateral injection of this vector into the VMH in female mice, the expression levels of ER- α as well as the estrogen-inducible progesterone receptor (PR) were profoundly reduced despite the continued presence of this receptor elsewhere in the brain. Thus, our manipulation was validated. Functionally, silencing of ER- α in the VMH abolished female lordosis behavior and courtship behaviors while enhancing rejection behaviors by the female. These AAV-mediated long-term knockdown of genes can be used to delineate their effects on complex behav iors in a specific nerve cell group. Working with my imaginative friend, the Norwegian professor Anders Ågmo, we invented “seminatural environments” to extend my earlier findings (Snoeren et al. 2015). We investigated the role of ER- α in the VMH, the preoptic area (POA), the medial amygdala (MePD), and

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the bed nucleus of stria terminalis (BNST) in sociosexual behavior in female rats. The idea was to study behav ior precisely in a larger and more complicated environment than is usually done. The rats were housed in groups in the seminatural environment for 8 days. A group of rats consisted of four females and three males, and all were unfamiliar with each other and sexually inexperienced. Before each group was introduced, the floor in the open area, the tunnels, and the nest boxes were covered with approximately 2 cm of aspen wood chips. Approximately 2 kg of food pellets were put on the floor, close to a corner in the open area. Twelve aspen wood sticks were randomly distributed in the open area, and three red polycarbonate huts were irregularly placed closed to the middle. In addition, six pieces of a small square mat of nonwoven hemp fibers were put in each nest box in the burrow area. To distinguish among the rats on the video record, rectangles were carefully shaved on the back of the rats the day of experimentation. We performed two sets of experiments: the VMH and POA were investigated in the fi rst set, and the MePD and BNST in the second set. In the first, we used a shRNA encoded within an AAV vector directed against the ERα gene to reduce the expression of ERα in the VMH or POA. As mentioned, in comparison to traditional test setups, the seminatural environment provides an arena in which the rats can express their full behavioral repertoire, which allowed us to investigate multiple aspects of social and sexual behavior in groups of rats. As expected, a reduction of ERα expression in the VMH or POA diminished the display of paracopulatory behaviors. This suggests that ERα gene expression in the VMH and POA plays an important role in intrinsic sexual motivation. The reduction in ERα did not affect the general social behavior of the females, suggesting some behavioral specificity to the effect. In the second experiment, the expression of ERα in the MePD and BNST, on the other hand, played no role in these sociosexual behaviors, indicating neuroanatomical specificity of our results. The kinds of locomotion used in courtship behav iors by the females were also of interest (Ogawa et al. 2003). For example, estrogens are known to increase running wheel activity of rodents primarily by acting on the medial preoptic area (mPOA). The mechanisms of this estrogenic regulation of running wheel activity are not completely

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understood. In par tic u lar, little is known about the separate roles of two types of estrogen receptors, ER- α and ER-β, both of which are expressed in mPOA neurons. In this study the effects of continuous estrogen treatment on running wheel activity were examined in male and female mice specifically lacking either the αERKO or the ER-β (βERKO) gene. The mice were gonadectomized and later implanted with either an estradiol benzoate (EB) or with a placebo control pellet. The same mice were also tested for open field activity before and after EB implants. In both female and male αERKO mice, the running wheel activity was not dif ferent from that in the corresponding wild-type (αWT) mice in placebo control groups. But in both females and males it was increased by EB only in αWT, not αERKO, mice. This was the main result: the type of locomotor activity used in courtship behaviors could not be increased by estrogens when the gene for ER- α was knocked out. This result just described was gene specific. In βERKO mice both doses of EB equally increased running wheel activity in both sexes just as they did in βWT mice. Absolute numbers of daily revolutions of EBtreated groups, however, were significantly lower in βERKO females compared with βWT females. Before EB treatment, αERKO female were significantly less active than αWT mice in open field tests, whereas βERKO females tended to be more active than βWT mice. This same pattern did not show up in the open field, indicating some behavioral specificity for these results. The locomotor activity that we mea sured is necessarily brought into play during the search for and approach to a potential partner. An shRNA encoded, as described previously, within an AAV vector directed against the ER- α gene (or containing a nonsense base sequence as a control treatment) was injected bilaterally into the VMH or the posterodorsal amygdala (MePDA) of female rats (Spiteri et al. 2010). After an 80  percent reduction of the expression of ER- α in the VMH, sexual approaches to the male were totally absent even after treatment with estradiol and progesterone. We also replicated the loss of lordosis, mentioned earlier in Sonoko Ogawa’s work, when ER- α expression was suppressed. Suppression of the ER-α in the MePDA lacked these effects, thus showing the neuroanatomical specificity of the VMH result.

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At least part of the requirement for a normal ER- α gene comes from the necessity for ER- α expression in the mPOA. Cooperating again with my Norwegian friend Anders Ågmo, we blocked ER- α expression in the POA specifically, and showed that females with reduced preoptic ER- α expression failed to show enhanced locomotor activity after treatment with EB (Spiteri et al. 2012). What about the nutritional requirements that go along with reproductive cycles? ER- α plays a pivotal role in the regulation of food intake and energy expenditure by estrogens. Although we had documented that a disruption of ER- α signaling in αERKO mice leads to an obese phenotype (Geary et al. 2001), the sites of estrogen action and mechanisms underlying this phenomenon had to be worked out. All we knew was that αERKO worked and βERKO had no effect. We then exploited RNA interference mediated by the AAV vectors mentioned previously to achieve focused silencing of ER- α in the VMH, a key center of energy homeostasis (Musatov et al. 2007). After ER- α expression in the VMH had been suppressed, female mice and rats developed a phenotype characteristic for the metabolic syndrome, marked by obesity, hyperphagia, impaired tolerance to glucose, and reduced energy expenditure. This phenotype persisted despite normal ER- α levels elsewhere in the brain. We think that VMH neurons integrate metabolic regulation appropriate for reproduction with the actual performance of reproductive behavior. Elena Choleris, now a professor at the University of Guelph in Ontario, Canada, was most interested in the processes of social recognition by the female that go along with successful reproduction (Choleris et al. 2003). That is, estrogens regulate many physiological and behavioral processes, some of which are connected to reproduction. These include a variety of social behaviors. We implicated no less than four gene products in a “micronet” required for mammalian social recognition, through which an individual learns to recognize other individuals. Female mice whose genes for the neuropeptide oxytocin (OT) or the ER-β or ER- α had been selectively knocked out were deficient specifically in social recognition and social anxiety. There was a remarkable parallelism among results from these three separate gene knockouts. The data, taken together, showed the involvement in social recognition of the four genes coding for ER- α, ER-β, OT, and the OT receptor (OTR). We thus proposed the four-gene micronet, which links hypotha-

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lamic and limbic forebrain neurons in the estrogen control over the OT regulation of social recognition. In our model, estrogens act on the OT system at two levels: through ER-β they regulate the production of OT in the hypothalamic paraventricular nucleus, and through ER- α they drive the transcription of the OTR in the amygdala. The proper operation of a social recognition mechanism allows for the expression of appropriate social behaviors, be they aggressive or sexual. To confirm and extend that extensive set of experiments, we followed them up with a binary social discrimination assay, in which the animals are given a simultaneous choice between a familiar and a previously unknown individual (Choleris et al. 2006), thus offering a more direct test of social recognition than in the 2003 study. The 2003 results were confi rmed. Differently from their WT controls, when given a choice the knockout mice showed either reduced (βERKO) or completely impaired (OTKO and αERKO) social discrimination. Detailed behavioral analyses indicated that all of the knockout mice had reduced anxiety-related stretched approaches to the social stimulus, with no overall impairment in horizontal and vertical activity, nonsocial investigation, and various other behav iors such as self-grooming, digging, and inactivity. Thus, the gene knockout results were specific to the social domain. Our four-gene micronet model for social recognition still stands. Maternal behavior is still another crucial behavior in the chain of behaviors needed for successful reproduction. We used small interfering RNA (siRNA) silencing of gene expression in neurons of the POA (Ribeiro et  al. 2012). The mPOA has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding ER- α protein is expressed in POA neurons (Chapter 1), and a very dense immunoreactive field of ER- α is found in the preoptic region. ER- α knockout animals show deficits in maternal care (Figure 4.2) and sexual behavior, and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the 2012 study, we used viral-vector–mediated RNA interference to silence ER- α expression specifically in the POA of female mice and mea sured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. The massive reduction in the expression of ER- α in preoptic neurons was validated.

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B

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Figure 4.2. Microinjection of a viral vector encoding a small-interfering RNA that blocks expression of the estrogen receptor-alpha gene (AAV.H1.ER1) into medial preoptic area causes failure of maternal behavior. (A) Lack of licking and nursing the pups. (B) Increased latency to pup retrieval. *, p> ♂’s 9.

Occurs rapidly, before Lordosis10. TR blocks ENK and Lordosis11,12. Antisense DNA reduces Lordosis13.

E → ENKmRNA↑3 through ENK promoter binding4 causing transcription ↑5 in VMN specifically6. Opioid δ receptor ↑ too7.

(Induced by E1,

enkephalin work to produce lordosis behavior in females but not males. (Adapted from Pfaff 1999; for references, see p. 129.)

in the estrogen- primed female rat but not the male. (For references, see Pfaff, 1999, p. 124.) Right: Transcription and function of the neuropeptide

Figure 9.1. Left: At several mechanistic levels, we see how transcription and function of the progesterone receptor (PR) work to produce lordosis behavior

IN VM HYPOTHAL

has PR11, PRB form mRNA12 E induced13 correlated with behavior14

DNA binding10a stronger in ♀’s

in ♀’s not ♂’s17)

(in Estrogen-primed rats,

PR knockout blocks.8 P needs new synthesis.9 P induces new mRNAs.10

behaviors blocked by PR antisense DNA1 or RU4862

INDUCING NEW GENES

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P

only one leads to certain complications. Of course, the male has only the maternal X, whereas the female may have either the maternal or the paternal X to be inactivated in the ser vice of “dosage compensation.” X-inactivation is a complex process. Some genes may actually escape X-inactivation and because many cells will have dosage-sensitive response systems, the inactivation escape can be crucial. In other cases, dif ferent neurons in the brain may have a balanced representation of genes from the maternal or the paternal X; or the representation could be unbalanced, with one or the other vastly predominating. Third, Arthur Arnold reminds us of the pioneering work of Barry Keverne at the University of Cambridge who was the fi rst to show parent- of- origin allelic imprinting of specific genes in the brain. In these cases, DNA methylation of a gene on the maternal X would prevent that gene from being expressed in at least some nerve cells in the male’s brain, whereas in the female the gene could be expressed from the paternal X. DNA methylation of the paternal X yields a simpler situation because the male does not even have it, whereas for a specific gene in the female brain, some neurons may be affected and others not. Fourth, we ask the question of what the inactivated X chromosome is actually doing in neurons of the female brain. If it is attracting epigenetic factors such as histone-modifying enzymes, then the concentrations of those factors on all the other chromosomes may be altered, with considerable consequences for behavior. It is important to think about this alternative set of mechanisms in the context of sexually differentiated brain disorders because the genetic thinking may suggest novel therapeutic approaches.

Biological Import of Sex Differences Biologists legitimately ask the question, “Why do we have two sexes, anyway?” There are many theories out there, and it is beyond the scope of this book to examine them in extenso. However, there seem to be three main lines of thought, which are not mutually exclusive. First, some reasoning focuses on evolution. Regarding natu ral selection of the fittest, mixing strings of the male’s DNA with the female’s offers a much greater set of potential combinations than if such mixing did not occur. There are two results, both beneficial. 1) The off-

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spring have a greater range of characteristics with which to meet the present environment. 2) As the environment changes, members of the species can change accordingly to better meet the challenges of the new environment. Second, most genetic mutations are deleterious. If there is only one copy of that gene, the individual is more susceptible to disease. Having more than one copy protects, especially since a recessive mutation will not be expressed after sexual reproduction during which the healthy gene copy from the sexual mate is dominant. Third, sexual reproduction requires that potential mates seek each other out. Male-male competition, combined with active mate choice by females, helps to guarantee reproduction that produces babies who will compete well in the next generation.

Maladies: Neurodevelopmental Disorders and Autoimmune Disorders It has been popu lar in recent years to discuss the possibility of sex differences in a wide variety of animal and human behaviors. To avoid the possibility of error and to remain close to the physiological and molecular mechanisms, in my laboratory I have avoided wandering into areas of brain and behavior research for which the claimed sex difference is much less impressive than the overlap between sexes.

Autism Many apparent sex differences in behavior are narrowly statistical, with large overlaps between male and female. One exception is autism, which is about four times more frequently found in males than females. Further, in addition to the psychological pain felt by many families who have an autistic child, the lifetime cost to society of that child will be as great as $4 million. These factors led me to seek collaborations with the developmental psychologist Sylvie Goldman and the pediatric neurologist Isabelle Rapin, who are experts in autism. A careful review of the literature led to our “three-hit” theory of the sex difference in autism (Pfaff, Rapin, and Goldman 2011). Brief ly, 1) one hit is the sex difference of being a male; 2) the second hit is the

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large body of evidence that various forms of early stress predispose the child to autism; and 3) the third hit is the classic observation that monozygotic twins have much higher concordance rates for autism than dizygotic twins or just siblings, which points to genetic causes. I will cover all three topics briefly. Sexual differentiation. When I first began thinking about the sex difference in autism, I concentrated on the effects of testosterone on the brain, but Rockefeller postdoctoral researcher Sara Schafsma (2014), who was influenced by the pioneering thinking of Arthur Arnold, brought up other potential causal routes that were not mutually exclusive. As previously mentioned, Arnold had pointed to the incontrovertible fact that an additional genetic route to sex difference was derived simply from the fact that the female has two X chromosomes and the male only one. The resulting process that occurs only in females of X-inactivation and so-called dosage compensation is complex and allows for considerable variability. Further, we must consider the phenomenon of parent-of-origin allelic imprinting of specific genes, through which only the paternal copy or only the maternal copy of a specific gene is preferentially expressed in some nerve cells but not others. Finally, there is the possibility of a more subtle phenomenon called the “heterochromatin sink.” This idea poses that the inactivated X-chromosome binds up epigenetic factors whose concentrations in the cell nucleus, as a result, are reduced, thus affecting gene expression from other chromosomes. All four of these sex differentiation routes could contribute to the large sex difference in autism. Early stress. Growing evidence suggests that exposure to prenatal adversity leads to neurological changes that underlie lifetime risks for mental illness (reviewed in Davis and Pfaff 2014). Beginning early in gestation, males and females show differential developmental trajectories and responses to stress. It is likely that the sex-dependent organization of neural circuits during the fetal period influences the differential vulnerability to mental health problems. Genetic mutations. At the beginning we all hoped that some small number of gene mutations would account for the heritability of autism. 234

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The postsynaptic anchoring protein Shank 3 was thought to be one strong candidate, as was the gene for the nerve cell membrane CTNAP2. But it turned out that mutations in these genes accounted only for an extremely small percentage of cases of autism. Recently, as reviewed by Washington University’s Evan Eichler, when counting de novo mutations it must be concluded that hundreds of genes’ mutations can contribute to the etiology of autism. Nevertheless, we wanted to test our three-hit theory of the sex difference in autism, and got the mouse bearing the CTNAP2 mutation from Daniel Geschwind’s laboratory at the University of California–Los Angeles. In one series of experiments, Sara Schaafsma and I tested the influence of each of the three hits on several measures of social and communicative behavior (Schaafsma et  al. 2016). Hit one was male versus female. Hit two was prenatal stress versus control. Hit three was the CTNAP2 mutation versus wild type. Indeed, several of our behavioral results indicated that animals bearing all three hits (male, stressed, and mutation) had poorer communicative or social behavior than animals with no hits (female, nonstressed, and wild type). By saving brain tissue from every animal with behavioral mea surements, we were able to link poorer behavioral results to deficiencies of corticotropin-releasing hormone receptor 1 (CRHR1) expression in the left hippocampus, and we found that the mRNA result in turn linked to epigenetic changes, acetylation on histone H3 and trimethylation on histone Hlysine4. We consider these results as constituting just one strand of evidence contributing to a much larger picture of molecular neurobiological changes contributing to autism in males.

Autoimmune Disorders Females are more susceptible to autoimmune disorders, sometimes in the ratio of eight or nine to one. In such disorders, for example, either antibodies or T cells can attack self antigens, causing loss of function. There can be implications for behavior as well. Chronic fatigue syndrome, Sjögren’s syndrome, and fibromyalgia, for example, all affect a female’s behavior not only through pain but also through a loss of behavioral energy. An especially devastating condition is multiple sclerosis (MS). MS is a demyelinating disease that can be diagnosed via behavioral changes SEX DIFFERENCE

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with or without brain imaging of lesions. Clinically, the loss of muscular control is obvious, but the disease is not necessarily progressive in a linear manner. Long periods of relapse may occur. The immunology involved is complicated, and heightened expression of certain immune phenotypes in females is thought to be involved, though this is not proven. Mouse models of experimental allergic encephalitis are being used to try to discover the most important underlying causes. Moreover, some data support Arthur Arnold’s approach, saying that simply having an XX sex chromosome complement (independent of ovarian versus testicular development) is part of the problem—that is, it is disease promoting. Principle inferred: The sex difference is strong for sex behaviors, and mechanisms of the difference are partly understood. For many more complex behaviors, sex differences may be claimed, but I have avoided studying them. An exception is autism, for which the sex difference is large and likely of medical importance.

Further Reading Davis, E.  P., and D.  W. Pfaff. 2014. “Sexually Dimorphic Responses to Early Adversity: Implications for Affective Problems and Autism Spectrum Disorder.” Psychoneuroendocrinology 49: 11–25. Fink, G., D. W. Pfaff, and J. Levine. 2012. Handbook of Neuroendocrinology. San Diego: Academic Press / Elsevier. Gagnidze, K., D. W. Pfaff, and J. Mong. 2010. “Gene Expression in Neuroendocrine Cells during the Critical Period for Sexual Differentiation of the Brain.” Progress in Brain Research 186: 97–111. Geary, N., L. Asarian, K. S. Korach, D. W. Pfaff, and S. Ogawa. 2001. “Deficits in E2-Dependent Control of Feeding, Weight Gain and Cholecystokinin Satiation in ER- α Null Mice.” Endocrinology 142 (11): 4751–4757. Lauber, A. H., G. J. Romano, and D. W. Pfaff. 1991. “Sex Difference in Estradiol Regulation of Progestin Receptor mRNA in Rat Mediobasal Hypothalamus as Demonstrated by in Situ Hybridization.” Neuroendocrinology 53: 608–613. Musatov, S., W. Chen, D. W. Pfaff, M. G. Kaplitt, and S. Ogawa. 2006. “RNAiMediated Silencing of Estrogen Receptor α in the Ventromedial Nucleus of Hypothalamus Abolishes Female Sexual Behav iors.” Proceedings of the National Academy of Sciences of the United States of Amer ica 103: 10456–10460.

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Ogawa, S., J. Chan, J.-A. Gustafsson, K. S. Korach, and D. W. Pfaff. 2003. “Estrogen Increases Locomotor Activity in Mice through Estrogen Receptor Alpha: Specificity for the Type of Activity.” Endocrinology 144: 230–239. Ogawa, S., V. Eng, J. Taylor, D. Lubahn, K. Korach, and D. W. Pfaff. 1998. “Roles of Estrogen Receptor-Alpha Gene Expression in Reproduction-Related Behav iors in Female Mice.” Endocrinology 139: 5070–5081. Ogawa, S., U. E. Olazabal, I. S. Parhar, and D. W. Pfaff. 1994. “Effects of Intrahypothalamic Administration of Antisense DNA for Progesterone Receptor mRNA on Reproductive Behavior and Progesterone Receptor Immunoreactivity in Female Rat.” Journal of Neuroscience 14: 1766–1774. Ogawa, S., J. Taylor, D. B. Lubahn, K. S. Korach, and D. W. Pfaff. 1996. “Reversal of Sex Roles in Genetic Female Mice by Disruption of Estrogen Receptor Gene.” Neuroendocrinology 64: 467–470. Pfaff, D. W. 1999. Drive: Neurobiological and Molecular Mechanisms of Sexual Motivation. Cambridge, MA: MIT Press. Pfaff, D. W., I. Rapin, and S. Goldman. 2011. “Male Predominance in Autism: Neuroendocrine Influences on Arousal and Social Anxiety.” Autism Research 4 (3): 163–176. Pfaff, D. W., and R. E. Zigmond. 1971. “Neonatal Androgen Effects on Sexual and Nonsexual Behav ior of Adult Rats Tested under Various Hormone Regimes.” Neuroendocrinology 7: 129–145. Ribeiro A. C., S. Musatov, A. Shteyler, S. Simanduyev, I. Arrieta- Cruz, S. Ogawa, and D. W. Pfaff. 2012. “siRNA Silencing of Estrogen Receptor- α Expression Specifically in Medial Preoptic Area Neurons Abolishes Maternal Care in Female Mice.” Proceedings of the National Academy of Sciences of the United States of Amer ica 109 (40): 16324–16329. Romano, G. J., A. Krust, and D. W. Pfaff. 1989. “Expression and Estrogen Regulation of Progesterone Receptor mRNA in Neurons of the Mediobasal Hypothalamus: An in Situ Hybridization Study.” Molecular Endocrinology 3: 1295–1300. Schaafsma, S., and D. W. Pfaff. 2014. “Etiologies Underlying Sex Differences in Autism Spectrum Disorders.” Frontiers in Neuroendocrinology 35: 255–271. Schaafsma, S., A. Reyes, K. Gagnidze, and D. W. Pfaff. 2016. “Testing a 3-Hit Theory of the Sex Difference in Autism: Experiments with CNTNAP2 Mutant Mice.” Proceedings of the National Academy of Sciences of the United States of Amer ica (in press).

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10 SU M M A RY

This book summarizes how one field of science, neurobiology, has come to emerge as a scientifically important field. Within neuroscience, neuroendocrinology has proven to be the most effective approach to linking neurobiology to modern molecular biology. Putting some of our work together, we see how exploiting the discovery of hormone receptors in the brain contributed to working out the first circuit for a biologically crucial vertebrate behavior and into the discovery of hormone effects on gene expression in the brain. In turn, analyzing an instinctive reproductive behavior turned out to be the best approach for revealing clear gene / behavior relationships. Fifty years of data have proven the reliability of the initial discovery and its subsequent developments. Throughout, rather than asking how the brain works, the book demonstrates a more successful approach: explaining how a biologically important function is physically realized. And the particu lar behavior that we have explained constitutes a mammalian social behavior.

Brief Summary The book bridges explanations of how individual genes in single neuronal types participate in a newly understood neural circuit, to the

production of an entire mammalian behavior. By linking genetics to neuroanatomy to physiology to behavior in a series of causal bridges, this book has offered a complete solution to the problem of how a vertebrate behavior can be produced and regulated. Our work began by discovering exact cellular targets for steroid hormones in the brain 50 years ago, with an emphasis on female-typical sex steroids. The limbic / hypothalamic system discovered is universal among vertebrate brains. Secondly, the estrogen-binding neurons in ventromedial hypothalamus (VMH) were useful in providing one anchoring point (together with sensory inputs and motor outputs) for working out the lordosis behav ior circuit, the fi rst for any vertebrate behav ior. Third, since the nuclear hormone receptors we study are ligandactivated transcription factors we could use applied molecular biological techniques to demonstrate effects of estrogens on gene expression in behaviorally relevant neurons, especially in VMH (Chapter 3). That work now extends to work in the lab concerning estrogenic effects on histone modifications, epigenetic changes useful for understanding the hormone / behavior relationship. Fourth, we used molecular pharmacological manipulations of the VMH and other neurons to prove the gene / behavior causal relations recounted in Chapter 4. Taken together, these four advances proved for the first time exactly how specific chemicals acting in specific parts of the vertebrate brain determine a complete behavioral response.

Reliability Of course, I am pleased that a discovery made 50 years ago not only has held up but has expanded into the physiological and molecular explanations of how a vertebrate behav ior is regulated. At the molecular level, various discoveries were superimposed on each other to constitute a solid body of understanding. For example, the molecular roles of progesterone receptor (PR) gene expression and its consequences were made clear at the hormone binding level by the estrogenic induction of PR messenger RNA (mRNA) and the subsequent PR gene promoter analysis, a phenomenon that is strongly sexually

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differentiated (Pfaff 1999, figure  9.1, p.  125). Further, as transcription factors PRs induce new mRNAs, and PR knockout abolishes lordosis behav ior. DNA-binding phenomena at the PR promoter are stronger in females than males. The neuroanatomy is right as well: PR is strongly bound in the VMH, and induction of such binding is correlated with lordosis behav ior. There, too, estrogens induce PR in females but not males. At these multiple levels of evidence, the molecular biology of estrogen-induced PR gene expression is beyond doubt. The neuropeptide enkephalin features the same type of convincing overlay of data (Pfaff 1999, figure  9.1, p.  129); in the VMH, estrogens induce enkephalin mRNA expression through clear promoter binding phenomena. And the VMH result is neuroanatomically specific. Enkephalin’s δ-opioid receptor is induced as well. DNA binding results are stronger in females than males, and the mRNA induction is stronger in females as well. The enkephalin gene induction by estrogen occurs rapidly, before lordosis behav ior. Antisense reduction of enkephalin mRNA in the VMH reduces lordosis behavior, and molecular competition through thyroid hormone receptor systems blocks both enkephalin mRNA induction and lordosis behav ior. The sexually differentiated aspects of these results are correlated with lordosis. Following from Chapters 3 and 4, we can say that multiple levels of evidence for the enkephalin gene show us the reliability of this topic in the developing field of neuroscience.

Ethology When I entered neuroscience, two schools of thought competed with each other. Ethology, founded mainly by prominent Eu ropean biologists, emphasized the noninvasive study of natu ral behav ior patterns in natu ral environments. Biological psychology, imitating physics and mainly starting in the United States, emphasized controlled experimentation in the laboratory. We combined the two in our work. We chose a complete natu ral behav ior that could be naturally evoked in the laboratory, and used neuroanatomical, electrophysiological, and molecular techniques to demonstrate its physical realization.

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Overdetermined Neural systems that govern reproductive behavior are not allowed to fail. To put it another way, species for which reproductive regulations were shaky would not last. In the ser vice of rock-hard, reliable regulation, lordosis behavior performance is overdetermined in five ways. First, both with respect to lordosis and the ovulatory release of luteinizing hormone from the pituitary, progesterone massively amplifies the estrogen effect (reviewed in Fink, Pfaff, and Levine 2014). Thus, estrogens foster transcription of PR (Chapter 3), and progestins bind to PR, and, as a result, acting both in the VMH and in the pituitary, help to guarantee the timely performance of the reproductive behavior exactly at the time it is needed with respect to ovulation. This produces a strong and stable system. Second, not just one but several transcriptional systems (Chapter3) display a dynamic pattern. (a) Estrogen treatment increases transcription for that par tic u lar gene, and (b) the gene product facilitates lordosis behav ior; therefore, (c) it is logical to infer that the par tic u lar transcriptional system represents one (of several) ways in which neuronal nuclear effects of estrogens drive the behav ior. The results summarized in Chapter 3 show how the probability of reliable behavioral performance is increased by the several transcriptional systems’ separate but converging contributions. Thus, for example, as shown in Chapter 3, adrenergic inputs to VMH neurons would help to guarantee the effects of adequate central ner vous system (CNS) arousal for sexual behaviors that require arousal. And the effect of enkephalins acting through δ-opioid receptors would help to guarantee sexual behavior performance under anxiety-producing environmental circumstances. Third, several neurotransmitter inputs to VMH neurons that cause increased electrical excitability of those neurons overdetermine their adequate electrical signaling to the lordosis behavioral circuit. I have mentioned adrenergic inputs acting through adrenergic α1 receptors, but muscarinic inputs, histaminergic inputs, and glutaminergic inputs are effective as well. Fourth, reliability and stability are shored up by coordinated activity among at least three non-neural organs in addition to nerve cell groups.

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That is, under the influence of the hypothalamus, 1) the anterior pituitary sends gonadotropins into the bloodstream in such a way that 2) cells in the ovary will produce estrogens (and later in the cycle progestins). Estrogens not only affect 3) the uterus (with its attendant innervation) but also feedback to the pituitary and the hypothalamus. The peak of estrogen levels, just before ovulation, is amplified by ovarian progesterone such that the ovulatory discharge of gonadotropins from the pituitary is synchronized with the performance of lordosis behavior. Fifth, though not emphasized here, I have written before about hormone-dependent behavioral funnels by which reproductively competent males and reproductively competent females can be brought to the same place at the same time (Pfaff 1999, 2010). Long ago, my laboratory illustrated this concept with data from hamsters. The male hamster’s testosterone-dependent flank marks (pheromones) attract the female hamster if and only if she has high estrogens. In turn, she deposits estrogen-dependent thick vaginal fluids (pheromones). These odors diffuse in time and space. Both animals go up the pheromone gradient, thus getting closer to each other. At that time, hormone-dependent ultrasounds provide signaling between male and female. Because ultrasounds do not go far (due to absorption on environmental surfaces) and are highly directional, the male and female can easily get together and then use cutaneous signals to direct behavior. Thus, hormone-dependent distance signals (or sexual readiness) lead the animals to get close for hormonedependent proximal signals to occur, and they in turn to come in physical contact with each other for the male to mount and female’s lordosis to permit fertilization.

Symmetry, Hierarchy Our lordosis behavior mechanisms are left-right symmetric, but modules along the anterior-posterior axis are strongly hierarchical. Hypothalamic hormone-dependent cells regulate the excitability of midbrain central grey neurons. In turn these neurons excite both lateral vestibulospinal and medial reticulospinal neurons, which in turn regulate the responses of lordosis-relevant motor neurons to behaviorally relevant sensory inputs. Thus, our neural network is modular and hierarchical.

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The accomplished ethologist Richard Dawkins (1976) sees great advantage in such an organization of mechanisms. In his chapter “Hierarchical Organization: A Candidate Principle for Ethology” he finds such systems to be efficient and stable. His argument fits with my “Overdetermined” section above, as I talked about reliability and stability in lordosis behavior mechanisms.

Universals Discovered In Chapter  1, I explained that the limbic-hypothalamic system of sex steroid-binding neurons discovered in the rat brain turned out to be universal among vertebrates. Likewise, gonadotropin-releasing hormone undergoes neuronal migration during development (Chapter 5) from the olfactory epithelium into the preoptic area / hypothalamus. In Chapter 7 there is a formulation of principles applicable to certain instinctive behavioral regulations. True for vertebrates in general, the principles deal with endocrine logic and the temporal and spatial features of hormone action on behav ior. Most importantly, they say that neural mechanisms have been conserved to provide adaptive body / brain / behav ior coordination in humans as in laboratory animals.

Sex Differences Obviously, sex differences are prominent in the expression of reproductive behav iors (Pfaff 2010). What my laboratory showed (Chapter  9) was that testosterone levels just before and just after birth are critical for the sexual differentiation of behavior, and that the behavioral changes are substantial and permanent. Effectively we are considering the physiological side of libido (Pfaff 1999), analogous to Eric Kandel’s and Joseph LeDoux’s consideration of emotional states. In fact, Sonoko Ogawa’s study showed that we can use genetic manipulation to reverse behavioral sex roles (Ogawa et al. 1996). That said, when considering generalized CNS arousal (Pfaff 2006), I do not expect differences between sexes. The generalized drive that leads to sexual approaches should be the same in female and male.

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Emphasis on the Analyses of Function The approach to the vertebrate brain’s regulation of behavior embodied in this book and espoused for the future has two major features. First, the fi ndings have proven extraordinarily durable; the discovery of hormone receptors in the brain was accomplished 50 years ago, and the ensuing findings have been heavily replicated. With modern methods we systematically followed a neo-Sherringtonian logic to work out the neural circuitry and then delve into detailed cell nuclear chemistry with the tools of molecular biology. Second, in working out the circuitry for a behavior our approach avoided a major mistake which is threatening current neuroscience: confusion about the definitions of nerve cell types. I recently read an article in Science magazine by Emily Underwood entitled “The Brain’s Identity Crisis: Will new tools for classifying neurons put a 150-year-long debate to rest?” Underwood was writing about the efforts of some other wise very smart neuroscientists to classify interneurons. The meeting convened to try this took place, in a poetically correct choice, in the home town of the founder of modern neuroanatomy, the Spanish neuroanatomist Ramon y Cajal. Cajal used the silver nitrate stain discovered by the Italian neurologist Camillo Golgi to describe comprehensively many of the “types” of neurons in the brain. The “Identity Crisis” meeting was convened to define and name “neuronal cell types.” The meeting was a disaster, according to Underwood’s summary. A phrase used: “confusion was on full display.” In the words of Giorgio Ascoli from George Mason University “It was like the Tower of Babel. We thought we are all rational people, we all believe in data, we will create labels and codes and agree to them, and make the Babel tower issue go away. But by lunchtime on the first day, he says, it was clear that the task with which they were charged was, simply put, impossible.” These veteran neuroscientists were going about things the wrong way. For one thing, they were ignoring the subtle epigenetic signals imposing on individual neurons in the most intimate and singular way during development. That is, consider that Inna Tabansky, Joel N. H. Stern, and I (2016) attacked the fallacy of false equivalence among developing cells, for example, the mistaken assumption of identity among cells that look alike. As a highly skilled stem cell biologist and develop-

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mental biologist, Tabansky knew that starting with newly derived neurons that look alike, various ones among them would follow trajectories that would separate them into dif ferent functionally equivalent groups. Her view was not static, but dynamic. We quoted recent discoveries using single cell RNA sequencing, studies that revealed differences in gene expression among cells that might other wise have been (mistakenly) considered identical. Thus, we envision the study of epigenetically influenced trajectories through space and time as the best way to characterize a neuron’s eventual functional identity. Tabansky et  al (2016) is an essay in developmental neurobiology while, in parallel, this book demonstrates the success of approaching the vertebrate brain, likewise asking not “How does the brain work?” but instead asking: HOW is a specific FUNCTION of the central ner vous system accomplished? Approaching the CNS in our way reveals the “identity” of individual neurons not a priori in the form of a dictionary sure to conflate neuronal apples with neuronal oranges, but instead it reveals each neuron’s type of specific contributions in action, as we show how a specific behavioral function is accomplished. That’s the best way to do it.

Public Health It might be considered that, since sexual drives are connected to other instinctive behaviors such as aggression and parental behaviors, and since mechanisms for these primitive behaviors tend to be conserved (Pfaff, 1999, cf. LeDoux, 1996 and Kandel, 2012), understanding exactly how they work will be of use for application to questions in public health.

Further Reading Dawkins, R. 1976. “Hierarchical Organ ization: A Candidate Principle for Ethology.” In Growing Points in Ethology, ed. P. Bateson and R. Hinde. Cambridge: Cambridge University Press, 7–54. Fink, G., D. W. Pfaff, and J. Levine. 2014. Handbook of Neuroendocrinology. San Diego: Academic. Kandel, E. R. 2012. The Age of Insight: The Quest to Understand the Unconscious in Art, Mind, and Brain, from Vienna 1900 to the Present. New York: Random House.

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LeDoux, J. 1996. The Emotional Brain: The Mysterious Under pinnings of Emotional Life. New York: Simon & Schuster. Ogawa, S., J. Taylor, D. B. Lubahn, K. S. Korach, and D. W. Pfaff. 1996. “Reversal of Sex Roles in Genetic Female Mice by Disruption of Estrogen Receptor Gene.” Neuroendocrinology 64: 467–470. Pfaff, D. W. 1980. Estrogens and Brain Function. Heidelberg: Springer. ———. 1999. Drive: Neurobiological and Molecular Mechanisms of Sexual Motivation. Cambridge, MA: MIT Press. ———. 2006. Brain Arousal and Information Theory. Cambridge, MA: Harvard University Press. ———. 2010. Man and Woman: An Inside Story. New York: Oxford University Press. Tabansky, I., J. N. H. Stern, and D. W. Pfaff. 2015. “Implications of Epigenetic Variability within a Cell Population for ‘Cell Type’ Classification.” Frontiers in Behavioral Neuroscience 9: 342. Underwood, E. 2015. “The Brain’s Identity Crisis.” Science 349: 575–577.

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ACKNOWLEDGMENTS

INDEX

ACKN OW L ED GM EN TS

This book, which reflects on the parallel development of my career and of neuroscience, was over fifty years in the making. That is, it has been fifty years since my discovery of hormone receptors in the brain. If I were to thank everyone who has influenced this story of molecular biology built on neurophysiology built on neuroanatomy and behavior, these acknowledgments would be prohibitively long. So suffice it to say that no one’s career in science just happens without the support of a great many people. I am grateful to them all, and throughout the text I try to give credit wherever it is due. Nonetheless, a few people cannot go without mention here because they had a direct effect on the quality of this book. In this regard, I would first like to thank Dr. Sandra Sherman, who helped me clarify my language—at least to the extent that anything so complicated as neuroscience can be “clarified.” Her object was not just to make my presentation clear to the average reader, but to ensure that its intended readers could fully appreciate the development of neuroscience that I seek to describe. Sandra has helped me on previous books, but this one presented a unique challenge. How do you summarize fifty years of hard science, for a group of hard scientists, and still make sure that the

main points do not fail to impress? To the degree that this book does justice to the field, I wish to thank Sandra for all of her help. I next wish to thank Janice Audet, editor at Harvard University Press. With her perspicacity, Janice understood at once the importance of telling a story of fi fty years during the development of this exciting field and making this book valuable to researchers across a rather broad intellectual spectrum. Also appreciated are the anonymous reviewers whose suggestions I have followed, to the benefit of the fi nal manuscript. Finally, I would like to thank the scientists formerly in my laboratory with whom I have spent so much time over the years. You will frequently see the name of Lee-Ming Kow, who still works with me in my laboratory at Rockefeller, and Sonoko Ogawa, who is now a professor at Tsukuba University in Japan. The wonderful research teams we have formed over the years account for the crescendo of scientific proofs— from hormone receptors in the brain, to neuroanatomy, to neurophysiology, to molecular biology—to offer a unified story of how the vertebrate brain regulates behavior. The creative atmosphere at the Rockefeller University allows this kind of ambitious research program to be initiated, maintained, and brought to fruition.

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INDEX

Alpha-adrenergic receptors, 89–91 Amygdala: androgen- concentrating neurons in, 22; connections to, 35; ER- α and OTR transcription in, 137; ER- α and OTR transcription in VMH and POA, 223–224; ER- α gene in medial (MePD), 133–134; estrogen-binding neurons in medial nucleus, 13–14, 17, 19, 20–21, 27; limbic-hypothalamic system, 97; posterodorsal (MePDA), 135–136; vlVMH projections to, 42 Antisense DNA technology: antisense riboprobes for neuronal NOS, 101–102; causal role of enkephalin gene, 110; against ER- α, 186; masculinization, 147; oligodeoxynucleotide (ODN), 149, 185; against OT receptor mRNA, 97; PR blocker for, 88; against PR mRNA, 141, 144, 145f, 229 Arcuate nucleus: estrogen binding in, 14; estrogen treatment effects on OT mRNA, 97; kisspeptin neurons

in, 202; labeled neurons in, 23–24, 26; mRNA encoding for leptin receptor, 197; neuroendocrine functions of, 16; pituitary controls by, 11; sex hormone concentration in, 29 Arginine-vasopressin (AVP): locations and functions, 180–181. See also Oxytocin (OT) Ascending sensory pathways, 55–58. See also Cutaneous / epithelial sensation Autism: genetic mutations, 234–235; sex differentiation factors in, 233–234 Autoimmune disorders, 235–236 Bed nucleus, stria terminalis: amygdala / diencephalon communications, 13; ER- α gene in, 133–134 Behavior- ovulation coordination, 72–73 Brain-body relations: behavioral components, 198–201; body-wide

Brain-body relations (continued) coordination, 195–198; GnRH neuron roles in, 200; leptin, 196–197; principles of behavioral regulation, 207–212; time, 202–207 Cutaneous / epithelial sensation: ascending pathways for, 55; convergence of pathways for, 71–72; dorsal horn processing, 69; estrogen effects on promoters, 55; hindbrain reticular neurons to midbrain, 57; initiation of lordosis, 53–55; neural pathways to spinal cord, 199–201; receptors in genital tissues, 198–199 Descending pathways: hypothalamus to MCG, 58–60; lower brainstem, 60–64; lower brainstem module, 69; medullary reticular formation (NGC), 58–60; medullary / spinal pathway damage, 63; motor neurons in spinal cord, 66f; motor output / motor neurons, 64–67; spinal module for, 69–71 Electrophysiology studies: dorsal root ganglion recordings, 54–55; hypothalamic and estrogensupported action potentials, 34; lateral vestibular nucleus stimulation and lesions, 61–62; MCG lesions and stimulation, 51–53; MCG stimulation with estrogen treatment, 52f; medullary reticular formation stimulation, 62f; motor neuron recordings, 64–65; NGC lesions, 63; NGC neurons to cutaneous stimuli, 56; NGC stimulation, 60–61; OT actions, 97–98; oxytocin receptor (OTR) system, 183–184; stimulation / lesioning of VMH, 45–51; VMH

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neurons, 43, 45–49; VMH stimulation, 50f Endocrine-neural link: brain impact of, 34–37; in vertebrates, 16–29 Enkephalin gene expression: fear response and, 150; in female sexual behav ior, 149; induction by estrogen, 240; in VMH for lordosis, 101, 110. See also Preproenkephalin (PPE) gene Epigenetic changes: in developing brain, 117; by histone modification, 112; stress, 143; X and Y chromosome expression in neurons, 230–232 Estradiol-binding neurons: α1 receptor adrenergic signaling, 89–90; binding capacity of, 13; fear response and, 222–223; neuron groups, 11; nuclear activity of, 10; preoptic implants and maternal behav ior, 185–186; ventromedial hypothalamic neurons, 15 Estrogen: in CNS arousal, 222, 224; effect on muscarinic receptors, 45–46; effects on adrenergic systems, 42, 92; facilitation of gene expression, 85f; feedback regulation by, 29–30; neuronal growth in VMH, 104–105; nNOS transcription enhancement, 119; protein synthesis induced with, 83–84; rRNA synthesis in VMH neurons, 104–105; transcription from NOS gene, 103 Estrogen-binding neurons: amphibians, 18–19; dorsal horn of spinal cord, 55; fish brain, 18; hamster brain, 16; localization of, 10–13; mink, 26–27; primate brains, 27; quantification of activity, 14–15; reptiles, 21–22 Estrogen receptor (ER): α-βERKO behavioral effects, 141; binding, 9;

ER- α and ER- β behav ior comparison, 150; ER-β cloning, 14; in female rat brain, 12f; function, 29; neurons in amphibians, 19f; protein products of, 30–32; as transcription factors, 79; in VMH hypothalamus of monkey, 28f Estrogen receptor α (ER- α): behavioral effects of blocking, 131f; BNST, 133–134; courtship behav ior, 223–224; expression for femalespecific sex behav ior, 229–230; in generalized arousal, 220–221, 222, 224; knockout (αERKO) and lordosis, 130; masculinization of brain, 116–117; maternal behav ior after silencing of, 137; maternal behav ior negated by reduction in, 206; medial amygdala (MePD), 133–134; nutritional requirements, 136; oxytocin-producing neurons and, 15; protein product distribution, 30; sexual incentive behav ior, 204; social recognition gene requirements, 136; VMH, 133–134 Estrogen receptor β (ER- β): βERKO and sexual behav ior, 142; βERKO on courtship locomotion, 135–136; cloning of, 30; lordosis behav ior, 130; mPOA neurons, 135; reproductive behav ior regulation, 139–140; social recognition gene requirements, 136; VMH metabolic regulation, 136 Estrogen response element (ERE): competitive DNA binding, 109–110; competitive TR / ER binding, 115–116; TR / ER interactions, 111–112 Estrogen-responsive genes, 9, 85 Estrogen-sensitive electrical activity, 34 Estrogen-stimulated synthetic events: α-adrenergic receptors, 89; enkeph-

alin and δ- opioid receptor, 98–101; gonadotropin-releasing hormone (GnRH), 94–96; growth process in hypothalamic neurons, 103–109; histamine, 92; hypothalamic neurons, 80–81; muscarinic cholinergic receptor, 92–94; neuronal nitric oxide synthase (nNOS), 101–103; oxytocin (OT) and oxytocin receptor, 96–98; progesterone receptor, 84–88; transcription factor competition, 109–112 Estrogen treatment: in behavioral effects of GnRH, 173; histone modification, 112; ultrastructural changes with, 105–109, 106f Female behav ior: ER- α expression requirement, 141–142. See also Sex differences Forebrain module, 72 γ-aminobutyric acid (GABA) neurons: in estrogenic control of GnRH transcription, 202; estrogenic effects on GnRH mRNA, 176 Gene / behav ior relationships: GnRH knockout effects, 147; for neuropeptides, 146–150; pathology, 128–130; reproductive behav ior, 138–139. See also Kallmann’s syndrome Generalized arousal (GA): arousalrelated transmitters, 216; characteristics of, 218–219; genes for regulation of, 220; ion channels in, 216–217; links to sexual arousal, 222–224; mechanisms of, 219–222; for reproductive behav ior, 215–218 Genes regulating behav ior: causal relationships, 151–152; enkephalin, 149–150; epigenet ics, 143–144; ER- α, 130–139; ER-α and ER-β comparison, 150; ER-β, 139–143; gonadotropinreleasing hormone (GnRH), 147;

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253

Genes regulating behav ior (continued) human behav ior, 151–152; modification of expression in brain, 132; oxytocin, 147–148; progesterone receptor mRNA, 144–146; SRY gene, 150–151; TR isoform effects, 146 Genomic stress response, 143 Genotype / age interactions: in aggressive behav ior, 140–141; ER- β gene expression, 142 Glutamatergic neurons: ER- α expression colocalization with, 15; excitation of GnRH neurons, 175; hormonal synergy with, 15; NO action with, 101; NO effects, 119 GnRH neurons: characteristics of, 175; developmental migration of, 159–160; estrogenic effects on mRNA in, 176; migration in human brain, 164–169; migration in mouse, 162f, 163f; migration in salmon, 164f; migration in vertebrates, 160–164 Gonadotropin-releasing hormone (GnRH): behavioral effects of, 173–174; behavioral effects of knockout, 147; chemistryphysiology-behavior link, 169–176; development migration of GnRH neurons, 159; genes expressing, 5; neuromodulatory action of, 172–173; neuronal migration in development, 152; promotion of mating behav ior / LH surge, 119 Hippocampus: corticosterone binding in, 14, 28; estrogen effects on OTR mRNA, 97; estrogen receptive cells in, 13; gene expression related to autism, 235; labeled pyramidal neurons in, 17, 27; sex hormone concentrating neuron projection to, 35

254

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Histamine (HA): ion channels for depolarization, 47–48; VMH neurons, 92 Histone modification: in developing brain, 117; estrogen-regulated modification in VMH, 143–144; transcription modified by, 113 Hormone-binding neurons: amphibians, 18–21; birds, 23–27; direct hormonal action on neurons, 9; fi sh, 17–18; hamster brain, 16; human brain, 28–29; mouse brain, 17; neuroanatomical connections of, 34–36; primate brains, 27–28; rat brain tissue, 11; reptiles, 21–23 Hormone- dependent behavioral funnel, 212 Hormone receptors: in brain, 10; corticotropin-releasing hormone receptors, 100; discovery of, 4, 9–10; entry point for neural circuit, 41; importance for behav ior, 32–34; ligand-activated transcription factors as, 80; neuronal networks with, 34–37; replication at mRNA level, 29–30; replication at protein level, 30–31; thyroid hormone receptors (TR), 80 Hormones: gene expression in brain, 79–127; genomics, 128–157; impact on brain, 8–39 Hypothalamic neurons: anterior pituitary regulation by, 8; estrogentriggered growth processes in, 103–109; outflow, 41–53; synthetics events in, 80; transcriptional system in, 112–120; transcription factor competition, 109–113 Hypothalamus: anteroventral periventricular nucleus, 228; estrogen- dependent output of, 4–5; labeled neurons in anterior area of, 11. See also specific nuclei of

In situ hybridization: adrenergic- α1 mRNA expression in VMH, 89; enkephalin gene expression, 101; ER gene expression in hypothalamic neurons, 29–31; gene expression related to lordosis, 73; GnRH gene expression, 94–95; GnRH neuronal migration, 160–164; mRNA for ER- α in VMH neurons, 84; NOS, 101–102; OT gene expression, 96–97, 110, 185; PPE mRNA expression, 98, 149, 217–218; PR mRNA levels in VMH, 86–87; rRNA synthesis in VMH neurons, 104 Ion channels: α1 receptor adrenergic agonists, 89; calcium channels, 90; generalized arousal (GA), 216–217; glutamate agonist, 48–49; histamine (HA), 92; link to neural circuitry, 80; muscarinic cholinergic receptors, 90; norepinephrine, 216–217; potassium channel, 47–48 Kallmann’s syndrome: associated mutations in, 168; migration of GnRH neurons in, 151 Kisspeptin, 202, 203 Limbic-hypothalamic system: anatomical connections of, 34–35; central grey extensions of, 11; ER- α protein in, 30–31; OT system in, 96–97, 137; sex hormone receptors in, 10; social recognition behav ior, 136–137; in vertebrates, 26–27, 29, 31, 239, 243 Lordosis behav ior: absence in male, 117; αERKO, 130, 132; antisense DNA against OTR, 148f; antisense vector against PR mRNA, 145f; anxiolysis for, 119; causal estrogeninfluenced gene expression, 83–84; descending systems for regulation, 59; ER- α expression requirement in

females, 141; estrogenic binding for, 32–33; estrogen-sensitive link for, 15; GnRH direct effects on, 170–171; GnRH injection elevation of, 170f; GnRH injection in MCG, 172f; GnRH knockout effects, 147; hierarchical motor control in, 71; histone modification in VMH, 144f; hypothalamic outflow regulation, 49–51; motor neuron effects for, 63; motor response component, 64–66; necessary / sufficient condition for, 41; NOS / NO system in, 102–103; OT effects of stress on, 189–190; OT receptor activity for, 97; oxytocin effects, 184; PR expression for, 88; protein synthesis in VMH, 83; PR transcription in production of, 231f; quantification of response, 67–68; requirements for regulation, 34; sex differences in, 228; somatosensory and VMH convergence, 57; somatosensory initiation of, 53–55; specific genes involved in, 5; supraspinal control required for, 56; thyroxine effects on, 114; transcriptional activity for, 114; ultrastructural correlation with, 105–109; ventromedial nucleus of hypothalamus, 29. See also Neural circuit for reproductive behav ior; Reproductive behav ior Lower brainstem module, 69–70 Luteinizing hormone-releasing hormone (LHRH). See Gonadotropin-releasing hormone (GnRH) Male behav ior. See Sex differences Maternal behav ior: failure with block of expression of ER- α, 138f; mPOA gene expression silencing, 137; OTR agonist reduction of, 186; oxytocin (OT) in, 205–206; pathways for,

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255

Maternal behav ior (continued) 204–205; preoptic estradiol implants and, 185; reduced ER- α expression in preoptic area, 206 Metabolic syndrome, 136 Midbrain central grey (MCG): estrogen effects on, 58; hypothalamic projections to, 43; hypothalamic-brainstem reticular formation interaction, 72; hypothalamic-reticular formation relay, 59–60; VMH projections to, 172 Midbrain module, 71–72 Motor mechanisms. See Descending pathways Muscarinic cholinergic receptors, 62–63, 92–94 Neural cell adhesion molecules (NCAM): GnRH neuronal migration, 166–168, 202; initiation of puberty, 202 Neural circuit for reproductive behav ior: ascending pathways, 55–58; descending pathways, 58–64; entry point for, 15, 238; hypothalamic outflow, 41–53; launching points for analysis, 70f; modular, hierarchical nature of, 69–72; motor mechanisms, 64–68; reticulospinal link, 60; sensory inputs for initiation, 53–55; ventral horn of spinal cord, 66f Neural circuit modules: forebrain, 72; hypothalamic, 43, 72–73; lower brainstem module, 69–70; midbrain, 71–72; pathways of, 70f; spinal, 69–71 Neurotransmitter receptors, 89 Nitric oxide (NO): as gaseous transmitter, 101; retrograde messenger, 102 Nitric oxide synthase (NOS), 101

256

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Norepinephrine: GnRH neuronal suppression by, 175; ion channels, 216–217 Nucleus gigantocellularis (NGC): amplification of motor neuron response, 60–61; axial musculature activation, 60–64; axonal distributions from, 221f; in generalized arousal, 220–221; neurons regulating cortical arousal, 220f; projections to MCG, 57, 58; somatosensory stimuli response, 56; spinal projections to, 57; VMH input to, 60 Overdetermination of reproductive behav ior, 118–120 Oxytocin (OT): brain-behavior (gene coexpression), 182–185; chemistry / molecular biology, 179–182; distribution and actions of, 179–180, 181; estrogen effects on promoters, 183f; estrogen sensitivity of, 182; genes expressing, 5; knockout effects on aggression, 187; OTR facilitation of sexual behav ior, 147; overcoming stress effects, 186; projections from paraventricular nucleus, 180; reduction of stress effects, 187–191; social recognition gene requirements, 136 Oxytocin receptor (OTR): antisense infusion effects, 147; coexpression of ER mRNA, 182; electrophysiology studies, 183–184; estrogen regulation of transcription, 182; protein kinase C (PKC) signaling by isoforms, 183–184 Paraventricular nucleus (magnocellular portion), 11 Polycomb group, 203 Preoptic area (POA): ER- α expression in medial, 138f; ER- α gene in

medial, 133–135; estrogen-binding neurons in medial, 11; suprachiasmatic portion, 11, 13 Preproenkephalin (PPE) gene: effects of estrogen treatment on, 217; expression and lordosis behav ior, 149; hypothalamic expression and lordosis, 149; knockout of PPEKO, 149–150; sex differences in expression, 230. See also Enkephalin gene expression Progesterone receptor (PR): amplification of estrogen effect, 119; estradiol transcription induction via promoter, 117–118; estrogenic induction, 86–87; with estrogen receptor binding, 30; induction of, 84–89; mRNA manipulation for, 143–144; prevention of stress effects on lordosis, 190 Protein synthesis: estrogen induced, 83–84; for gene expression, 81–82 Puberty: DNA and histone modification in, 203; excitatory and inhibitory inputs with, 201–202; genotype / age interactions, 142; GnRH release with, 173; kisspeptin and, 202; leptin and, 196–197 Reproductive behavior: behavioral chain of, 206; body-wide coordination of nonbehavioral components, 195–198; ER- α expression requirement, 142; ER- α gene, 134; ER- α gene knockout effects, 132; female locomotion in courtship, 134–135; harmonization by GnRH, 176; normal chain of events for, 206; OT prevention of stress effects, 190; OTR mRNA interference effects on, 147, 148f; oxytocin binding in, 182; patterns of gene expression for, 133; PR mRNA reduction and, 144, 145f; social recognition gene require-

ments, 136. See also Lordosis behavior Reticulospinal system: motor neuron excitability, 59; synergy with vestibulospinal system, 62–63 Sex differences: autism, 233–234; autoimmune disorders, 235–236; biological import of, 232–233; in brain, 116–117; early testosterone concentrations and, 228–229; ER- α gene disruption in male, 142; ER mRNA regulation by feedback, 29–30; hypothalamic and POA gene expression, 150–151; mechanisms of differentiation, 228–230; protein kinase C (PKC) signaling by isoforms, 183–184; PR promoter, 117–118; stress inhibition of sexual behav ior, 118; X and Y chromosome expression in, 230–232 Sex hormones: lordosis behav ior with, 9–10; receptors in limbichypothalamic system, 4; sleep-wake cycle regulation, 222 Sexual behav ior. See Reproductive behav ior Signaling systems, 6, 8–9, 36 Social behav ior: estrogen / oxytocin linkage in, 186–187; genetic influence on human, 152; mea surement techniques, 148; oxytocin binding in, 182; with TR knockout,  146. See also Reproductive be hav ior Somatosensory stimuli. See Cutaneous / epithelial sensation Spinal module, 69 SRY gene, 150–151 Testosterone: genotype / age interactions in aggression, 140–142; Kallmann’s syndrome, 152, 165; labeled cells in vertebrate brain,

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257

Testosterone (continued) 18–26; progesterone supplementation of, 9; uptake of tritiated, 13 Thyroid hormone receptors (TR), 14, 80; effects on ER-α transcription, 146; opposing effects of isoforms, 146 Transcriptional systems: convergence of pathways for, 119; estrogenregulated in VMH, 113–114; repression, 202–203; VMH, 119–120 Transcription factor competition, 109–113 Ultrastructural studies, 105–109 Ventral premammillary nucleus, 11 Ventrolateral portion of VMH (vlVMH): ER mRNA expression in, 30–31; oxytocin effects on electrical activity, 184

258

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Ventromedial hypothalamus (VMH): antisense treatment of, 101; ascending projections from, 42; AVP neurons in, 181; descending projections from, 42–43; effects of arousal-related neurotransmitters, 216; ER, PR, and TR presence in, 116; ER- α gene, 133–134; estrogen sensitivity of transcriptional systems, 118–120; histamineinduced change in membrane potential, 217f; histone modifications in, 114; NO and NADPH diaphorase in, 102; patterns of gene expression, 133; PPE mRNA expression in vlVMH, 98; projections to MCG, 42–44; ventrolateral subdivision of, 42 Vestibulospinal system: synergy with reticulospinal system, 59, 62–63