Estrogen-Induced Remodeling of Hypothalamic Neural Circuitry

Frontiers in Neuroendocrinology 21, 309 –329 (2000) doi:10.1006/frne.2000.0204, available online at http://www.idealibrary.com on

Estrogen-Induced Remodeling of Hypothalamic Neural Circuitry Loretta M. Flanagan-Cato Department of Psychology and Institute for Neurological Sciences, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6196

For decades, sexual behavior has been a valuable model system for behavioral neuroscientists studying the neural basis of motivated behaviors. One striking example of a change in motivation is the binary switch in sexual receptivity that occurs during the estrous cycle in female rats. Investigations of the neural basis of this change in behavior have fundamentally advanced our understanding of both behaviorally relevant neural pathways and basic mechanisms of steroid action in the brain. These advances have made this behavioral model system a staple of neuroendocrinology. A challenge that remains before us, given our current understanding of the circuitry and chemistry, is to develop a coherent model of how neural plasticity in the hypothalamus contributes to the dependence of this behavior on motivational state. This review will focus on the ventromedial nucleus of the hypothalamus, especially its ventrolateral subdivision. First, the anatomical, neurochemical, and functional aspects of the macro- and microcircuitry of this brain region will be discussed, followed by a discussion of the likely mechanisms of estrogen action within the ventrolateral VMH. Then, the evidence for estrogen-induced neural plasticity will be considered, including a comparison with the effects of estrogen on synaptic organization in other brain regions. Finally, a working model of neural plasticity within the ventrolateral VMH microcircuitry will be presented as a starting point for future experiments to verify or, more likely, revise and expand. KEY WORDS: estrogen; dendritic spines; lordosis; pseudorabies virus; ventromedial hypothalamus. ©

2000 Academic Press

BACKGROUND

Unlike males, female mammals experience cycles in their fertility. These cycles are generated by neurosecretory cells in the hypothalamus, whose secretion controls not only gamete maturation, but also ovarian steroidogenesis. As a consequence of the pattern of hypothalamic–pituitary secretions, females experience concomitant fluctuations in circulating ovarian steroids, specifically estrogen and progesterone. These steroids exert three complementary actions: (1) trophic effects on the female reproductive tract to promote gestation, (2) positive and negative feedback effects on gonadotropin secretion to time ovulation, and (3) numerous effects in the brain to synchronize reproAddress correspondence and reprint requests to L. M. Flanagan-Cato, Department of Psychology, University of Pennsylvania, 3815 Walnut Street, Philadelphia, PA 19104-6196. Fax: 215-8987301. 309

0091-3022/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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ductive status with behaviors that promote mating. This review will focus on the neurobehavioral effects of estrogen. The constellation of behavioral changes that accompany the increased estrogen levels around the time of ovulation include decreased ingestive behaviors (11), increased general locomotor activity (7), decreased behavioral indications of anxiety (100), and altered learning and memory functions (117, 126). Thus, ovarian hormones have global effects on brain function, influencing competing motivated behaviors, emotion, and cognitive functions, and each of these effects may promote reproductive success. Female rodent sexual behavior has served as an illuminating model system to explain the cellular mechanisms underlying these diverse neurological effects of estrogen (97). A useful feature of this behavior is that the effect of estrogen on sexual behavior is quite pronounced, especially when compared with its more modulatory effects on other behaviors. In rodents, sexual receptivity is typified by a reflexive lordosis posture in response to mounting attempts by males. Ovariectomy abolishes this component of female sexual behavior, and estrogen replacement is sufficient to facilitate receptive behavior, although the addition of progesterone treatment is needed to reinstate maximal lordosis response and solicitious behavior (27). Given the robust effects of estrogen on the lordosis reflex, and the salience and biological importance of this behavior, this paradigm of ovariectomy and estrogen replacement has been extensively used to investigate the neural mechanisms of estrogen action.

THE MACROCIRCUITRY OF FEMALE SEXUAL BEHAVIOR

The macrocircuitry governing the motor component of the lordosis reflex has been studied extensively using neurophysiological, electromyographical, lesion, knife cut, and tract tracing experiments, as previously reviewed (97). In brief, the epaxial muscles that execute the lordosis posture are innervated by motor neurons in the lumbar ventral horn. Partial spinal transections demonstrated that descending medullary reticulospinal projections, but not corticospinal projections, are critical for the lordosis reflex. These medullary premotor neurons, in turn, receive input from the periaqueductal gray, which also influences this behavior. The periaqueductal gray is innervated by the hypothalamic ventromedial nucleus (VMH). Behavioral studies have shown that within this hierarchical network, the VMH is a key site for estrogen to facilitate this behavior. The bulk of anatomical evidence in support of this neural pathway for the lordosis response has been obtained in a stepwise fashion with traditional retrograde tracers. However, because centrally injected retrograde tracers can be transported from neighboring, but functionally unrelated, terminal fields, such tracers are susceptible to false-positive results, that is, labeled neurons that are not synaptically connected with the lordosis motor pathway. To avoid this confound, the serial connectivity of the lordosis pathway was examined

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using a transneuronal tracing technique. Specifically, pseudorabies virus (PRV) was injected into the lumbar epaxial muscles, and the presence of PRV in the CNS was assayed at various intervals after infection. The ventral horn of the spinal cord, the medullary reticular formation, the periaqueductal gray, and the VMH were sequentially labeled with PRV (18). Quantification of PRV-labeled cells in the subdivisions of the VMH indicated that the majority of labeled neurons resided in the ventrolateral subdivision of the VMH. This finding is consistent with the data, discussed below, implicating this region specifically in the control of the lordosis response. Thus, the time course of CNS labeling of transneuronal tracer placed in the lumbar epaxial muscles is fully consistent with serial connectivity of the pathway previously implicated in the lordosis reflex, namely, ventrolateral VMH 3 caudal ventrolateral periaqueductal gray 3 medullary reticular formation 3 lumbar ventral horn 3 lordosis-producing muscles. The neurochemical phenotype of the VMH neurons that project to the periaqueductal gray has been explored with traditional tracers, and several peptides, including enkephalin, substance P, and prolactin, have emerged as candidates, as previously reviewed (97). Each of these peptides has been localized to neurons in the ventrolateral VMH. In addition, central application of each of these peptides facilitates the lordosis response. It is not known whether any of these peptides are colocalized with each other or with other neurotransmitters. Thus, it remains uncertain how these peptides may interact to influence sexual behavior. The projection to the periaqueductal gray is recognized as being especially critical for lordosis behavior; however, the VMH has numerous other projection targets (14). A subset of these projections provide direct and indirect connectivity to the nucleus accumbens and the subpallidal region, which may relate to goal-directed motor activity. The VMH also maintains direct projections to the central and lateral nuclei of the amygdala, which may influence emotionally relevant learning and memory processing. VMH projections to the hindbrain and medial amygdala may serve to modulate sensory input received by the VMH. Although these plausible functions have been proposed for VMH projections (14), the specific contributions of these VMH projection targets to sexual behavior have not been well studied. The details of the lordosis-relevant macrocircuitry have been most extensively studied in laboratory rats; however, research in other species indicates that this pathway has been conserved across evolution. In particular, the importance of the VMH for female sexual behavior has been documented in other mammals, ranging from guinea pigs (37), hamsters (70, 118, 121), and cats (61) to sheep (8). Studies in reptiles, such as whiptail lizards (53, 104), have indicated that this neural mechanism is phylogenetically old. Furthermore, electrophysiological recordings in nonhuman primates have supported an active role of the VMH in female sexual behavior in primates (4). Thus, it seems safe to say that the connectivity and estrogen modulation of VMH circuitry in rats may apply to diverse other species.

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Anatomical tracing techniques have identified numerous forebrain afferents to the VMH (26, 54, 67). In general, anterograde and retrograde studies agree that the VMH receives dense projections from the medial preoptic area, the anterior hypothalamic area, the corticomedial amygdala, the ventral subiculum, the peripeduncular nucleus, and the dorsomedial nucleus of the hypothalamus. Substantial inputs also arise from the bed nucleus of the stria terminalis, lateral septum, medial parvocellular paraventricular nucleus (PVN), ventral premamillary nucleus, and periaqueductal gray. In terms of the hindbrain, serotonin fibers arise from the pontine and mesencephalic raphe nuclei (128), the noradrenergic projection presumably originates in A1 and/or A2, and a cholecystokinin (CCK) projection arises from the superior part of the lateral parabrachial nucleus (35, 45, 47, 134). Brain regions that send projections to the VMH share several features. First, they all have estrogen receptorcontaining neurons (21, 96, 116, 119). Second, these forebrain regions all are activated by female sexual behavior (98, 99), although the hindbrain afferents have not been as well studied in this regard. And third, the VMH maintains reciprocal projections with most of these regions (14). Thus, VMH afferents form a distributed network of estrogen-responsive neurons that participate in mating behavior. A plausible function for these afferents would be to relay male-originating sensory information, including chemosensory, flank, and vaginal– cervical cues. Electrophysiological studies showed that VMH units responded to somatosensory and olfactory cues. Furthermore, estrogen treatment increased the percentage of cells responding to these stimuli (12, 16). Studies mapping the induction of immediate early gene expression also have demonstrated that VMH neurons are activated by olfactory, flank, and vaginal– cervical stimulation (23, 98, 99). Flank stimulation induces a modest expression of Fos in the VMH, whereas vaginal– cervical stimulation and mating induce robust expression of Fos in the VMH, regardless of estrogen treatment (98). As an alternative to integrating male-originating sensory cues, afferents to the VMH may relay environmental cues, such as circadian phase or photoperiod and/or energy status cues. At least one source of vaginal– cervical information is the noradrenergic projection to the neuropil surrounding the VMH, which may be directly responsive to estrogen (43, 110). As previously reviewed (25), this pathway appears to relay vaginal– cervical information to the VMH. During mating encounters, copulation itself, but not visual, olfactory, flank, or perineal stimulation, increases extracellular norepinephrine levels in the VMH. However, the effect of vaginal– cervical stimulation on norepinephrine release in the VMH depends on the motivational state, as it only occurs in the presence of estrogen. One of the best-characterized channels of sensory information received by the VMH is pheromonal, as reviewed previously (24). The VMH is needed for male-originating cues, such as pheromones, to enhance lordosis (103). Vome-

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ronasal information may be carried by the significant projections from the medial amygdala and the preoptic area to the VMH, with gonadotropin releasing hormone (GnRH) serving as one of the neurotransmitters in the pathway from the preoptic area. Infusions of a GnRH agonist into the VMH facilitate lordosis and increase the firing rate of VMH neurons. Messenger RNA for the GnRH receptor has been localized to the VMH (48) and GnRH fibers are found in the neuropil of the ventrolateral VMH (76). Thus, for this lordosis-relevant exteroceptive cue, there is a well-described neural pathway with at least one known neurotransmitter. Other projections to the VMH that specify somatosensory and/or visceral information remain uncertain. One candidate is the CCK projection from the superior part of the lateral parabrachial nucleus (PBN). CCK is expressed in PBN neurons with projections to the ventrolateral VMH (35, 45, 47, 134). CCK levels and CCK receptor binding activity in the VMH are regulated by estrogen, perhaps through an indirect mechanism (78). Behavioral studies indicate that CCK action in the VMH inhibits lordosis (5). Although the neuroanatomy, neurochemistry, and regulation of this pathway have been well-studied, the physiological information it provides to the VMH remains conjectural. Several other behaviorally relevant pathways have been neurochemically identified, including a serotonergic projection from the hindbrain (66, 128) and oxytocinergic and enkephalinergic pathways from the PVN (28, 132). In each case, the actions of the neurotransmitters on VMH neuronal activity correlates with their effects on sexual behavior (59) but the physiological signal remains unclear. In summary, for a few of the afferents to the VMH, the neurotransmitter, behavioral effect, and likely physiological signal have been well characterized. However, the functions and chemical mediators of certain forebrain connections, including the major inputs from the lateral septum, the bed nucleus of the stria terminalis, and the anterior hypothalamus are the least understood. Investigations of the specific physiological information these pathways carry, how they are chemically coded, and where they synapse within the dendritic arbor of VMH neurons will provide insight into the integration that occurs within the VMH.

ESTROGEN ACTION IN THE VMH

The genomic mechanism of estrogen action in the VMH was inferred from the nuclear location of the receptor protein and the fact that behavioral effects were disrupted by drugs that blocked messenger RNA or protein synthesis (102, 130). In addition, estrogen treatment markedly up-regulates the cellular machinery for protein synthesis, including increases in nuclear area and in stacked rough endoplasmic reticulum (52). Later, when the estrogen receptor was cloned (55), its membership in the superfamily of steroid receptors provided further details about its mechanism of action (6, 15). In particular, ligand binding is thought to induce a conformational change in the protein that

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allows the receptor to interact with specific DNA sequences, estrogen response elements, which transactivationally increases the activity of polymerase on the expression of the adjacent reading frame. These considerations have lead to a host of studies on the effects of estrogen treatment on VMH gene expression. Estrogen treatment is associated with changes in the expression of several dozen proteins in the VMH (50). Although the identities of all the estrogenregulated proteins have not been completely determined, many examples are related to chemical neurotransmission, including peptide neurotransmitters, neurotransmitter receptors, and synapse-related proteins. Relatively recently, a novel subtype of the estrogen receptor was discovered (60, 86), leading to the designations ER␣, the original, and ER␤, the more recent. For the purposes of this review, it is important to note that the VMH seems to express the ER␣ subtype exclusively (115). Therefore, female transgenic mice with disrupted expression of the ER␣ sustain a major deficit in sexual behavior (93). However, such a deficit is not apparent in transgenic animals with a null mutation for ER␤ (92). In addition to the well-established role for the genomic sequelae of ER␣ activation in the VMH, nongenomic mechanisms may participate as well. As reviewed previously, in certain other brain areas estrogen may alter neuronal firing rate and neurotransmitter release with a latency of less than 2 min, which is considered too rapid to be secondary to changes in gene expression (29). In some cases, such effects of estrogen have been blocked by drugs that interfere with membrane-associated signaling pathways (40, 77). Such findings are intriguing in the light of recent studies that have claimed membrane localization of the classical estrogen receptor (91, 105). Such nongenomic mechanisms may extend to the role of the VMH in sexual behavior. In particular, in the VMH slice recordings, estrogen causes a change in potassium ion conductance within 3 to 5 min of application (81). Thus, it remains possible that nonnuclear estrogen receptor action may provide additional signaling that promotes this behavior. Estrogen-induced changes in gene expression also alter neurophysiology in the VMH, as reviewed previously (97). A time course study showed that certain electrophysiological effects of estrogen did not occur until after 3 days of continuous estrogen exposure, which correlated with changes in sexual behavior (122). In addition, blockade of action potentials with the local application of tetrodotoxin to the VMH prevents estrogen-mediated lordosis behavior (41). The estrogen-induced increase in the excitability of VMH neurons may be in part secondary to changes in gene expression that affect neuronal sensitivity to various neurotransmitters known to promote sexual behavior, including norepinephrine, GnRH, and oxytocin (58, 59). In general, neurotransmitters that enhance lordosis increase the firing rate of VMH neurons. Because estrogen treatment increases both the rate of action potentials and the responsiveness to lordosis-relevant sensory stimuli and neurotransmitters, it has been proposed that excitation in the VMH, rather than inhibition, is critical to mediate the effect of estrogen on lordosis (95). Furthermore, neurotransmitters arising from different afferents to the VMH converge on an intracellular final common

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pathway for the production of lordosis (95). Specifically, a striking proportion of the neurotransmitter–receptor systems that both promote lordosis and increase the firing rate of VMH neurons share a common signaling pathway involving inositol trisphosphate formation and intracellular calcium mobilization (56, 82). This “molecular 3 electrical 3 behavior” model of the neurobehavioral effects of estrogen has been an important synthesis in explaining the transduction of a steroid signal to a neural signal and, ultimately, a behavioral response. A cluster of studies have suggested that in addition to an estrogen-induced neural activation that is correlated temporally with the appearance of lordosis, some immediate activating effect of estrogen also occurs. In particular, the administration of either a ␥ aminobutyric acid (GABA)-A receptor agonist anesthetic or an N-methyl-D-aspartate (NMDA) receptor antagonist at the time of estrogen administration blocks the effect of estrogen on sexual behavior (30, 107). Both of these results suggest that at a time before estrogen is behaviorally effective, and perhaps before many of the genomic effects have occurred, some neuronal excitation is present, which is necessary for the behavior to ultimately occur. In fact, this neural activation appears to mediate some of the genomic effects of estrogen (101). Such findings hint at a cascade of effects, perhaps reverberating, across multiple neuronal elements within the VMH. However, the details remain sketchy about the series of cellular and intercellular events that sets the stage for sexual behavior to occur, as well as the interrelationships between estrogen, neuronal activation, and gene expression. Because of the abundance of estrogen receptor-containing neurons in the VMH, it has been common to interpret various effects of estrogen as being mediated within the neurons that express estrogen receptors. However, it is important to consider that various afferents to the VMH also express estrogen receptors, including projections from the hindbrain, preoptic area, and amygdala, allowing for indirect effects. Furthermore, considering the evidence for multiple neuronal elements within the local VMH network, local effects also may be transsynaptic. Therefore, when considering the cascade of estrogen action in the VMH, it will be important to distinguish direct intracellular effects of estrogen in the VMH from those that are secondary to changes in local interneuron activity. A better understanding of the synaptic organization of the VMH would aid in unraveling these likely combinatorial effects of estrogen. In addition to the evidence that neuronal excitation in the VMH promotes lordosis, behavioral pharmacology studies also have revealed a role for disinhibitory mechanisms. In particular, local infusion of an NMDA receptor agonist at the time of behavioral testing inhibits the lordosis response (57, 73). Conversely, a similar infusion of a GABA-A receptor agonist into the VMH promotes sexual behavior (74). As reviewed previously, estrogen regulates the GABAergic system in the VMH (72). Immunocytochemistry studies have detected GABAergic neurons within the primate VMH (62). There are several possible reasons that electrophysiological studies have not revealed an inhib-

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itory circuit within the VMH. First, the function of this network may be altered by either anesthetics or the denervations intrinsic to slice recordings. Second, many recording studies focused on projection neurons, whereas the inhibitory elements instead may involve local interneurons. Although electrophysiological experiments have not delineated inhibitory components within the VMH, the behavioral pharmacology experiments have indicated the need for their inclusion in models of VMH function. The findings that estrogen treatment can affect the electrical properties of VMH neurons within 3 min (81), increase gene expression within 30 min (49), and alter protein levels in a matter of 2 h (51) are somewhat incongruous with the 18- to 24-h latency for estrogen to promote sexual behavior (39). One explanation for this temporal discrepancy would be secondary or tertiary waves of genomic effects that produce necessary changes in neural activity. An alternative, although not mutually exclusive, explanation would be that primary changes in gene expression may produce subsequent changes in local connectivity that are required for behavioral receptivity, as discussed below. At any rate, the temporal discrepancy between nongenomic and genomic effects of estrogen and the onset of behavioral receptivity suggests some sort of cascade (97), but the cellular details remain elusive. In summary, the genomic mechanisms implied by the general function of steroid receptors may have been deceptively simple. In addition to the welldocumented genomic mechanisms, there may be concurrent nongenomic mechanisms and numerous effects secondary to changes in neural activity. Furthermore, there may be simultaneous effects on a parallel inhibitory local circuit. Thus, an understanding of the lordosis-relevant microcircuitry within the VMH would be helpful in disambiguating the multiplicative effects of estrogen that together so profoundly change behavior.

MICROCIRCUITRY OF THE VMH

Electrophysiological and behavioral pharmacology studies have suggested that multiple elements in the local VMH neural circuit control the lordosis response. However, due to the tight clustering of hypothalamic neurons, their apparent nondescript morphology, and the diffuseness of the VMH afferents, it has been difficult to define the lordosis-relevant microcircuitry of the VMH. As a starting point, the VMH has been subdivided into the dorsal and ventrolateral regions on both neurochemical and anatomical grounds. For instance, the ventrolateral VMH selectively expresses estrogen receptor, progesterone receptor, oxytocin receptor, enkephalin, substance P, and prolactin (2, 9, 19, 42, 96, 131), whereas the dorsal VMH selectively expresses androgen receptor, the transcription factor SF-1, corticotropin releasing hormone type 2 receptors, and neurotensin fibers (46, 64, 69, 112). More importantly, female sexual behavior selectively induces immediate early gene expression in the ventrolateral VMH (28, 98, 99, 125). Finally, recent behavioral and anatomical studies have supported a role for the caudal ventrolateral periaqueductal gray

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in the lordosis reflex (17, 65), which receives dense terminal fields from the ventrolateral, not dorsal, VMH (14). Collectively, these data suggest that the ventrolateral VMH, rather than the dorsal VMH, plays a special role in the estrogen-dependent lordosis response. Studies using various anatomical techniques agree that the VMH maintains a large number of intranuclear connections (14, 79, 90, 123). The caudal ventrolateral VMH is unique, however, because it does not seem to receive input from the other VMH subdivisions (123). Instead, the caudal ventrolateral VMH may receive a disproportionately large amount of the afferents to the VMH and, in turn, relay such information to other VMH regions (26). It is notable that electrophysiological detection of periaqueductal gray-projecting neurons in the VMH tends to occur in the more rostral pole of the VMH (1, 109). Thus, there may be a topographical organization of the VMH, such that much of the extranuclear afferent information funnels into the caudal ventrolateral VMH, which integrates and relays this information to the rostral ventrolateral VMH, which in turn sends descending projections to the midbrain. Investigations of cellular morphology have provided rudimentary information about the synaptic organization of the VMH. Golgi impregnation studies indicated that the VMH includes a dense core of cell bodies, surrounded by a neuropil, also referred to as the fiber plexus or the shell (79). The neurons within the core have simple dendritic arbor, usually with two to three dendrites, some of which extend into the VMH shell (80). The shell contains axonal processes from other brain regions, containing various neurotransmitters, including norepinephrine, serotonin, GnRH, and oxytocin (76, 111, 120). There also are a few neurons found in the shell (80). To further investigate the synaptic organization of the VMH, the transneuronal tracer PRV was injected into the back muscles that produce the lordosis posture (17). Four days after the tracer injection, there was modest labeling of the ventrolateral VMH. This initial labeling may be considered first order with respect to the VMH in the sense that the virus has not yet infected local interneurons, thus defining some portion of the lordosis-relevant projection neurons of the nucleus. To determine whether these PRV-defined projection neurons were directly estrogen responsive, they were double labeled for ER␣ using immunohistochemistry. The majority of the PRV-labeled neurons were anatomically segregated from the dense cluster of ER␣-containing neurons, and only about 3% of the PRV-labeled neurons expressed nuclear ER␣. Although the lack of double labeling in infected neurons must be interpreted cautiously, this result suggests that the ventrolateral VMH microcircuitry has at least two distinct elements, lordosis-relevant projection neurons and ER␣containing neurons. Previous studies using traditional tracers also indicated that only a minority of VMH neurons projecting to the periaqueductal gray possess estrogen binding activity (3, 85). This anatomical evidence for at least two components of the VMH microcircuitry may help reconcile the behavioral effects of acute local administration of glutamatergic and GABAergic drugs by allowing a site for disinhibition. These observations have contributed to a

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FIG. 1. Working model of microcircuitry in the ventrolateral VMH that controls the lordosis response. Shown on the left are afferents to the VMH which are likely to terminate in the neuropil (shaded area). There may be two types of interneurons (blue), those with estrogen receptor and those without. Estrogen treatment induces spines on the short primary dendrites of the neurons without estrogen receptor. Either type of interneuron may innervate the projection neurons (red), which generally do not express estrogen receptors. The neurons that exhibit estrogen-induced spines also may be projection neurons. Abbreviations used: 5HT, serotonin; CCK, cholecystokinin; dend., dendrite; E, estrogen; enk, enkephalin; ER, estrogen receptor; GABA, ␥ aminobutyric acid; glu, glutamate; GnRH, gonadotropin releasing hormone; NE, norepinephrine; OT, oxytocin; PAG, periaqueductal gray; PR, progestin receptor; PRL, prolactin; SubP, substance P.

working model of VMH synaptic organization (see Fig. 1). It is now important to understand how the projection neurons and the estrogen receptor-containing neurons are connected, both anatomically and neurochemically. In summary, very little is known about the synaptic organization of the lordosis circuit embedded within the VMH, although it is likely to reside in the ventrolateral subdivision. The segregation of the PRV-labeled projection neurons from the estrogen receptor-containing neurons suggests that estrogen does not simply have direct effects on the excitability of the final common pathway for the lordosis response. Given the abundant electrophysiological evidence for estrogen-induced changes in the final common pathway, estrogen must influence the projection neurons transynaptically. However, further studies are needed to elucidate the connectivity between the estrogen receptorcontaining neurons and the projection neurons.

NEURAL PLASTICITY IN THE VMH

Electron microscopy studies showed that estrogen treatment promotes the development of axodendritic synapses in the VMH, with a trend toward increased axospinous synapses (34, 90). Due to the constraints of electron microscopic analysis, however, little has been learned about the phenotype of the

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neurons undergoing these estrogen-induced changes in terms of neurochemistry, morphology, or function within the lordosis circuit. Nevertheless, it is necessary to know the relevant connectivity within the VMH before such changes in synaptic configuration can be appreciated. Later studies, therefore, turned to Golgi impregnation techniques which allowed the entire neuronal profile to be visualized. Although synapses could not be resolved, dendritic spines, small protrusions which form specialized sites of synaptic contact, could be viewed. Such studies found that spine density in the VMH increased twofold after treatment with estradiol (32). In addition, the density of dendritic spines in the VMH fluctuated during the estrous cycle, with an increased density occurring on proestrus compared with diestrus. Despite the advantage of visualizing the entire neuron, various limitations of the Golgi technique have prevented further revelations about estrogen-induced neural plasticity. For instance, spine density analysis using camera lucida is time intensive, and therefore researchers only analyzed an arbitrarily chosen subset of dendrites. Also, Golgi impregnation is not compatible with other methods, such as tract tracing or immunocytochemistry, which might reveal the projections, receptors, or neurotransmitters of the impregnated neurons. Thus, to circumvent these constraints, individual VMH neurons were iontophoretically injected with Lucifer Yellow and analyzed morphologically with confocal laser scanning microscopy (13). Unlike Golgi impregnation, this technique is compatible with concomitant labeling for fluorescent tracers and immunohistochemically stained proteins, making it possible to identify functional characteristics of the neurons that exhibit estrogen-induced changes in spine density. Such analysis may provide insight into the neural circuitry context in which changes in spine density occur. The basic morphological features of VMH cells filled with Lucifer Yellow were remarkably consistent with those described for Golgi-impregnated VMH neurons, in terms of soma size, number of dendrites, and length of dendrites. In particular, these were small neurons with simple dendritic trees, usually with only two to three dendrites (13). The analysis of spine density verified previous reports that estrogen induced dendritic spines in this brain region. These experiments extended previous studies by showing that this effect was specific to the ventrolateral, not dorsal, VMH. Thus, the localization of neural plasticity is correlated with the region of VMH implicated in sexual behavior, which supports the hypothesis that this neural plasticity might be relevant for behavior. This study also explored the mechanism of estrogen-induced spines in the VMH. First, it was found that the effect on spine density was dendrite specific. In particular, estrogen treatment induced spines on the short, but not the long, primary dendrites. This result seems more compatible with a transynaptic, rather than a direct, mechanism. An interesting hypothesis arising from these observations would be that estrogen-sensitive terminals selectively innervate the short primary dendrites. The fact that most synapses in the VMH survive a circumscribing knife cut (90) supports the notion that the estrogen-sensitive input arises locally. Second, subsequent immunostaining for ER␣ indicated

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that none of the filled neurons expressed detectable nuclear ER␣. This result makes it even less likely that spines were induced by a direct genomic action of estrogen and instead suggests a transsynaptic mechanism. Although the function of the filled neurons was not known in that study, it seems reasonable to propose that they are involved in sexual behavior. In fact, more than three quarters of the VMH neurons that express an immediate early gene in response to vaginal– cervical stimulation do not express nuclear ER␣ (124). Although estrogen-induced spines occurred specifically in the ventrolateral VMH, the hypothesis that the induced spines are relevant for lordosis remains to be directly proven. Nevertheless, it remains an exciting hypothesis that part of the estrogen-induced change in sexual motivation is mediated by a change in the weighting of specific synaptic contacts. The possibility that the different VMH primary dendrites have different tasks in processing lordosis-relevant information suggests a level of synaptic organization that had not been previously appreciated. Based on their length and orientation, it seems reasonable to propose that the short dendrites receive input primarily from local estrogen receptor-containing interneurons, whereas the long dendrites, with access to the neuropil, receive the bulk of extranuclear input. The need to elucidate the configuration of local and extrinsic information that interfaces with the lordosis microcircuitry is underscored by the possible importance of afferent activity in mediating the effects of estrogen in the VMH. In summary, the cell filling technique has been useful for assembling detailed information about the synaptic organization of VMH neurons. In addition, these studies have suggested that the mechanism of estrogen-induced spines in the VMH is transsynaptic. Furthermore, this analysis has suggested differential innervation of the dendritic arbor of VMH neurons and at least two classes of lordosis-relevant VMH neurons. These observations have contributed to a preliminary model of VMH synaptic organization (see Fig. 1).

ESTROGEN EFFECTS ON MICROCIRCUITRY IN OTHER BRAIN REGIONS

In addition to its effects on the lordosis network in the VMH, the effects of estrogen on several other neural systems in the adult brain have been well studied. For example, estrogen positive feedback is crucial for the GnRH surge in rats (106). A possible site of estrogen action to trigger the GnRH surge is the arcuate nucleus (89). Estrogen-induced changes in synaptic morphology in the arcuate nucleus include a decrease in axosomatic and axodendritic synapses (36). This effect of estrogen may be mediated by altering the contacts between neurons and glia, with a net effect of decreasing the GABAergic inputs to the cell bodies of arcuate neurons (44, 94). This reduced GABA input may alter the potency of estrogen negative feedback, thereby allowing positive feedback to occur. Although the causal events are not fully elucidated in either region, the data suggest some probable differences in mechanism of action between the arcuate and VMH. In particular, unlike the VMH, estrogen seems to decrease

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synaptic contacts in the arcuate nucleus. During development, the mechanisms of estrogen-induced sexual differentiation also differ between the arcuate and the VMH (83). Thus, the effect of estrogen on neural plasticity in the arcuate nucleus and the VMH may rely on different mediators. The effects of estrogen on synaptic organization have been most vigorously studied in the CA1 region of the dorsal hippocampus in female rats. As recently reviewed (129), estrogen treatment increases spine density in the apical dendrites of pyramidal neurons despite the fact that nuclear estrogen receptors of either subtype have not been detected in these neurons. In vivo and in vitro studies have implicated glutamate activity and NMDA receptors in mediating the spine induction, and the spine induction is correlated with increased sensitivity to NMDA receptor activation. In addition to increased excitatory mechanisms, in vitro studies also indicated that estrogen attenuated GABA activity (87). A concomitant decrease in inhibitory signals and increase in glutamate signaling may trigger spine formation by a cascade of both increased intracellular calcium release and increased cyclic AMP signaling (88). Based on the available data, there are several parallels between the mechanisms of estrogen-induced spines in the CA1 hippocampus and VMH. For instance, in both regions, dendritic spines are induced in neurons that do not express nuclear estrogen receptor, which suggests a transynaptic mechanism. In addition, in both regions, the estrogen receptor-containing neurons appear to be GABAergic (10, 62, 87, 127). Although the role of glutamate in spine induction in the VMH has not been tested, glutamate receptors are present in the VMH (20, 75). It will be interesting for future studies to determine whether the mechanisms of estrogen-induced neural plasticity in the VMH really are divergent from those in the adjacent arcuate and instead truly similar to those in the CA1 hippocampus.

SEXUAL DIMORPHISM OF THE VMH

Given the prominent role the VMH plays in a sexually dimorphic behavior, it is not surprising that sexual dimorphisms are apparent in the VMH at several levels of analysis. Sex differences include the volume of the VMH (22), estrogen-inducible electrophysiological changes (108), the density of spine and shaft synapses (71), the regulation of spine density (33, 63), and the levels of key neurotransmitters and synapse-associated proteins, such as GAP-43 (31, 38, 68, 113, 114). It is plausible that differences in neurotransmitter activity may maintain sex differences in behaviorally relevant connectivity. This hypothesis is supported by the observation that manipulating certain afferents to the VMH unmask the lordosis response in male rats (84, 133). To the extent that dendritic spines are induced and maintained by neuronal activity, the sex difference in spine density in the VMH implies intrinsic differences in either connectivity, basal synaptic activity, or both. Future investigations of the components of the local circuitry of the VMH in both sexes would potentially

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explain the neurological underpinnings of the sexual dimorphism of this behavior.

FUTURE DIRECTIONS

In summary, there is a substantial database on the macrocircuitry of the VMH, including its afferent and efferent connections with the hindbrain, hypothalamic, and limbic regions. For a few of the afferent and efferent pathways, the function and neurochemistry have been worked out. In addition to extranuclear afferents and descending projections, anatomical and behavioral pharmacological evidence suggest that intranuclear connections are an integral component of the lordosis circuitry. Despite the tremendous progress in our knowledge of this model system, several key issues remain unresolved, including the physiological information and chemical coding of hypothalamic and limbic afferents, the contribution of VMH projections to the learning that occurs with sexual experience, the possible contribution of nongenomic effects of estrogen for this behavior, and how the sex specificity of this behavior is maintained at the level of synaptic organization. The synaptic organization of the functional elements within the VMH has remained enigmatic for several reasons, including the lack of distinguishing morphological or topographical features of the interneurons and projection neurons and the lack of a known topographical organization of the axons arriving into the VMH shell. Recent investigations have made some inroads into clarifying local circuitry in the VMH. The use of a functional tracer, PRV, has helped delineate the topography of lordosis-relevant projection neurons within the VMH, with a notable segregation from the estrogen receptorcontaining neurons. In addition, cell filling techniques have been employed to address the identity of the neurons that display estrogen-induced changes in connectivity. These results have been used to construct a working model of the lordosis-relevant microcircuitry of the VMH (see Fig. 1). At present, this model is necessarily simple because of uncertainty about various connections and neurotransmitters. Ongoing studies are designed to elaborate this model to provide a sharper definition of the synaptic organization within the VMH which may serve as a framework for understanding how estrogen engages a behavioral switch at the level of synapses within a confined neural circuit. Thus, it should be possible to expand the “molecular 3 electrical 3 behavior” model of estrogen action in the VMH to a “molecular 3 electrical % rewiring 3 behavior” model. Not only is the VMH a natural brain region for the study of motivated behavior, it also provides an alternative venue for studying the regulation of dendritic spines. Spines have been vigorously studied in higher brain structures as possible substrates for learning and memory processes. Such studies have revealed features of the intracellular signaling involved in spine induction. However, an investigation of spines in the VMH may reveal that spines represent a substrate not only for learning, but also for motivation. Because

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spine density in the VMH fluctuates with the estrous cycle, the VMH presents a convenient model system for studying not only the physiological induction of dendritic spines, but also the physiological dismantling of spines. Therefore, it seems likely that investigations of estrogen-induced spines in the VMH will continue to inform us about fundamental processes in the brain.

ACKNOWLEDGMENTS The author is supported by National Institutes of Health Grants MH54712, MH43787, and DK52018. The editorial comments of Lyngine Calizo and Derek Daniels are gratefully acknowledged. Derek Daniels provided the graphics for Fig. 1.

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