Arcuate nucleus kisspeptin response to increased nutrition in rams.

Rams respond to acute nutritional supplementation by increasing the frequency of gonadotrophin-releasing hormone (GnRH) pulses. Kisspeptin neurons may mediate the effect of environmental cues on GnRH secretion, so we tested whether the ram response to nutrition involves activation of kisspeptin neurons in the arcuate nucleus (ARC), namely kisspeptin, neurokin B, dynorphin (KNDy) neurons. Rams were given extra lupin grain with their normal ration. Blood was sampled before feeding, and continued until animals were killed for collection of brain tissue at 2 or 11h after supplementation. In supplemented rams, LH pulse frequency increased after feeding, whereas control animals showed no change. Within the caudal ARC, there were more kisspeptin neurons in supplemented rams than in controls and a higher proportion of kisspeptin cells coexpressed Fos, regardless of the time the rams were killed. There were more Fos cells in the mid-ARC and mid-dorsomedial hypothalamus of the supplemented compared with control rams. No effect of nutrition was found on kisspeptin expression in the rostral or mid-ARC, or on GnRH expression in the preoptic area. Kisspeptin neurons in the caudal ARC appear to mediate the increase in GnRH and LH production due to acute nutritional supplementation, supporting the hypothesised role of the KNDy neurons as the pulse generator for GnRH.

Impact of psychosocial stress on gonadotrophins and sexual behaviour in females: role for cortisol?

This review focuses on the importance of cortisol in mediating the inhibitory effects of psychosocial stress on reproduction in females. In particular, we have summarized our research in sheep where we have systematically established whether cortisol is both sufficient and necessary to suppress reproductive hormone secretion and inhibit sexual behaviour. Our findings are put into context with previous work and are used to develop important concepts as well as to identify productive further lines of investigation. It is clear that cortisol is necessary to inhibit some, but not all, aspects of reproduction in female sheep. These actions vary with reproductive state, and there are important interactions with gonadal steroids. The impact of cortisol on the tonic secretion of gonadotrophin-releasing hormone and luteinizing hormone has been investigated extensively, but less is known about the surge secretion of these hormones and their effects on sexual behaviour. Furthermore, there are separate effects of cortisol in the brain (hypothalamus) and at the anterior pituitary, illustrating that there are different mechanisms of action. Thus, although cortisol is important in mediating some of the effects of stress on reproduction, we need to look beyond cortisol and investigate some of the other mechanisms and mediators that relay the effects of stress on reproduction. In this regard, we propose that a group of neurons in the hypothalamus that co-synthesize kisspeptin, neurokinin B and dynorphin, termed KNDy cells, play important roles in mediating the effects of cortisol on reproduction. This hypothesis needs to be rigorously tested.

Altered hematopoiesis, behavior, and sexual function in mu opioid receptor-deficient mice.

The mu opioid receptor is thought to be the cellular target of opioid narcotics such as morphine and heroin, mediating their effects in both pain relief and euphoria. Its involvement is also implicated in a range of diverse biological processes. Using a mouse model in which the receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in a number of physiological functions. Mice homozygous for the disrupted gene developed normally, but their motor function was altered. Drug-naive homozygotes displayed reduced locomotor activity, and morphine did not induce changes in locomotor activity observed in wild-type mice. Unexpectedly, lack of a functional receptor resulted in changes in both the host defense system and the reproductive system. We observed increased proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in both bone marrow and spleen, indicating a link between hematopoiesis and the opioid system, both of which are stress-responsive systems. Unexpected changes in sexual function in male homozygotes were also observed, as shown by reduced mating activity, a decrease in sperm count and motility, and smaller litter size. Taken together, these results suggest a novel role of the mu opioid receptor in hematopoiesis and reproductive physiology, in addition to its known involvement in pain relief.

Importance of neuroanatomical data from domestic animals to the development and testing of the KNDy hypothesis for GnRH pulse generation.

Work during the last decade has led to a novel hypothesis for a question that is half a century old: how is the secretory activity of GnRH neurons synchronized to produce episodic GnRH secretion. This hypothesis posits that a group of neurons in the arcuate nucleus (ARC) that contain kisspeptin, neurokinin B (NKB), and dynorphin (known as KNDy neurons) fire simultaneously to drive each GnRH pulse. Kisspeptin is proposed to be the output signal to GnRH neurons with NKB and dynorphin acting within the KNDy network to initiate and terminate each pulse, respectively. This review will focus on the importance of neuroanatomical studies in general and, more specifically, on the work of Dr Marcel Amstalden during his postdoctoral fellowship with the authors, to the development and testing of this hypothesis. Critical studies in sheep that laid the foundation for much of the KNDy hypothesis included the report that a group of neurons in the ARC contain both NKB and dynorphin and appear to form an interconnected network capable of firing synchronously, and Marcel's observations that the NKB receptor is found in most KNDy neurons, but not in any GnRH neurons. Moreover, reports that almost all dynorphin-NKB neurons and kisspeptin neurons in the ARC contained steroid receptors led directly to their common identification as "KNDy" neurons. Subsequent anatomical work demonstrating that KNDy neurons project to GnRH somas and terminals, and that kisspeptin receptors are found in GnRH, but not KNDy neurons, provided important tests of this hypothesis. Recent work has explored the time course of dynorphin release onto KNDy neurons and has begun to apply new approaches to the issue, such as RNAscope in situ hybridization and the use of whole tissue optical clearing with light-sheet microscopy. Together with other approaches, these anatomical techniques will allow continued exploration of the functions of the KNDy population and the possible role of other ARC neurons in generation of GnRH pulses.

Medial nucleus of the amygdala mediates chemosensory control of male hamster sexual behavior.

Bilateral lesions restricted to the medial nucleus of the amygdala eliminate mating behavior in the male hamster and severely diminish the male's sniffing and licking investigation of the female hamster's anogenital region. The results suggest that olfactory and vomeronasal sensory information critical to male mating behavior is processed in the medial nucleus, which is an androgen-binding brain area. Thus the medial nucleus may act as a relay through which chemosensory information influences activity in the medial preoptic-anterior hypothalamic junction and the bed nucleus of the stria terminals, areas important in the mediation of male sexual behavior.

Transplantation: a new tool in the analysis of the mammalian hypothalamic circadian pacemaker.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of pacemaker cells that generate circadian rhythmicity in mammals. Transplantation of the nucleus into animals whose own nucleus has been ablated results in the restoration of overt rhythmicity to the arrhythmic host. By using donors and hosts with genetically different circadian characteristics, the unambiguous recognition of the donor rhythm expressed in a transplant recipient is possible. The reappearance of a rhythm indicates that not only has the grafted tissue survived the transplantation procedure, but that pacemaker cells that generate circadian rhythms were included in the graft; this is essential in interpreting results of such transplantation experiments. The restoration of circadian function by neural transplantation has become an important tool for studying the generation and expression of biological rhythms in mammals, and is being used in the investigation of basic questions in this field.

Immunocytochemical colocalization of GABA-B receptor subunits in gonadotropin-releasing hormone neurons of the sheep.

GABA has been shown to play an important role in the control of gonadotropin-releasing hormone (GnRH) and luteinizing hormone secretion in many mammals. In sheep, seasonal differences in the ability of GABA-B receptor antagonists to alter pulsatile luteinizing hormone secretion have led to the hypothesis that this receptor subtype mediates the increased inhibitory effects of estradiol on GnRH and luteinizing hormone pulse frequency seen during the non-breeding season (anestrus). The aim of the present study was to use multiple-label immunocytochemistry to determine if ovine GnRH neurons contain the GABA-B receptor subunits R1 and/or R2, and to determine whether there are seasonal differences in the colocalization of these subunits in GnRH neurons. A majority of GnRH cells in the preoptic area, anterior hypothalamic area, and medial basal hypothalamus of both breeding season and anestrous ewes contained either GABA-B R1 or R2 subunits; a subset of GnRH neurons in breeding season (42%) and anestrous ewes (60%) contained both subunits. In contrast to colocalization within cell bodies, GnRH fibers in the median eminence did not colocalize GABA-B receptor subunits. Although the percentage of GnRH neurons expressing GABA-B receptor subunits tended to be higher in anestrus than in the breeding season, there were no significant seasonal differences in R1 and R2 subunit colocalization in GnRH cell bodies. Thus, while GABA may act directly on GnRH cell bodies via GABA-B receptors in the sheep, any role that GABA-B receptors may play in seasonal reproductive changes is likely mediated by other neurons afferent to GnRH cells.

Colocalisation of dynorphin a and neurokinin B immunoreactivity in the arcuate nucleus and median eminence of the sheep.

Dynorphin A (DYN)-containing cells play a key role in conveying the negative feedback influence of progesterone upon pulsatile gonadotrophin-releasing hormone (GnRH) secretion in the ewe. A very high percentage of DYN cells in the arcuate nucleus express the progesterone receptor; another population of arcuate nucleus cells that also express steroid receptors in the sheep are those that express the tachykinin peptide, neurokinin B (NKB). Both DYN and NKB fibres have been shown to form close contacts with ovine GnRH cells. Therefore, the present study tested the hypothesis that neurones expressing NKB and DYN represent the same neuronal population in the arcuate nucleus. Confocal microscopic analysis of brain sections processed for dual immunofluorescence revealed that a large majority of DYN neurones in the arcuate nucleus were also immunoreactive for NKB. Likewise, a similar majority of NKB neurones in the arcuate nucleus were immunoreactive for DYN. By contrast, DYN cells in the preoptic area and anterior hypothalamus did not colocalise with NKB, nor did DYN cells in the paraventricular or supraoptic nuclei. Fibres that stained positively for both DYN and NKB were seen in the arcuate nucleus, where they formed close appositions with DYN/NKB-positive neurones, and in the external zone of the median eminence. Taken together with previous findings, these data suggest that a subpopulation of arcuate nucleus neurones coexpressing DYN and NKB mediate the negative feedback influence of progesterone on pulsatile GnRH secretion in the ewe and may also be involved in other feedback actions of gonadal steroids.

Surge-Like Luteinising Hormone Secretion Induced by Retrochiasmatic Area NK3R Activation is Mediated Primarily by Arcuate Kisspeptin Neurones in the Ewe.

The neuropeptides neurokinin B (NKB) and kisspeptin are potent stimulators of gonadotrophin-releasing hormone (GnRH)/luteinsing hormone (LH) secretion and are essential for human fertility. We have recently demonstrated that selective activation of NKB receptors (NK3R) within the retrochiasmatic area (RCh) and the preoptic area (POA) triggers surge-like LH secretion in ovary-intact ewes, whereas blockade of RCh NK3R suppresses oestradiol-induced LH surges in ovariectomised ewes. Although these data suggest that NKB signalling within these regions of the hypothalamus mediates the positive-feedback effects of oestradiol on LH secretion, the pathway through which it stimulates GnRH/LH secretion remains unclear. We proposed that the action of NKB on RCh neurones drives the LH surge by stimulating kisspeptin-induced GnRH secretion. To test this hypothesis, we quantified the activation of the preoptic/hypothalamic populations of kisspeptin neurones in response to POA or RCh administration of senktide by dual-label immunohistochemical detection of kisspeptin and c-Fos (i.e. marker of neuronal activation). We then administered the NK3R agonist, senktide, into the RCh of ewes in the follicular phase of the oestrous cycle and conducted frequent blood sampling during intracerebroventricular infusion of the kisspeptin receptor antagonist Kp-271 or saline. Our results show that the surge-like secretion of LH induced by RCh senktide administration coincided with a dramatic increase in c-Fos expression within arcuate nucleus (ARC) kisspeptin neurones, and was completely blocked by Kp-271 infusion. We substantiate these data with evidence of direct projections of RCh neurones to ARC kisspeptin neurones. Thus, NKB-responsive neurones in the RCh act to stimulate GnRH secretion by inducing kisspeptin release from KNDy neurones.

Distribution of preprodynorphin mRNA and dynorphin-a immunoreactivity in the sheep preoptic area and hypothalamus.

Endogenous opioid peptides (EOP) are important modulators in a variety of neuroendocrine systems, including those mediating reproduction, energy balance, lactation, and stress. Recent work in the ewe has implicated the EOP, dynorphin (DYN), in the inhibitory effects of progesterone on pulsatile gonadotropin releasing hormone secretion. Although DYN is involved in a number of hypothalamic functions in the sheep, little is known regarding the localization of preprodynorphin (PPD) expression and its major product DYN A (1-17). In this study, we determined the distribution of PPD mRNA and DYN A-containing cell bodies in the brains of ovary-intact, luteal ewes. To detect PPD mRNA, an ovine PPD mRNA was subcloned by reverse transcription-polymerase chain reaction from sheep hypothalamus and used to create a (35)S-labeled riboprobe for in situ hybridization. Neurons that expressed PPD mRNA and DYN A immunoreactivity were widely distributed in the ovine preoptic area and hypothalamus. PPD mRNA-expressing cells were seen in the supraoptic nucleus, paraventricular nucleus, preoptic area, anterior hypothalamus area, bed nucleus of the stria terminalis, ventromedial nucleus (VMN), dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. All of these regions also contained DYN A-positive cell bodies except for the VMN, raising the possibility that PPD is preferentially processed into other peptide products in the VMN. In summary, based on the expression of both mRNA and peptide, DYN cells are located in a number of key hypothalamic regions involved in the neuroendocrine control of homeostasis in sheep.

Restoration of circadian rhythmicity by transplants of SCN "micropunches".

Although it is widely accepted that the suprachiasmatic nuclei (SCN) of the hypothalamus serve as biological pacemakers regulating circadian rhythmicity, a number of studies suggest that some circadian rhythms may be controlled by extra-SCN structures. Transplantation of fetal anterior hypothalamic tissue containing the SCN restores circadian locomotor rhythms in SCN-lesioned hosts. Such transplants, however, contain substantial extra-SCN hypothalamic tissue. In the present study, the authors examined the recovery of circadian locomotor rhythms in animals implanted with small grafts harvested by taking "micropunches" from vibratome-sectioned brain slices. Micropunches were taken from three areas of the hypothalamus known to receive retinal input: the SCN, the subparaventricular zone, and the supraoptic nucleus. The results indicate that transplants restricted to the SCN region are necessary and sufficient for restoration of circadian locomotor activity rhythms and that micropunches of tissues from other sources are ineffective.

Culture and transplantation of the mammalian circadian pacemaker.

In transplantation studies using the tau mutation in the golden hamster, it has been demonstrated that suprachiasmatic nucleus (SCN) pacemaker cells and mechanisms of communication with the host brain are retained even after tissue dissociation and maintenance for many weeks in primary cell culture. Brain grafts of cultured SCN cells are capable of restoring overt rhythms of locomotor activity, and preliminary studies where cells from two tau genotypes are combined in a single graft demonstrate that pacemaker cells may communicate with each other to produce coherent rhythms with intermediate periods. The opportunity is presented, therefore, to study pacemaker-pacemaker communication in circadian chimeras produced by SCN transplantation. Immunocytochemical analysis of graft-host interactions requires the positive identification of host versus donor cells. Although grafted blocks of tissue are easily recognized during immunocytochemical analysis, implants of dissociated and cultured cells may be more diffusely located and are not as readily identified. Unless distinct strain- or species-specific markers are available, it is difficult to identify connections that may carry timing information to the host organism. We have taken an anatomical approach that utilizes cell-labeling techniques for hamster tissue along with foreign protein expression in transgenic mice to identify patterns of communication among graft and host cells, focusing specifically on SCN-SCN communication. The data indicate the usefulness of these transgenes as markers in transplantation studies where communication between graft and host is addressed.

Regulation of the phase and period of circadian rhythms restored by suprachiasmatic transplants.

The influence of exogenous signals on circadian rhythms restored by transplants of the suprachiasmatic nucleus (SCN) of the hypothalamus has received little study. The authors tested the responsiveness of hamsters bearing SCN transplants to photic and pharmacological treatments. Light intensities as high as 6,500 lux were insufficient to produce entrainment, although masking was observed frequently. Triazolam failed to produce statistically significant phase shifts when administered during the subjective day, but 2 animals bearing functional SCN grafts responded to this benzodiazapine during the subjective night. The authors next tested the hypothesis that the host can retain circadian aftereffects that influence the period of the circadian system reconstituted by the graft. Intact hamsters were entrained to light:dark cycles of short (23.25-h) and long (25-h) period (T) for at least 3 months. Control hamsters released into constant darkness exhibited profound and long-lasting aftereffects of entrainment to T cycles. Hamsters that received SCN lesions after exposure to these T cycles and SCN grafts 3 weeks later exhibited marginal but statistically significant aftereffects that disappeared within 3 months. On subsequent transfer to constant light, tau lengthened by 0.25 +/- 0.6 h in hamsters with intact SCN (p < .05). Animals bearing SCN grafts continued to free run in constant light but differed from intact animals in that circadian period did not lengthen. Functional SCN grafts contained vasoactive intestinal polypeptide (VIP), neurophysin (NP), and cholecystokinin (CCK) immunoreactive (ir) cells. Inputs of neuropeptide Y-and serotonin-ir fibers from the host brain to grafted SCN peptide cell clusters were variable. Limited observations using retrograde and anterograde tracers do not support the existence of extensive input to the graft. Retinal input overlapped only rarely with clusters of VIP-ir, CCK-ir, or NP-ir cells. The authors conclude that the circadian system reinstated by SCN transplants is relatively impervious to photic influences that exert parametric and nonparametric influences in intact hamsters. The transient expression of aftereffects induced in the host before transplantation indicates that extra-SCN systems of the host can influence the period of the reconstituted circadian system to at least a limited degree.

Evidence for Changes in Numbers of Synaptic Inputs onto KNDy and GnRH Neurones during the Preovulatory LH Surge in the Ewe.

Kisspeptin neurones located in the arcuate nucleus (ARC) and preoptic area (POA) are critical mediators of gonadal steroid feedback onto gonadotrophin-releasing hormone (GnRH) neurones. ARC kisspeptin cells that co-localise neurokinin B (NKB) and dynorphin (Dyn), are collectively referred to as KNDy (Kisspeptin/NKB/Dyn) neurones, and have been shown in mice to also co-express the vesicular glutamate transporter, vGlut2, an established glutamatergic marker. The ARC in rodents has long been known as a site of hormone-induced neuroplasticity, and changes in synaptic inputs to ARC neurones in rodents occur over the oestrous cycle. Based on this evidence, the the present study aimed to examine possible changes across the ovine oestrous cycle in synaptic inputs onto kisspeptin cells in the ARC (KNDy) and POA, and inputs onto GnRH neurones. Gonadal-intact breeding season ewes were perfused using 4% paraformaldehyde during either the luteal or follicular phase of the oestrous cycle, with the latter group killed at the time of the luteinising hormone (LH) surge. Hypothalamic sections were processed for triple-label immunodetection of kisspeptin/vGlut2/synaptophysin or kisspeptin/vGlut2/GnRH. The total numbers of synaptophysin- and vGlut2-positive inputs to ARC KNDy neurones were significantly increased at the time of the LH surge compared to the luteal phase; because these did not contain kisspeptin, they do not arise from KNDy neurones. By contrast to the ARC, the total number of synaptophysin-positive inputs onto POA kisspeptin neurones did not differ between luteal phase and surge animals. The total number of kisspeptin and vGlut2 inputs onto GnRH neurones in the mediobasal hypothalamus (MBH) was also increased during the LH surge, and could be attributed to an increase in the number of KNDy (double-labelled kisspeptin + vGlut2) inputs. Taken together, these results provide novel evidence of synaptic plasticity at the level of inputs onto KNDy and GnRH neurones during the ovine oestrous cycle. Such changes may contribute to the generation of the preovulatory GnRH/LH surge.

Prenatal testosterone excess decreases neurokinin 3 receptor immunoreactivity within the arcuate nucleus KNDy cell population.

Prenatal exposure of the female ovine foetus to excess testosterone leads to neuroendocrine disruptions in adulthood, as demonstrated by defects in responsiveness with respect to the ability of gonadal steroids to regulate gonadotrophin-releasing hormone (GnRH) secretion. In the ewe, neurones of the arcuate nucleus (ARC), which co-expresses kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy cells), play a key role in steroid feedback control of GnRH and show altered peptide expression after prenatal testosterone treatment. KNDy cells also co-localise NKB receptors (NK3R), and it has been proposed that NKB may act as an autoregulatory transmitter in KNDy cells where it participates in the mechanisms underlying steroid negative-feedback. In addition, recent evidence suggests that NKB/NK3R signalling may be involved in the positive-feedback actions of oestradiol leading to the GnRH/luteinising hormone (LH) surge in the ewe. Thus, we hypothesise that decreased expression of NK3R in KNDy cells may be present in the brains of prenatal testosterone-treated animals, potentially contributing to reproductive defects. Using single- and dual-label immunohistochemistry we found NK3R-positive cells in diverse areas of the hypothalamus; however, after prenatal testosterone treatment, decreased numbers of NK3R immunoreactive (-IR) cells were seen only in the ARC. Moreover, dual-label confocal analyses revealed a significant decrease in the percentage of KNDy cells (using kisspeptin as a marker) that co-localised NK3R. To investigate how NKB ultimately affects GnRH secretion in the ewe, we examined GnRH neurones in the preoptic area (POA) and mediobasal hypothalamus (MBH) for the presence of NK3R. Although, consistent with earlier findings, we found no instances of NK3R co-localisation in GnRH neurones in either the POA or MBH; in addition, > 70% GnRH neurones in both areas were contacted by NK3R-IR presynaptic terminals suggesting that, in addition to its role at KNDy cell bodies, NKB may regulate GnRH neurones by presynaptic actions. In summary, the finding of decreased NK3R within KNDy cells in prenatal testosterone-treated sheep complements previous observations of decreased NKB and dynorphin in the same population, and may contribute to deficits in the feedback control of GnRH/LH secretion in this animal model.

Herpes simplex virus as a transneuronal tracer.

Determining the connections of neural systems is critical for determining how they function. In this review, we focus on the use of HSV-1 and HSV-2 as transneuronal tracers. Using HSV to examine neural circuits is technically simple. HSV is injected into the area of interest, and after several days, the animals are perfused and processed for immunohistochemistry with antibodies to HSV proteins. Variables which influence HSV infection include species of host, age of host, titre of virus, strain of virus and phenotype of infected cell. The choice of strain of HSV is critically important. Several strains of HSV-1 and HSV-2 have been utilized for purposes of transneuronal tract-tracing. HSV has been used successfully to study neuronal circuitry in a variety of different neuroanatomical systems including the somatosensory, olfactory, visual, motor, autonomic and limbic systems.

Do gonadotropin-releasing hormone, tyrosine hydroxylase-, and beta-endorphin-immunoreactive neurons contain estrogen receptors? A double-label immunocytochemical study in the Suffolk ewe.

We used double label immunocytochemistry to examine the brains of ovariectomized ewes and determine whether GnRH, tyrosine hydroxylase-(TH), and beta-endorphin-immunoreactive (IR) neurons contain IR-estrogen receptors (ER). Because of their possible importance as a target for the feedback actions of estradiol, we also examined the presence of nuclear ER in LH-IR cells of the pars tuberalis of the pituitary. Although preoptic GnRH neurons were frequently in close proximity to ER-IR cells, only one out of approximately 1000 GnRH cells examined was found to coexpress ER. In contrast, in the arcuate nucleus and vicinity, 3-5% of TH cells and 15-20% of beta-endorphin cells contained ER. Virtually all LH-IR cells, seen predominantly in the ventral portion of the pars tuberalis, coexpressed ER. These results suggest that in sheep as in rodents, the influence of estradiol on the reproductive neuroendocrine system is not directly mediated by GnRH neurons, but instead is conveyed to GnRH cells via presynaptic afferents. Subsets of TH- and beta-endorphin-IR cells which coexpress ER are two candidates for relaying gonadal steroid signals to GnRH cells. At the level of the pituitary, the feedback actions of estradiol may be expressed directly upon the gonadotroph.

Neurons that migrate from the olfactory epithelium in the chick express luteinizing hormone-releasing hormone.

In several mammalian species, luteinizing hormone-releasing hormone (LHRH) neurons have been shown to migrate from nasal regions to the brain during early development. Using immunocytochemistry, we have identified LHRH containing neurons in developing chick embryos. In embryonic day 4 (E4) and E5 animals, a small group of LHRH immunoreactive (IR) neurons were found just ventral to the olfactory pit. LHRH-IR neurons were also found within the immunoreactive (IR) neurons were found just ventral to the olfactory pit. LHRH-IR neurons were also found within the nasal epithelium. In E6 and E7 animals, many more LHRH-IR neurons were observed in nasal epithelium, in close association with the olfactory nerve, and within the telencephalon. These data are consistent with the hypothesis that LHRH neurons in chicks originate within nasal structures and migrate into the brain.

Fos expression during the estradiol-induced gonadotropin-releasing hormone (GnRH) surge of the ewe: induction in GnRH and other neurons.

The protein product of the protooncogene c-fos was used as a marker of cellular activation in an attempt to identify those neurons in the preoptic area and hypothalamus that participate in generation of the estradiol-induced surge of GnRH in the ewe. GnRH- and Fos-expressing cells were identified immunocytochemically, and the percent of coexpression was determined in three states: mid-luteal phase (low GnRH release, n = 6); short-term ovariectomy (high episodic GnRH release, n = 6); and induced GnRH surge (high sustained release, n = 8). To induce the GnRH surge, a follicular phase rise in circulating estradiol was simulated in a physiological model for the estrous cycle. Serum LH was measured as an indicator of GnRH release. In the luteal phase, LH was basal, indicating low GnRH secretion. Few cells expressed Fos; these were not GnRH cells. Despite high intermittent GnRH release in short-term ovariectomized ewes, GnRH cells did not express Fos. During the surge (sustained high GnRH release), 41 +/- 8% of GnRH cells expressed Fos; these cells were dispersed throughout the field of distribution of GnRH neurons. In addition to Fos in GnRH-positive cells, many more non-GnRH cells in the preoptic area, anterior hypothalamus, and ventrolateral hypothalamus expressed Fos during the surge than in the luteal phase or after ovariectomy. We suggest that Fos expression in GnRH cells is markedly increased by the positive feedback action of estradiol (surge), whereas short-term removal of negative feedback (ovariectomy) has little, if any, effect, despite increased GnRH release in both states. Since estradiol induces Fos expression in far more than GnRH neurons, our results also suggest that estradiol activates other cells, some of which may be part of a neuronal chain leading to GnRH surge generation, and some of which may be related to other neural actions of estradiol, such as estrous behavior.

Estrogen receptors in dendrites and axon terminals in the guinea pig hypothalamus.

The ultrastructural localization of steroid hormone receptors has been made possible by the development of immunocytochemical procedures using monoclonal antibodies. Estrogen receptor-immunoreactivity (ER-IR) in the brain is present most abundantly in neuronal nuclei when observed with light microscopy. However, we have also observed ER-IR in the perikarya and cytoplasmic processes of neurons. To determine the organelles with which the cytoplasmic ER-IR is associated, we developed a technique for ultrastructural visualization of ER-IR. Ovariectomized guinea pigs were perfused, brains vibratome-sectioned, and estrogen receptors immunostained by either an immunoperoxidase-diaminobenzidine technique or by an immunogold-streptavidin procedure, each followed by silver intensification. Electron microscopic analysis confirmed distribution of ER-IR throughout cell nuclei, but ER-IR was also observed in proximal and distal dendrites and rough endoplasmic reticulum. Most surprisingly, however, ER-IR was found in many axon terminals containing predominantly round, and in some cases, flattened clear synaptic vesicles. Parallel experiments examining the distribution of progestin receptors confirmed the localization at the same subcellular sites as for estrogen receptors. The results of this experiment corroborate our earlier findings of extranuclear steroid receptor-immunoreactivity in the brain, and they suggest potential nongenomic sites of action for estradiol and progesterone in dendrites and axon terminals.