Hypothalamic prolactin stimulates the release of luteinizing hormone-releasing hormone from male rat hypothalamus

Previous works from our laboratory and others have shown that there is a PRL-like immunoreactive protein with immunological, chromatographic, and biological characteristics identical to those of pituitary PRL, and this is widely distributed in the rat central nervous system. Since pituitary PRL is important in controlling hypothalamic LHRH release, we have hypothesized that hypothalamic PRL-like immunoreactive protein might serve a similar role, that of an endogenous neuromodulator influencing hypothalamic LHRH release. To this end, we have examined the effect of PRL antiserum and normal rabbit serum on the release of immunoreactive LHRH from rat hypothalamic fragments cultured in vitro. In the first experiment, LHRH release from hypothalami of intact rats, bathed in PRL antiserum (1:200 in Krebs-Ringer bicarbonate buffer), was significantly lower than that from hypothalami bathed in normal rabbit serum (1:200 in Krebs-Ringer bicarbonate buffer) for 90 min of incubation. It was, however, possible that the PRL, immunoneutralized in the first experiment, was material that represented contamination from pituitary PRL. Therefore, we repeated the experiment using hypothalami from animals that had been hypophysectomized 2 weeks before death. Again, PRL antibody significantly inhibited the release of LHRH compared with that by hypothalami incubated in normal rabbit serum. Since testosterone is important to LHRH synthesis, a third experiment was carried out using hypothalami from hypophysectomized male rats that had been implanted sc with testosterone-containing capsules 72 h before death. By 72 h serum testosterone levels had normalized. PRL antibody added to medium containing hypothalamic explants from these animals substantially inhibited in vitro LHRH release, a pattern essentially similar to that seen in intact and hypophysectomized animals without testosterone replacement. From these studies we have concluded that hypothalamic PRL is an important neuromodulator that promotes the release of LHRH from the hypothalamus. Testosterone, at least under the experimental conditions employed, appears not to be essential in this hypothalamic PRL-LHRH interaction.     

In vitro release of luteinizing hormone-releasing hormone from the hypothalamus of old male rats

The present experiments tested the ability of the hypothalamus of old male rats to release LHRH in vitro. Mediobasal hypothalami (MBH) of 18-month-old and 6-month-old male rats were perfused for a total of 5 h and 5 min; the amounts of LHRH released, both in basal conditions and after stimulation with high extracellular K+ (110 mM, applied for 5 min every 30 min), were measured in the effluent. Our results show that the basal secretion of LHRH from the perfused MBH of young and old male rats is quantitatively similar. Moreover, the MBHs of young and old animals are capable of responding to the repeated administration of a K+ stimulus with repeated bursts of LHRH hypersecretion. From a quantitative point of view, the MBHs of old animals respond to the K+ stimulus in a fashion similar to that found in younger animals; even if the responses to K+ of the hypothalami of old animals appear to be somewhat lower than those of the hypothalami of younger animals, the differences recorded were not significant. LHRH content (as measured by RIA) of the MBH before the perfusion period was similar in young and old animals. At the end of the experiment, the content of LHRH of the perfused hypothalamus was not significantly different in the old and young groups. Serum and intrapituitary levels of LH and FSH were significantly lower in 18-month than in 6-month-old animals. These data suggest that the alterations of the function of the hypothalamic-pituitary-gonadal complex observed in aged male rats are not due to an intrinsic age-related defect of LHRH-synthesizing neurons.     

Neuropeptide Y stimulates growth hormone and gonadotropin release from the goldfish pituitary in vitro

The effects of neuropeptide Y (NPY) on release of growth hormone (GH) and gonadotropin (GTH) from the goldfish pituitary in vitro were investigated. Exposure of perifused pituitary fragments, taken from female goldfish at late stages of gonadal recrudescence, to 5-min pulses of human NPY resulted in a rapid dose-dependent stimulation of GH and GTH release, with half-maximal effective dosages of 0.51 +/- 0.24 and 2.37 +/- 1.05 nM for GH and GTH, respectively. Repeated treatments with pulses of NPY (10 nM for GH, 5 nM for GTH) at 55-min intervals did not significantly alter the responsiveness of pituitary fragments to NPY; however, prior exposure of pituitary fragments to pulses of higher doses of NPY (50 nM GH, 10 nM for GTH) significantly reduced the subsequent hormone responses. When given at 85-min intervals repeated treatment with NPY did not blunt hormone responses to the second and third stimulations at these higher dosages. These results indicate that NPY acts at the pituitary level to stimulate GH and GTH secretion in female goldfish. The GTH response and, to a lesser extent, the GH response become desensitized to further stimulation by NPY in dose- and time-dependent manners. NPY should be considered as one element in the multifactorial systems regulating the GH and GTH secretion in goldfish.     

Neuropeptide Y is a neuromodulator of pulsatile luteinizing hormone-releasing hormone release in the gonadectomized rhesus monkey.

In a previous study, we have demonstrated that infusion of neuropeptide Y (NPY) into the stalk-median eminence (S-ME) of gonadectomized rhesus monkeys stimulated LHRH in a dose-dependent manner. This finding led us to address the following questions: 1) What are the characteristics of NPY release in vivo? 2) How does NPY release relate to LHRH release? 3) Is endogenous NPY essential to pulsatile LHRH release? To answer these questions, three experiments using push-pull perfusion were performed in adult gonadectomized rhesus monkeys. Perfusate samples from the S-ME were collected at 10-min intervals for 6 to 12-h periods, and the concentrations of LHRH and NPY in perfusates were determined by RIA. In Exp I, the release pattern of NPY and LHRH in the S-ME was independently determined in a group of 11 conscious monkeys: NPY release in the S-ME was pulsatile with an interpulse interval of 44.9 +/- 3.3 min (n = 11). This interpulse interval was similar to that seen for LHRH release (43.8 +/- 1.1 min, n = 7). Exp II was designed to determine whether NPY pulses and LHRH pulses occur synchronously and to examine whether NPY release in the S-ME is correlated with circulating LH pulses. NPY and LHRH concentrations in aliquots of the same perfusate sample from the S-ME and circulating LH levels were concurrently measured in 8 monkeys sedated with Saffan. It was found that NPY pulses were temporally correlated (P less than 0.001) with LHRH pulses, which were also temporally correlated (P less than 0.001) with LH pulses. Moreover, NPY pulses were correlated (P less than 0.05) with LH pulses. NPY peaks preceded LHRH peaks by 4.5 +/- 0.6 min, LHRH peaks preceded LH peaks by 5.5 +/- 0.6 min, and NPY peaks preceded LH peaks by 9.7 +/- 0.8 min. In Exp III, the role of endogenous NPY in LHRH release was evaluated by infusing a specific antiserum to NPY into the S-ME during push-pull perfusion in 8 conscious monkeys. Infusion of a specific antiserum to NPY into the S-ME at 1:100 and 1:1000 dilutions suppressed pulsatile LHRH release significantly (P less than 0.05). Infusion of nonimmune serum as a control was without effect. These results are summarized as follows: 1) NPY release in the S-ME is pulsatile, 2) NPY pulses occur synchronously with LHRH and LH pulses, and 3) immunoneutralization of endogenous NPY in the S-ME suppresses pulsatile LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuropeptide Y enhances the release of luteinizing hormone (LH) induced by LH-releasing hormone

Depending upon the steroid hormonal milieu, centrally administered neuropeptide Y (NPY) exerts differential effects on the release of LH. Ovarian hormones also effect the concentrations of NPY in hypothalamic nuclei, and some of the changes are similar to those caused by LHRH. The present studies tested whether NPY acts directly on the pituitary gland, either alone or in combination with LHRH, to modify LH secretion. Hemipituitary fragments obtained from ovariectomized rats were incubated in medium 199, and the in vitro effects on LH release of LHRH, NPY, or the two peptides together were assessed. As expected, LHRH (10(-9)-10(-7) M) produced a dose-dependent release of LH, whereas NPY alone had a lesser stimulatory effect at concentrations of 10(-7) or 10(-6) M. On the other hand, 10(-6) M NPY significantly enhanced LH release in response to 10(-9) M LHRH. A potentiation by NPY of the LHRH-induced LH response was observed in an anterior pituitary cell culture system. Cells from the pituitaries of ovariectomized rats were dispersed and cultured for 3 days in medium 199 with BSA, gentamicin, horse serum, and fetal calf serum. During a 3-h incubation, NPY alone (10(-9)-10(-7) M) failed to affect LH release, but significantly potentiated the release induced by 10(-9) or 10(-8) M LHRH. These findings are in accord with the hypothesis that hypothalamic NPY neurons may participate in the regulation of LH secretion in the rat and indicate that one of the mechanisms of its action may be to increase the pituitary LH response to LHRH.     

Acetylcholine, melatonin, and potassium depolarization stimulate release of luteinizing hormone-releasing hormone from rat hypothalamus in vitro.

We have examined the release of radioimmunoassayable luteinizing hormone-releasing hormone (LH-RH) from fragments of rat medial basal hypothalamus. These fragments were cultured overnight in medium containing serum and then preincubated in groups of three for 10 min in medium resembling cerebrospinal fluid in its electrolyte constituents and containing bacitracin. This was followed by 30-min incubation periods during which some of the hypothalami were exposed to test substances. Potassium depolarization, effected by the addition of 56 mM potassium chloride to the incubation medium, caused a marked stimulation in LH-RH release, but only in the presence of calcium. Acetylcholine at 10 nM and the parasympathomimetic anticholinesterase agent neostigmine at 1 microM markedly stimulated LH-RH release. Hexamethonium, a nicotinic antagonist, at 1 microM abolished the acetylcholine-induced increment in LH-RH release. Melatonin, a pineal indolamine, caused significant stimulation of LH-RH release at a concentration as low as 10 nM. Bacitracin (21 microM) was employed in all these experiments. It had no effect on LH-RH release but did prevent the degradation of LH-RH in this system. We conclude that acetylcholine and melatonin are capable of inducing LH-RH release from the rat medial basal hypothalamus. These actions may account for some of the progonadotropic properties previously ascribed to these agents.     

Interleukin-1 inhibits the ovarian steroid-induced luteinizing hormone surge and release of hypothalamic luteinizing hormone-releasing hormone in rats

Interleukin-1 (IL-1), a polypeptide cytokine secreted by activated macrophages, has been postulated as a chemical messenger between the immune and endocrine systems. IL-1-immunopositive neurons and fibers have been visualized in the human and rat hypothalamus, and IL-1 receptors are present in the rat brain. We have examined the effects of human recombinant IL-1 (alpha- and beta-subtypes) on LH release in vivo and hypothalamic LHRH release in vitro. Ovariectomized rats were primed with estradiol benzoate, and progesterone was injected 48 h later to elicit a LH surge in the afternoon. IL-1 alpha and IL-1 beta were injected either intracerebroventricularly (icv) via a preimplanted cannula in the third ventricle of the brain or iv. Systemic injection of IL-1 alpha or IL-1 beta (58.8 pmol at 1300 and 1500 h) failed to influence the afternoon LH surge seen in saline-injected control rats. However, IL-1 beta (1.76 pmol) administered icv at 1300 and 1500 h or a single icv injection at 1300 h blocked the progesterone-induced LH surge. Similar icv injections of IL-1 alpha also significantly suppressed the afternoon LH surge compared to that in saline-injected control rats. However, IL-1 alpha was relatively less effective than the beta-subtype, since the LH surge was detected in some rats. To ascertain whether suppression of the LH surge was due to inhibition of LHRH release, the medial basal hypothalamus-preoptic area of estradiol benzoate-progesterone-treated ovariectomized rats was incubated with and without IL-1. Both IL-1 alpha and IL-1 beta, at concentrations of 0.1 nM and higher, significantly suppressed LHRH release in vitro from the medial basal hypothalamus-preoptic area. In contrast, IL-1 (10 nM) was completely ineffective in suppressing LHRH release from the microdissected median eminence. These results demonstrated an overall inhibitory effect of icv IL-1 on the LHRH-LH axis and suggest that suppression of the steroid-induced LH surge by IL-1 may primarily be due to inhibition of LHRH release at hypothalamic sites located within the blood-brain barrier.     

Stimulation of luteinizing hormone release from chicken pituitary cells by analogues of luteinizing hormone-releasing hormone.

The luteinizing hormone (LH) releasing activities of luteinizing hormone-releasing hormone (LH-RH) and four related analogues were compared using isolated chicken anterior pituitary cells. The analogues, des-Gly¹⁰-LH-RH and Phe⁵-LH-RH, exhibited a greater potency than LH-RH (150 and 237%, respectively), whereas LH-RH(OH) was much less active (1.1%). The potency of Phe⁵-LH-RH was reduced to 0.9% by the insertion of a tyrosine molecule at position 11, indicating that chain length is a significant feature of the biological activity of the molecule. des-Gly¹⁰-LH-RH and Phe⁵-LH-RH were more active in the present system, than is indicated by available information for the rat.     

Evidence that norepinephrine and epinephrine systems mediate the stimulatory effects of ovarian hormones on luteinizing hormone and luteinizing hormone-releasing hormone

Results from previous investigations have suggested an important role for central epinephrine (EPI) systems in mediating the stimulatory effects of ovarian hormones on LH release in ovariectomized female rats. The purpose of these experiments was 1) to test whether selective inhibition of EPI synthesis blocks the sequential accumulation and decline of LHRH concentrations in the median eminence that precedes the ovarian hormone-induced LH surge and 2) to test whether the stimulatory ovarian hormone regimen enhances the activity of EPI systems in the hypothalamus. Ovariectomized rats were treated with estradiol, followed 2 days later by progesterone. Animals were treated before progesterone administration with saline, one of the EPI synthesis inhibitors [SK&F 64139 (2,3-dichloro-tetrahydroisoquinoline HCl) or LY 78335 (dichloro-alpha-methylbenzylamine)], or the dopamine-beta-hydroxylase inhibitor FLA-63 (bis-4-methyl-1-homopiperazinyl thiocarbonyl disulfide), which inhibits NE and EPI synthesis. The catecholamine synthesis inhibitors blocked or delayed the afternoon LH surge. FLA-63 completely prevented the accumulation of LHRH in the median eminence that preceded the rise in LH release. However, selective EPI synthesis inhibition with SK&F 64139 only partially prevented this increase in LHRH. A second EPI synthesis inhibitor, LY 78335, delayed both the LH surge and the rise in LHRH. In a second experiment, the administration of estradiol and progesterone to ovariectomized rats increased the alpha-methyltyrosine-induced depletion of hypothalamic EPI, suggesting increased activity in this system during the LH surge. Further experiments localized this effect to the medial basal hypothalamus. The depletion of both NE and EPI after synthesis inhibition was also enhanced during an earlier period, approximating the time of LHRH accumulation. These results suggest that the ovarian hormones activate both NE and EPI systems to stimulate the early afternoon rise of LHRH in the median eminence and to induce the subsequent LH surge.     

Involvement of transforming growth factor alpha in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus.

Little is known about the presence of trophic factors in the hypothalamus and the role they may play in regulating the functional development of hypothalamic neurons. We have investigated the ability of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) to affect the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls reproductive development. We have also determined whether the genes encoding EGF and TGF-alpha are expressed in the prepubertal female hypothalamus. Northern blot analysis of poly(A)+ RNA utilizing a single-stranded EGF cDNA probe failed to reveal the presence of EGF mRNA in either the hypothalamus or the cerebral cortex at any age studied (fetal day 18 to postnatal day 36). In contrast, both a complementary RNA probe and a double-stranded TGF-alpha cDNA recognized in these regions a 4.5-kilobase (kb) mRNA species identical to TGF-alpha mRNA. The abundance of TGF-alpha mRNA was 3-4 times greater in the hypothalamus than in the cerebral cortex. Both EGF and TGF-alpha (2-100 ng/ml) elicited a dose-related increase in LHRH release from the median eminence of juvenile rats in vitro. They also enhanced prostaglandin E2 (PGE2) release. The transforming growth factors TGF-beta 1 and -beta 2 were ineffective. Only a high dose of basic fibroblast growth factor was able to increase LHRH and PGE2 release. Blockade of the EGF receptor transduction mechanism with RG 50864, a selective inhibitor of EGF receptor tyrosine kinase activity, prevented the effect of both EGF and TGF-alpha on LHRH and PGE2 release but failed to inhibit the stimulatory effect of PGE2 on LHRH release. Inhibition of prostaglandin synthesis abolished the effect of TGF-alpha on LHRH, indicating that PGE2 mediates TGF-alpha-induced LHRH release. The results indicate that the effect of EGF and TGF-alpha on LHRH release is mediated by the EGF/TGF-alpha receptor and suggest that TGF-alpha rather than EGF may be the physiological ligand for this interaction. Since in the central nervous system most EGF/TGF-alpha receptors are located on glial cells, the results also raise the possibility that--at the median eminence--TGF-alpha action may involve a glial-neuronal interaction, a mechanism by which the trophic factor first stimulates PGE2 release from glial cells, and then PGE2 elicits LHRH from the neuronal terminals.     

Alterations in luteinizing hormone-releasing hormone release from the mediobasal hypothalamus of ovariectomized, steroid-primed middle-aged rats as measured by push-pull perfusion

The spontaneous luteinizing hormone (LH) surge on proestrus as well as the steroid-induced LH surge and hypersecretion of LH following ovariectomy are attenuated in middle-aged female rats when compared to their young counterparts. It is generally assumed that the lower titers of serum LH measured in aging animals result, in part, from age-related alterations in luteinizing hormone-releasing hormone (LHRH) neurosecretion, yet no direct measurements of LHRH release from the hypothalamus of aging females are currently available. The present study utilized the push-pull perfusion technique to characterize and compare in vivo LHRH output from the mediobasal hypothalamus of ovariectomized middle-aged and young females during a steroid-induced LH surge. Twelve-minute perfusates were collected for a period of 6 h from middle-aged and young animals outfitted with push-pull cannulae resting in the mediobasal hypothalamus in close proximity to the median eminence. The overall pattern of LHRH release differed in the two age groups. Mean levels of LHRH measured in brain perfusates from middle-aged females were significantly lower than mean levels measured in young animals. Moreover, mean levels of LHRH detected in perfusates during the 1- and 2-hour intervals prior to and the 1-hour interval after the time of maximal LHRH output were lower in middle-aged animals, and a larger percentage of samples from middle-aged females contained levels of LHRH below the detectability of the assay. The mean amplitude of identified LHRH pulses and maximal LHRH output, however, did not differ in the two age groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of blockade of kappa-opioid receptors in the medial basal hypothalamus and medial preoptic area on luteinizing hormone release during midpregnancy in the rat.

The objective of this study was to determine if kappa-opioid receptors present in the medial preoptic area (MPOA) and medial basal hypothalamus (MBH) are involved in opioid peptide suppression of LH secretion during midpregnancy (day 13-16) in the rat. Nor-binaltorphimine (nor-BNI), a selective antagonist of brain kappa-opioid receptors, was applied directly to the MPOA or MBH for 3.5 h by means of push-pull perfusion. Nor-BNI perfusion in the MBH produced a dose-dependent increase in LH pulse frequency as well as increases in blood LH level. The effect on amplitude could not be determined, since too few pulses occurred in cerebrospinal fluid-treated control rats. Nor-BNI perfusion in the MPOA also increased LH pulse frequency. Moreover, in the majority (62%) of rats perfused with nor-BNI in the MPOA, the final 1.5 h of perfusion were unexpectedly characterized by an increase in LH that was of greater magnitude and more prolonged than an LH pulse and produced an elevation in blood LH levels. This delayed LH response did not occur in any rat perfused in the MBH. Perfusion with nor-BNI in the diagonal band of Broca had no significant effect on LH secretion. The LH responses observed during nor-BNI perfusion in the MPOA or MBH were not due to spread to the third ventricle and subsequent diffusion via the cerebrospinal fluid to another brain site, since perfusion with nor-BNI in an area of the ventral thalamus close to the third ventricle had no effect on LH release. These results provide support for the involvement of kappa-opioid receptor-mediated mechanisms in both the MPOA and MBH in the suppression of LH secretion during midgestation in the rat.     

Calcium and calmodulin mediation of growth hormone-releasing hormone release from the rat hypothalamus in vitro.

A major role for Ca2+ and calmodulin in stimulus-secretion coupling has been suggested for several neuropeptides; however, the cellular mechanisms of GH-releasing hormone (GHRH) release have been little investigated so far. We have used a previously validated acute rat hypothalamic explant system in order to elucidate whether Ca2+ acts as a second messenger in the regulation of GHRH release, and whether calmodulin-dependent pathways are involved. Calcium dependence of somatostatin (SRIH) release was assessed in the same experiments. Calmodulin dependence of SRIH was not investigated in detail, as it has been established previously. The calcium-entry antagonist, verapamil, antagonized K(+)-stimulated GHRH and SRIH release in a dose-dependent manner, with maximal inhibition shown at 10(-4) M. The calmodulin antagonist W7 also blocked K(+)-evoked GHRH release in a dose-dependent manner, with significant inhibition in the dose range 5 X 10(-5) M to 2 X 10(-4) M; similarly, a more specific calmodulin inhibitor, the W7 derivative 5-iodo-C8 (W8), reversed K(+)-stimulated GHRH release, showing slightly higher potency than W7. W7 also reversed GHRH release in response to the calcium-ionophore A23187, although verapamil had no effect on A23187-evoked GHRH or SRIH release. Thapsigargin, which increases the efflux of Ca2+ from calciosomes, did not affect either GHRH or SRIH release at 10(-5) M or 10(-4) M. The basal release of GHRH was clearly suppressed by W7 and W8 (10(-4) M), whereas verapamil had no effect. We conclude that calcium influx is crucial for depolarization-induced GHRH and SRIH release. Calcium entrance in response to A23187 appears to be independent of verapamil-sensitive calcium channels. The lack of effect of thapsigargin suggests that increased intracellular Ca2+ from intracellular stores is not equivalent to an increase in Ca2+ influx. Both basal and depolarization-induced release of GHRH in this system are calmodulin dependent.     

Alcohol inhibits luteinizing hormone-releasing hormone release by activating the endocannabinoid system

We hypothesized that ethanol (EtOH) might act through the endocannabinoid system to inhibit luteinizing hormone-releasing hormone (LHRH) release. Therefore, we examined the mechanism by which EtOH and anandamide (AEA), an endogenous cannabinoid, inhibit LHRH release from incubated medial basal hypothalamic explants. In previous work, we demonstrated that EtOH inhibits the N-methyl-D-aspartic acid-stimulated release of LHRH by increasing the release of two neurotransmitters: beta-endorphin and gamma-aminobutyric acid (GABA). In the present work, bicuculline, a GABAergic antagonist, completely prevented the inhibition of AEA (10(-9)M) on N-methyl-D-aspartic acid-induced LHRH release, but naltrexone, a micro-opioid receptor antagonist, had no effect. AEA also significantly increased GABA release but had no effect on beta-endorphin release. Therefore, AEA could inhibit LHRH release by increasing GABA but not beta-endorphin release. Because EtOH and AEA acted similarly to inhibit LHRH release, we investigated whether both substances would affect the adenylate cyclase activity acting through the same GTP-coupled receptors, the cannabinoid receptors 1 (CB1-rs). AEA and EtOH (10(-1)M) reduced the forskolin-stimulated accumulation of cAMP, but AM251, a specific antagonist of CB1-r, significantly blocked that inhibition. Additionally we investigated whether CB1-r is involved in the inhibition of LHRH by EtOH and AEA. AEA and EtOH reduced forskolin-stimulated LHRH release, but AM251 significantly blocked that inhibition. Also, we demonstrated that EtOH did not act by increasing AEA synthase activity to inhibit LHRH release in our experimental conditions. Therefore, our results indicate that EtOH inhibits the release of LHRH acting through the endocannabinoid system.     

A subset of gonadotropin-releasing hormone neurons in the ovine medial basal hypothalamus is activated during increased pulsatile luteinizing hormone secretion

GnRH neurons active in the preovulatory LH surge have been identified in several species using the early intermediate gene product, Fos, but the GnRH neurons active during episodic LH secretion remain unknown. In this study, we have used Fos and Fos-related antigens (FRA) to determine whether a subset of GnRH neurons is active when pulsatile LH secretion is acutely stimulated in sheep. In experiment 1, episodic LH secretion was stimulated in five of six ewes by injection of an opioid antagonist to luteal phase ewes. These five ewes had a 6-fold increase in the percentage of GnRH neurons in the medial basal hypothalamus (MBH) expressing Fos/FRA, compared with control ewes that had no LH pulses before death. Fos/FRA expression was not increased in GnRH neurons found in any other area. In experiment 2, episodic LH secretion was induced in rams by introduction of estrous ewes. This treatment increased Fos/FRA expression in MBH GnRH neurons approximately 10-fold compared with control rams. Again, this increase in Fos/FRA expression in GnRH neurons was limited to the MBH. This selective activation of MBH GnRH neurons could reflect the preferential inhibition of these perikarya by endogenous opioid peptides. It also raises the possibility that a subset of GnRH neurons in the MBH may be responsible for episodic GnRH secretion in sheep.