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.

Aspects of GnRH neurobiology conserved across vertebrate forms.

The decapeptide gonadotropin-releasing hormone (GnRH) came into prominence because of its roles in releasing luteinizing hormone and follicle-stimulating hormone and promoting reproductive behavior. At least three aspects of GnRH neurobiology have features which may be universal among vertebrates. First, the GnRH neuronal migration from the olfactory placode into the basal forebrain appears to hold true for forms ranging from fish to humans. Second, for proper agonist activity in the anterior pituitary, GnRH must be released in a pulsatile fashion. Since GT-1 neuronal cell cultures can demonstrate pulsatile release, it must be concluded that GnRH neuronal networks themselves can manage this type of pulsatility. Using a neuronal mathematical model with "minimalist" assumptions, we demonstrated that a network of identical neurons can achieve this self-organizing property without the use of, or spontaneous appearance of, "pacemaker cells." Indeed, since many parameter combinations worked, and since no information about species identity or chemical cell type is provided to the model, this conclusion could apply across many vertebrate forms and, perhaps, even for other neuroendocrine cell types. Third, Fernald and colleagues (this issue of General and Comparative Endocrinology) have demonstrated remarkable effects of social context on GnRH expression in fish. Reviewed here are some data in musk shrews suggesting that behavioral and social stimuli can also modify GnRH neurons in mammals. Therefore, although GnRH neuronal mechanisms are adapted to meet species-typical variations in environment and physiology, some of the important features of this system appear to be widely conserved.

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 among genes for transcription factors in hypothalamic neurons: implications for reproductive behaviors.

Gene products for nuclear hormone receptors, which are transcription factors, interact in the cell nuclei of neurons in a manner important for the hypothalamic control of instinctive behaviors. In doing so, the combinatorial logic of their competition or synergy may serve to integrate environmental and physiological constraints upon those behaviors.

Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells.

Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoids. Type I cells have a neuronal phenotype, extending many lengthy processes, and express neuronal, but not glial, markers. Type II cells show weaker or negative immunostaining for neuronal markers and exhibit fewer processes. The effect of glucocorticoids on gonadotropin-releasing hormone (GnRH) secretion and gene expression was compared in type I and type II GT1-7 cells. For secretion studies, cells were attached to Cytodex beads and perifused with control medium or medium containing dexamethasone (dex). The high level of GnRH secreted by type I cells was slightly enhanced in the presence of dex, whereas dex rapidly and profoundly decreased the already low level of GnRH secreted by type II cells. Immunocytochemistry for GnRH showed dark reaction product in the cell bodies and processes of type I cells and little or no immunoreactivity in type II cells. Both the endogenous mouse GnRH mRNA and the transcriptional activity of a mouse GnRH promoter luciferase reporter gene plasmid were suppressed to a greater extent in type II cells than in type I. In electrophoretic mobility shift assays, there was no difference between type I and type II nuclear extracts in the pattern of protein-DNA complexes formed on two previously identified negative glucocorticoid response elements located at -237 to -201 and -184 to -150 bp of the mouse promoter. Both cell types contained glucocorticoid receptors (GR) by Western blot analysis. Cytosols from type I or type II cells were incubated with [3H]dex to obtain GR binding parameters. Binding data were consistent with a one-site model for dex binding in each case. Small differences in Kd (1.7 nM, type I; 3.1 nM, type II) or Bmax (approximately 3600 sites/cell, type I; approximately 1800 sites/cell, type II) were not likely to account for the differential sensitivity to dex treatment. In conclusion, nuclear alterations in type II cells leading to greater transcriptional susceptibility to dex, coupled with low GnRH storage levels, may be reflected in exquisite sensitivity of GnRH secretion to glucocorticoid repression. This represents the first example of a steroid hormone acting directly on GnRH-producing cells to alter GnRH secretion.

Differential LHRH secretion, dye coupling, and protein expression in two morphologically distinct cell types identified in GT1-7 cultures.

The immortalized neuronal cell line, GT1-7, has been shown to secrete LHRH in a pulsatile manner and to possess many other characteristics of hypothalamic LHRH neurons in vivo, and thus provides a potential model system for studying biochemical and physiological mechanisms regulating LHRH secretion. In the present study, two morphologically and functionally distinct types of cells have been identified in GT1-7 cultures and each type purified to over 95% homogeneity. One type (N cells) appeared more neuronal with extended neurites and somewhat rounded cell perikarya, while the other type (G cells) had flatter cell perikarya that contained filopodia but no neurites. Growth properties of the two cell types also differed. The doubling time for proliferation of N cells was nearly two-fold shorter than that for G cells and N cells displayed 'piling up' whereas G cells exhibited contact inhibition. Functionally, N cells, but not G cells, were dye-coupled as measured by a fluorescence photobleaching assay. While both cell types expressed LHRH, N cells released significantly higher levels of LHRH into the culture media and exhibited more intense LHRH immunostaining. The two cell types also showed differences in immunostaining for other proteins. N cells, unlike G cells, immunostained positive for neuron-specific enolase (NSE), whereas G cells, unlike N cells, stained immunopositive for vimentin. Both cell types expressed SV-40 T antigen protein, indicating that they were derived from the same transgenic mouse hypothalamic tumour. The physiological significance of these two cell types in GT1-7 cultures remains to be determined, but elucidation of their morphological and biochemical properties is intended to contribute to better understanding and application of this experimentally important neuroendocrine cell line.

Gender-specific induction of pituitary RNA by estrogen and its modification by thyroid hormone.

Estrogen and thyroid hormones play important roles in the regulation of pituitary function. We presently show that pituitary weight and total cellular RNA levels were significantly decreased by ovariectomy in female rats and were significantly increased by castration in males, without alterations in pituitary DNA levels as compared to intact animals. Treatment with a single dose of estrogen produced a significant increase in pituitary RNA in ovariectomized females but not castrated males. This effect was more obvious following multiple doses of estrogen, and was blocked by pretreatment with cycloheximide, or surprisingly by concomitant administration of triiodothyronine (T3). Analysis of estrogen response element (ERE) binding activity in pituitary nuclear protein extracts revealed that estrogen produced a rapid induction of a slow mobility complex of ERE binding in ovariectomized females much greater than in castrated males. Thus, estrogen-induced increases in pituitary total RNA levels are dependent on new protein synthesis, are gender-specific, are inhibited by T3, and may be mediated via specific estrogen-induced changes in protein-DNA interactions.

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.

Hypothalamic cellular and molecular mechanisms helping to satisfy axiomatic requirements for reproduction.

In the absence of universal equations expressing neurobiological findings, the safest theoretical approach for the neuroendocrinologist is to start from axiomatic requirements for biologically adaptive neural mechanisms, in our case for reproduction. From this emerge two themes: the likely importance of interactions between internal (hormonal) and external signals in controlling gene expression relevant to reproductive functions; and, second, the vision of molecular interactions on DNA subserving environmental impacts on reproduction. The first theoretical notion has so far yielded data showing a role for synaptic inputs during the onset of estradiol actions for the hormone's induction of enkephalin mRNA, a finding which parallels earlier behavioral results. As well, noxious somatosensory inputs interact with estrogens and progesterone in their influence on enkephalin gene expression. The second theme led to novel investigations of thyroid influences on reproductive molecular biology and behavior, including the ability of exogenous or endogenous thyroid hormones to reduce female mating responses. Since elevated thyroid hormone levels could signal environmental cold, our experiments offer the possibility of explaining ethological facts at a molecular level. More generally, nuclear hormone receptor interactions on the surface of DNA may offer a new level of neural integration revealed first by hormone effects in neuroendocrine cells.

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.