GABA influences the development of the ventromedial nucleus of the hypothalamus.

The region that becomes the ventromedial nucleus of the hypothalamus (VMH) is surrounded by cells and fibers containing immunoreactive gamma-aminobutyric acid (GABA) by embryonic day 13 (E13), several days before the nucleus emerges in Nissl stains. As GABA plays many roles during neural development, we hypothesized that it influences VMH development, perhaps by providing boundary information for migrating neurons. To test this hypothesis we examined the VMH in embryonic mice in which the beta3 subunit of the GABA(A)-receptor, a receptor subunit that is normally highly expressed in this nucleus, was disrupted by gene targeting. In beta3 -/- embryos the VMH was significantly larger, and the distribution of cells containing immunoreactive estrogen receptor-alpha was expanded compared to controls. Using in vitro brain slices from wild-type C57BL/6J mice killed at E15 we found that treatment with the GABA(A) antagonist bicuculline increased the number of cells migrating per video field analyzed in the VMH. In addition, treatment with either bicuculline or the GABA(A) agonist muscimol altered the orientation of cell migration in particular regions of this nucleus. These data suggest that GABA is important for the organization of cells during VMH formation.

Disruption of the gene encoding SF-1 alters the distribution of hypothalamic neuronal phenotypes.

The ventromedial nucleus of the hypothalamus (VMH) in mice first emerges as a histologically distinct cell cluster around embryonic day 17 (E17). The earliest known marker for cells destined to form the VMH is the orphan nuclear receptor, steroidogenic factor 1 (SF-1), which can be detected in the hypothalamic primordium by E11. Strikingly, the VMH is absent in newborn SF-1 knockout mice, suggesting that SF-1 is essential for the development of VMH neurons. We reported previously that the VMH can be identified before it emerges as a histologically distinct nucleus (i.e., at E13) by the exclusion of cells that are immunoreactive for both gamma-aminobutyric acid (GABA) and the synthetic enzyme, glutamic acid decarboxylase (GAD67). Subsequently, by E15, the developing VMH is demarcated further by cells that are immunoreactive for neuropeptide Y, estrogen receptor alpha (ERalpha), and galanin. It is noteworthy that the normal exclusion of GABA from the developing VMH is not seen in SF-1 knockout mice, and cells that are immunoreactive for neuropeptide Y, ERalpha, and galanin also are distributed aberrantly in this region. Thus, the absence of SF-1 profoundly affects the cellular architecture of the VMH from early stages in its formation. These data suggest that, directly or indirectly, SF-1 plays important roles in determining the distribution of cells in the mediobasal hypothalamus.

The two thyroid hormone receptor genes have opposite effects on estrogen-stimulated sex behaviors.

The two genes coding for thyroid hormone receptors (TR) alpha 1 and beta have opposite effects on female sex behaviors. Deletion of TRalpha 1 reduced them, whereas deletion of TRbeta actually increased them. These results could not be attributed to altered levels of hormones in the blood, general alterations in estrogen responsiveness or altered general activity. Instead, they indicate a previously unknown molecular mechanism upon which the two TR genes exert opposite influences.

Effect of thyroid hormone administration on estrogen-induced sex behavior in female mice.

Effects of thyroid hormones on reproductive processes have been described in sheep, birds, and rats. To extend this subject to mice for eventual analysis with genetically modified animals, we looked for effects of thyroid hormone treatment on lordosis behavior of ovariectomized estrogen-treated female mice. High doses of thyroid hormones reduced lordosis behavior. Since we could not explain this result by pharmacokinetic or peripheral effects, we infer that it worked by a central mechanism. Future investigations must determine whether endogenous fluctuations within the thyroid's normal physiological range have any behavioral effects.

Thyroid hormone coadministration inhibits the estrogen-stimulated elevation of preproenkephalin mRNA in female rat hypothalamic neurons.

Expression of the enkephalin gene in ventromedial hypothalamus (VMH) of the female rat has been correlated with the performance of lordosis behavior. By antisense DNA evidence, it has been drawn into a causal role as well. Here, we explored whether, parallel to earlier molecular and behavioral results, thyroid hormone coadministration could disrupt the estrogenic induction of preproenkephalin (PPE) mRNA. As expected, estradiol benzoate treatment to ovariectomized rats led to a large and significant increase in PPE gene expression in the VMH. 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 form a direct parallel both to previous transcriptional measurements and to reproductive behavior assays in which thyroid hormones were able to oppose estrogenic facilitation. Previous evidence supports the notion of competitive DNA binding and protein/protein interactions providing mechanisms for nuclear thyroid hormone receptors to affect estrogen receptor function, but other, additional mechanisms cannot be ruled out. To date, both oxytocin and PPE gene expression represent potential hypothalamic systems by which thyroid hormones could interfere with estrogen-stimulated female rat reproductive behavior.

Thyroid hormones and estrogen affect oxytocin gene expression in hypothalamic neurons.

The oxytocin (OT) gene promoter has a composite hormone response element, such that several members of the steroid/thyroid hormone superfamily of nuclear receptors can interact at this response element in vitro. To investigate this in brain tissue, parallel to foregoing behavioural experiments, we used in situ hybridization histochemistry to seek interactions between estrogen and thyroid hormones on OT mRNA in the hypothalamus. In ovariectomized (OVX) rats, high doses of triiodothyronine (T3) elevated OT mRNA levels in the paraventricular (PVN) nucleus, while treatment with estradiol benzoate (EB) alone had no significant effect. In contrast, animals that were thyroidectomized (TX) in addition to OVX had dramatically elevated levels of OT gene expression in the PVN following EB treatment. That is, endogenous thyroid hormones interfered with EB-induction of gene expression. Moreover, in both OVX and TX/OVX animals, OT gene expression was reduced to values equivalent to controls when T3 was given together with EB. Particular subdivisions of the PVN responded differentially to T3 and EB treatment, demonstrating marked heterogeneity of OT-containing neurons in this nucleus. Thus, parallel to and perhaps related to the manner in which thyroid hormones reduced estrogen-stimulated behaviour, endogenous or exogenous thyroid hormones interfered with estrogen stimulation of OT mRNA. These data demonstrate competition between nuclear proteins, transcription factors, in hypothalamic neurons.

Olfactory bulb development is altered in small-eye (Sey) mice.

Small-eye (Sey) is a spontaneous, semidominant murine mutation that results from a point mutation in the Pax-6 gene. Both the eyes and the olfactory system fail to develop in homozygotes and these animals die neonatally. Heterozygotes (Sey/+) have different degrees of eye abnormalities including decreased lens size and cataracts. In the present study, we examined whether one mutated allele of Pax-6 also affects olfactory system development. By 42 days of age, main olfactory bulb volume was significantly decreased in Sey/+ animals compared with wild-type littermates, and this effect was even more dramatic in 70-day-old animals. In contrast, there was no effect on accessory olfactory bulb, olfactory epithelial, or vomeronasal organ development at any age in Sey/+ animals, demonstrating the specificity of the effect. In the main olfactory bulb, the largest differences in laminar volume were found in the glomerular and granule cell layers. These layers contain the olfactory bulb interneurons, and a subpopulation of these cells were found to be Pax-6 immunoreactive. Examination of the neurochemical consequences of this mutation showed that the number of both tyrosine hydroxylase (TH)- and gamma-aminobutyric acid (GABA)-immunoreactive profiles were dramatically decreased in Sey/+ animals as compared with controls. In contrast, neither calretinin nor calbindin immunoreactivity was affected by this mutation. Dual-labeling immunohistochemistry showed that nearly all TH-immunoreactive cells and a subpopulation of GABA-immunoreactive cells coexpressed Pax-6. However, calretinin- and calbindin-immunoreactive cells were not Pax-6 immunopositive. These data indicate that two normal alleles of Pax-6 are required for normal olfactory bulb development and, as part of this effect, this gene may be involved in the development of specific neurotransmitter systems.

The gonadotropin-releasing hormone system does not develop in Small-Eye (Sey) mouse phenotype.

This study examined the development of the gonadotropin releasing-hormone (GnRH) system in a spontaneous mouse mutation, Small-Eye (Sey). This phenotype is due to a point mutation in the developmental control gene Pax-6 and results in failed development of the eye and olfactory placodes in homozygous (Sey/Sey) embryos and a variety of eye abnormalities in heterozygotes (Sey/+). Therefore, Sey/Sey embryos provided a naturally occurring olfactory placode ablation to ask whether all of the GnRH neurons found in the adult mouse forebrain arise from the olfactory epithelium. In Sey/Sey embryos, GnRH-immunoreactive neurons were not present in either the presumptive nasal regions or in any area of the brain at any embryonic age. In contrast, in Sey/+ embryos, there was no apparent effect on either GnRH cell proliferation or migration. These data support and extend the hypothesis that GnRH neurons in mice originate in the olfactory placodes and also demonstrate that two normal alleles of Pax-6 are not required for GnRH system development.

Interactions between hormonal and environmental signals on hypothalamic neurons molecular mechanisms signaling environmental events.

It is axiomatic that the central nervous system must manage the integration of several environmental factors with steroid hormonal influences for the biologically adaptive performance of reproductive behavior. Launching from established behavioral investigations and from hormonal influences on gene function in the brain, we review here studies on how synaptic inputs and sex hormone influences codetermine hypothalamic gene expression. A particularly exciting implication of results on the ability of thyroid hormone receptors to interfere with estrogen receptor-dependent neuroendocrine function is that environmentally stimulated changes in thyroid hormone levels could influence hypothalamic transcriptional mechanisms important for behavior. If so, this would unite naturalistic environmental thinking with molecular neurobiological thinking important for the hypothalamic control of reproduction. (Trends Endocrinol Metab 1997;8:111-115). (c) 1997, Elsevier Science Inc.

Thyroid hormone and estrogen interact to regulate behavior.

Environmental perturbations that increase plasma thyroid hormone (T3) concentrations also profoundly affect female reproductive behavior and physiology. We explored whether these effects were mediated by interactions between T3 receptor (TR) and estrogen receptor (ER). This hypothesis was of interest because the half-site of a consensus T3 response element DNA sequence is identical to an ER response element (ERE), and TRs bind to a consensus ERE. Molecular data presented in the accompanying paper [Zhu, Y.-S., Yen, P.M., Chin, W.W.& Pfaff, D.W. (1996) Proc. Natl. Acad. Sci. USA 93, 12587-12592] demonstrate that TRs and ERs are both present in rat hypothalamic nuclear extracts and that both can bind to the promoter the hypothalamic gene preproenkephalin and that interations between liganded TRs and ERs affect preproenkephalin transcription. In this paper, we show that molecular interactions between TRs and ERs are sufficient to mediate environmental effects on estrogen-controlled reproductive behavior. Ovariectomized (OVX) rats treated with high doses of T3 showed significantly lower levels of lordosis behavior in response to estradiol benzoate (EB) compared with OVX females treated with EB alone. Conversely, thyroidectomized/OVX females treated with EB showed significantly greater levels of lordosis behavior compared with OVX females treated with EB, showing the effect of endogenous T3. Thyroid hormone interference with EB-induced behavior could not be explained by a reduction in plasma E2 concentrations or by a general reduction in responsiveness of EB-sensitive tissues. Moreover, numbers of hypothalamic ER-immunoreactive cells increased dramatically following T3 treatment. These data suggest that T3 may reduce EB-dependent sexual behavior through interactions between TR and ER in the nuclei of behaviorally relevant hypothalamic neurons, envisioning for the first time a functional consequence of interactions between two nuclear hormone receptors in brain. These results also open up the possibility of molecular interactions on DNA encoding environmental signals, a new field for the study of neuronal integration.

Interactions with males promote rapid changes in gonadotropin-releasing hormone immunoreactive cells.

It is well known that hormones can regulate behaviors. However, the reciprocal interaction, the effects of behavior on hormones, has received less direct experimental attention. Dramatic changes in hormones and behaviors occur at puberty and some of these changes can be triggered by modification of the social environment. Interactions with males accelerate production of pulsatile release of gonadotropins and steroid hormones which, in turn, initiate estrous cycles, ovulation, and sexual behavior in females. Ultimately all of these actions are controlled by changes in production and secretion of gonadotropin-releasing hormone (GnRH). Little is known about how behavior affects GnRH-producing neurons. In female musk shrews, the first mating initiates the onset of puberty. Musk shrews lack a behavioral estrous cycle and they become receptive within minutes after their first contact with a male. As soon as 1 h after interactions with males there is a significant increase in the numbers of GnRH-immunoreactive (GnRH-ir) neurons in specific brain regions. In the present study, we examined changes in GnRH-ir cell number during the initial mating bout. We found dynamic changes in the numbers of GnRH-containing cells, correlated with changes in behavior. Interactions with males for less than 30 minutes induced a significant increase in GnRH-ir neurons in specific olfactory-related forebrain nuclei. At the end of a mating bout, numbers of GnRH-ir neurons declined. Because behavioral interactions have rapid and pronounced effects on the neurons that produce GnRH, this model can be used to examine the behavioral regulation of neuronal plasticity.

Potential interactions between estrogen receptor and thyroid receptors relevant for neuroendocrine systems.

Environmental signals can profoundly affect reproductive behavior, physiology and responses to steroids. One consequence of nutritional or temperature stress is altered plasma concentrations of thyroid hormone. Recent in vivo and in vitro data indicate that manipulations of estrogen and thyroid hormone levels can alter each other's functions. One possible mechanism for interaction may be that thyroid and estrogen receptors bind to parts of the same hormone response elements of target genes and compete with each other, thus serving to integrate environmental signals with neuroendocrine responses.

Mating alters gonadotropin-releasing hormone cell number and content.

Female musk shrews (Suncus murinus) are induced ovulators, which lack a behavioral and ovarian estrous cycle. Females mate the first time they are introduced to a male, but a second or third mating, at least 24 h later, is usually required to induce ovulation. Because GnRH-immunoreactive (GnRH-ir) cell numbers increase during and after exposure to a male, we hypothesized that mating promotes synthesis of this important peptide. To test this hypothesis, we examined changes in GnRH-ir cell number and GnRH-ir content at select time points after mating and ovulation. One hour after mating, GnRH-ir cell numbers in olfactory-related regions of the forebrain were increased. By 15 h after mating, just before ovulation, GnRH-ir cell number and content were increased. Twenty-four hours after mating, GnRH-ir cell numbers in the tenia tecta and medial septum/diagonal band were lower in females that ovulated compared with females that did not ovulate. By 40 h postmating, females that ovulated had fewer GnRH-ir neurons and lower GnRH content in the entire brain than females that did not ovulate. In addition, we found significant negative correlations between plasma estradiol concentrations and both GnRH-ir cell numbers and content in the preoptic area of animals killed around the time of ovulation. Interestingly, significantly more GnRH-ir neurons and a greater content of GnRH peptide were observed in several forebrain nuclei of females that did not ovulate 40, compared to 24, h after mating. In contrast, numbers of GnRH-ir neurons in the midbrain declined 40 h postmating in ovulated females. These results suggest that mating stimulates activity in GnRH-ir neurons, and that ovulation is correlated with a decline in GnRH-ir cell number and content. In this species, mating can be used as an external trigger to activate GnRH neurons and examine the regulation and production of GnRH in heterogenic neuronal populations.

Co-localization of aromatase enzyme and estrogen receptor immunoreactivity in the preoptic area during reproductive aging.

Immunoreactive aromatase enzyme (AROM-IR) was studied in the preoptic and septal ares of the male Japanese quail brain relative to the age-related decline in endocrine and behavioral components of reproduction. Additional analyses were conducted to determine if the co-localization of AROM-IR and estrogen receptor immunoreactivity (ER-IR) in the medial preoptic area change during aging. Young, sexually active, male quail (6 months of age) were compared to aged sexually active or inactive, male quail (36 months of age). Testis size decreased in old, sexually inactive males, similar to our previous observations. The numbers of AROM-IR neurons in the medial preoptic area (POM) and the lateral septum (LS) decreased significantly with aging and sexual activity. The number of cells that co-localized both AROM-IR and ER-IR did not differ with age. As a consequence of the age-related change in AROM-IR cells, the relative percentage of dual labelled (AROM-IR and ER-IR) and single labelled cells (AROM-IR) increased in aged males. These data provide histochemical evidence that alterations in the aromatase enzyme system in the medial preoptic area may underlie behavioral and endocrine events associated with reproductive aging.

Gonadotropin-releasing hormone-immunoreactive cell numbers change in response to social interactions.

Puberty is characterized by either a steroid-dependent or independent increase in the production and secretion of neural GnRH. Several measures have been used to assess the pubertal activation of neurons that express GnRH. Some morphological changes in these cells occur at puberty, but no dramatic changes indicative of increases in the synthesis of GnRH have been noted. In the musk shrew, puberty is induced by mating. We hypothesize that because the first mating facilitates the display of female sexual behavior and subsequent ovulation, that mating may have direct and rapid effects on GnRH neurons. Significant changes in the number of GnRH-immunoreactive (GnRH-ir) neurons in the brains of prepubertal virgin females were note after contact with males. Different populations of GnRH-ir cells were affected by different types of interactions with males. Exposure to an adult male across a wire barrier for 48 h increased the number of GnRH-ir cells in olfactory-related nuclei. Mating increased GnRH-ir cell numbers in olfactory and ventral forebrain regions. Ovulation reduced GnRH-ir cell numbers in several forebrain nuclei. These rapid and quantitative responses of GnRH-ir neurons to social cues allow direct examination of the neuroendocrine changes that occur at puberty.

Distribution and steroid dependence of aromatase enzyme immunoreactivity in limbic nuclei of the female musk shrew brain.

In the female musk shrew (Suncus murinus) neural aromatization of testosterone to estradiol is critical for the expression of sexual behavior. To localize the brain regions capable of aromatization, we used immunocytochemistry to map the distribution of aromatase enzyme. Aromatase immunoreactivity (AROM-ir) has a discrete distribution primarily limited to the lateral septum (LS), central nuclei of the amygdala (Ce) and the bed nucleus of the stria terminalis (BST). In these nuclei the intensity of immunoreactivity varies with hormonal status. Ovariectomy (OVX) significantly reduces the optical density of AROM-ir neurons in all nuclei as compared with brains of normal females. Combined OVX and adrenalectomy (ADX) further reduces optical density readings in AROM-ir cells in the LS and BST, as compared with readings from the brains of OVX animals. Normal and ovariectomized females implanted with testosterone had qualitatively equivalent AROM-ir. High levels of aromatase activity have been measured in the preoptic area and hypothalamus in a number of mammals, including the musk shrew. However, in this experiment AROM-ir was absent in these areas. We present several hypotheses to account for this discrepancy between previously reported biochemical data and these histological data. In summary, these data suggest that limbic nuclei may play a role in the expression of sexual behavior in female musk shrews.

Presence and differential distribution of distinct forms of immunoreactive gonadotropin-releasing hormone in the musk shrew brain.

Gonadotropin-releasing hormone (GnRH) immunoreactive cells and fibers were revealed in olfactory regions, the ventral forebrain, and in the midbrain of the musk shrew (Suncus murinus). Immunoreactive neurons in olfactory and telencephalic areas were specific for the mammalian form of GnRH. Cell bodies in the midbrain, however, cross-reacted with an antibody specific for chicken-II GnRH. High-performance liquid chromatography and radioimmunoassay analyses confirmed these results; high levels of chicken II GnRH were present in the midbrain, and mammalian GnRH was detected in both forebrain and midbrain. In addition, a third, late-eluting form of GnRH was revealed using high-performance liquid chromatography in both forebrain and midbrain of the musk shrew. Midbrain neurons containing GnRH have not been reported previously in a mammal, although mesencephalic GnRH immunoreactivity within cell bodies is common among nonmammalian vertebrates. Likewise, while multiple forms of GnRH have been reported in nonmammalian vertebrates and several metatherian species of mammals, this is the first report on multiple forms of GnRH in the brain of a placental mammal. Taken together, the findings suggest that this primitive eutherian mammal has retained the ability to produce GnRH protein in the midbrain. This feature of the GnRH system has been conserved among nonmammalian vertebrates, but appears to have been lost in modern placental mammal species. The functional significance of this group of neurons has yet to be determined.

Neural distribution of estrogen receptor immunoreactive cells in the female musk shrew.

In this report we describe the neural distribution of estrogen receptor immunoreactivity (ER-IR) in the female musk shrew (Suncus murinus). The highest concentrations of neurons containing ER-IR were found in the preoptic areas, the lateral septum, the anterior arcuate and ventromedial nuclei of the hypothalamus, the medial nuclei of amygdala, and the midbrain central grey. Additional preoptic, hypothalamic, limbic and midbrain nuclei also contained ER-IR cells. The distribution of ER-IR was similar to that described in other mammals and birds, with several important differences. In the female musk shrew, there was little difference in ER-IR intensity or distribution when brains from gonadally intact and ovariectomized musk shrews were compared. In addition, long-term treatment with a supraphysiological dose of estradiol was required to detect a decrease in ER-IR intensity. Finally ER-IR was noted in both nuclear and cytoplasmic regions of cells in ovariectomized and gonadally intact musk shrews. The dense ER-IR noted in intact females as well as the presence of cytoplasmic stain may be due to the unusual relationship between estradiol, ovulation and sexual receptivity in this species.

Adrenal contribution to the induction of sexual behavior in the female musk shrew.

Sexually experienced female musk shrews (Suncus murinus) lack an ovarian, vaginal, and behavioral estrous cycle. Females, once induced by their initial contact with a male, are able to display copulatory behavior whenever a male is present (Rissman, Silveira, and Bronson, 1988). Based on plasma levels of steroids, and on hormone replacement studies conducted after ovariectomy (OVX), we have shown that testosterone (T) plays an essential role in the regulation of female sexual behavior (Rissman and Crews, 1988; Rissman, Clendenon, and Krohmer, 1990a; Rissman, 1991). To date we have only examined the potential contribution of adrenal steroids to female sexual behavior in a preliminary manner. After adrenalectomy, gonadally intact females display significantly lower levels of sexual behavior than controls (Rissman and Bronson, 1987). The following experiments were conducted to examine the role the adrenal steroids (in contrast to the medullary hormones) play in the induction of female sexual behavior in the musk shrew. In the first experiment gonadally intact females were treated with dexamethasone (DEX) to reduce the secretion of adrenal steroids. Significantly fewer females receiving DEX demonstrated sexual behavior as compared with controls. In the second study, OVX females received T-filled Silastic implants. When DEX was administered to OVX + T females at a dose that dropped circulating T levels to those found in ovary and adrenal intact females, no effect on sexual behavior was noted. The data show that the adrenals are a behaviorally important source of T and contribute toward the hormonal control of sexual behavior in these female mammals.

The bed nucleus-amygdala continuum in human and monkey.

The cytoarchitecture and distributions of seven neuropeptides were examined in the the bed nucleus of the stria terminalis (BST), substantia innominata (SI), and central and medial nuclei of the amygdala of human and monkey to determine whether neurons of these regions form an anatomical continuum in primate brain. The BST and centromedial amygdala have common cyto- and chemo-architectonic characteristics, and these regions are components of a distinct neuronal complex. This neuronal continuum extends dorsally, with the stria terminalis, from the BST and merges with the amygdala; it extends ventrally from the BST through the SI to the centromedial amygdala. The cytoarchitectonics of the BST-amygdala complex are heterogeneous and compartmental. The BST is parcellated broadly into anterior, lateral, medial, ventral, supracapsular, and sublenticular divisions. The central and medial nuclei of the amygdala are also parcellated into several subdivisions. Neurons of central and medial nuclei of the amygdala are similar to neurons in the lateral and medial divisions of the BST, respectively. Neurons in the SI form cellular bridges between the BST and amygdala. The BST, SI, and amygdala share several neuropeptide transmitters, and patterns of peptide immunoreactivity parallel cytological findings. Specific chemoarchitectonic zones were delineated by perikaryal, peridendritic/perisomatic, axonal, and terminal immunoreactivities. The results of this investigation demonstrate that there is a neuronal continuity between the BST and amygdala and that the BST-amygdala complex is prominent and discretely compartmental in forebrains of human and monkey.