Luteinizing Hormone-releasing Hormone Neurons Research Articles

Luteinizing Hormone-Releasing Hormone (LHRH) Neurons Maintained in Hypothalamic Slice Explant Cultures Exhibit a Rapid LHRH mRNA Turnover Rate

Evidence indicates that neuropeptide gene expression is tightly coupled to biosynthesis and secretion. Moreover, rhythmic gene expression often accompanies rhythmic secretion. Luteinizing hormone-releasing hormone (LHRH) neurosecretion, which regulates gonadal function, is pulsatile, with interpulse intervals of approximately 1 hr and pulse decays of <30 min in rats. As a basis for a rapid fall in peptide secretion, we hypothesize that LHRH mRNA levels rapidly decay. To address this hypothesis, we examined LHRH mRNA turnover in primary postnatal LHRH neurons maintained in long-term hypothalamic/preoptic area slice explant cultures, using in situ hybridization histochemistry (ISHH). Relative LHRH mRNA content per cell was quantitated by single-cell analysis after transcription inhibition with 5, 6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) or actinomycin D. Cultures were maintained in serum-free medium with tetrodotoxin to suppress spontaneous electrical activity and hence assess only intrinsic cellular activity. A plot of LHRH mRNA level per cell versus DRB treatment time showed a rapid initial decay of LHRH mRNA (t1/2, 5-13 min), followed by a slower decay rate (t1/2, 329-344 hr). LHRH cell number after drug treatment as determined by immunocytochemistry did not change. Comparison of mammalian LHRH mRNA 3'-untranslated regions showed two conserved regions. These data indicate that, in primary LHRH neurons, LHRH mRNA has an intrinsically high rate of turnover and a mRNA stabilization component. Foremost, decay of LHRH mRNA, the fastest reported for a neuropeptide to date, corresponds to the decay of LHRH peptide pulses.

Progesterone blockade of a luteinizing hormone surge blocks luteinizing hormone-releasing hormone Fos activation and activation of its preoptic area afferents

Progesterone is capable of facilitating or blocking the luteinizing hormone (LH) surge, depending on the timing of its administration. However, the precise targets of progesterone's actions are unknown. Since recent studies described the presence of a periventricular preoptic area (pePOA) neuron population afferent to LH-releasing hormone (LHRH) neurons that is co-activated to express c-Fos with LHRH neurons at the time of the LH surge, the present study was designed to determine if the pePOA neurons contain progesterone receptors (PRs) and whether progesterone inhibition is manifested by a failure of LHRH and pePOA neurons to become activated at the time of an LH surge. For progesterone facilitation, a group of immature rats each received a silastic capsule (1.57 mm i.d., 3.18 mm o.d., 1.5 cm long) containing estradiol-17 β (E 2) in peanut oil (150 μg/ml) at 09.00 h on postnatal day 28 followed 24 h later by a progesterone implant (crystalline, 1.57 mm i.d., 3.18 mm o.d., 1.5 cm long). For progesterone inhibition, a second group of rats received the estrogen capsule and a progesterone capsule (3.35 mm i.d., 4.65 mm o.d., 3.0 cm long) together at 09.00 h on day 28, and 24 h later received only a blank capsule. On the afternoon of postnatal day 29, all animals were anesthetized and perfused for localization of c-Fos and LHRH, PRs alone, or c-Fos and PRs. The present studies determined that following a progesterone-inhibition paradigm, along with blockade of the LH surge, both activation of LHRH and pePOA neurons was low or absent. Staining of PRs in progesterone-facilitated and progesterone-inhibited rats indicated that the pePOA neurons contained PRs in similar patterns. Double labeling of c-Fos and PRs in progesterone-facilitated rats indicated that nearly all the c-Fos-positive neurons of the pePOA (80±4.2%) co-expressed PRs; in progesterone-inhibited rats, only 32±12% of few c-Fos-positive neurons also contained PRs. In no instance were LHRH neurons found to contain PRs. Taken together, these data suggest that both progesterone facilitation and inhibition likely involve direct actions of progesterone on the pePOA neurons, and are consistent with a role for the pePOA neurons in transducing steroid effects on LHRH neurons.

Estradiol Enhances Prostaglandin E2Receptor Gene Expression in Luteinizing Hormone-Releasing Hormone (LHRH) Neurons and Facilitates the LHRH Response to PGE2by Activating a Glia-to-Neuron Signaling Pathway

Prostaglandin E2 (PGE2) mediates the stimulatory effect of norepinephrine (NE) on the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling reproductive function. In rodents, this facilitatory effect requires previous exposure to estradiol, suggesting that the steroid affects downstream components in the cascade that leads to PGE2-induced LHRH release. Because astroglia are the predominant cell type contacting LHRH-secreting nerve terminals, we investigated the involvement of hypothalamic astrocytes in the estradiol facilitation of PGE2-induced LHRH release. A subpopulation of LHRH neurons was found to express the mRNA encoding the PGE2 receptor subtype EP1-R, which is coupled to calcium mobilization. The LHRH-producing cell line GT1-1 also contains EP1-R mRNA and, to a lesser extent, the three alternatively spliced forms of EP3-R mRNA (alpha, beta, and gamma) that encode receptors linked to inhibition and stimulation of cAMP formation. Hypothalamic astrocytes treated with estradiol produced a conditioned medium that when applied to GT1-1 cells resulted in a selective upregulation of EP1-R and EP3gamma-R mRNAs. The conditioned medium also enhanced the LHRH response to EP1-R and EP3-R agonists and the cAMP response to EP3-R activation. Thus, one mechanism by which estradiol facilitates the effect of neurotransmitters acting via PGE2 to stimulate LHRH release is by enhancing the glial production of substances that upregulate PGE2 receptors on LHRH neurons. The existence of such a mechanism underscores the emerging importance of glial-neuronal communication in the control of brain neurosecretory activity.

Prader-Willi syndrome and the hypothalamus.

Dysfunction of various hypothalamic systems may be the basis of a number of symptoms in Prader-Willi syndrome. The often abnormal position of the baby in the uterus at the onset of labour, the high percentage of infants with asphyxia and the high proportion of children born prematurely or post-maturely may all be related to abnormal fetal hypothalamic systems, as the fetal hypothalamus plays a crucial role in labour. Abnormal luteinizing hormone-releasing hormone neurones are thought to be responsible for the decreased levels of sex hormones, resulting in non-descended testes, undersized sex organs and insufficient growth during puberty. A lack of growth hormone-releasing hormone may also contribute to the short stature of patients with Prader-Willi syndrome. In addition, the aberrant control of body temperature and daytime hypersomnolence may result from hypothalamic disturbances. The number of oxytocin neurones--the putative satiety neurones--in the hypothalamic paraventricular nucleus is markedly decreased in Prader-Willi syndrome. This is presumed to be the basis of the insatiable hunger and obesity of patients with the syndrome.

Primary cell culture of LHRH neurones from embryonic olfactory placode in the sheep (Ovis aries).

The aim of this study was to establish an in vitro model of ovine luteinizing hormone-releasing hormone (LHRH) neurones. Olfactory placodes from 26 day-old sheep embryos (E26) were used for explant culture. Cultures were maintained successfully up to 35 days, but were usually used at 17 days for immunocytochemistry. LHRH and neuronal markers such as neurofilament (NF) were detected by immunocytochemistry within and/or outside the explant. Three main types of LHRH positive cells are described: (1) neuroblastic LHRH and NF immunoreactive cells with round cell body and very short neurites found mainly within the explant, (2) migrating LHRH bipolar neurones with an fusiform cell body, found outside the explant, (3) network LHRH neuron, bipolar or multipolar with long neurites connecting other LHRH neurons. Cell morphology was very similar to that which has been described in the adult sheep brain. These results strongly suggest that LHRH neurones in the sheep originate from the olfactory placode. This mode may represent a useful tool to study LHRH neurones directly in the sheep.

Nitric oxide is involved in the genesis of pulsatile LHRH secretion from immortalized LHRH neurons.

Neuronal networks controlling endocrine events present synchronous activity which is required for maintaining physiological functions, including reproduction. Although pulsatile hormone secretion is of paramount importance, the mechanism(s) by which secretory episodes are generated remain largely unknown. Nitric oxide (NO) has become the prototype of a new family of signaling molecules in the body. Nitric oxide diffuses from the source cell and controls activity of neighboring neurons as well as itself as a retrograde messenger. Cells of the luteinizing hormone-releasing hormone (LHRH) neuronal network, the key component in the control of reproduction, are scattered and loosely arranged in the anterior hypothalamus. A diffusible neurotransmitter could provide a means for establishing synchronous activation of the LHRH neuronal network leading to physiologically-relevant pulsatile LHRH secretion. In this study, we demonstrate that immortalized LHRH-producing neurons (GT1-7 cells) express NO synthase (NOS) mRNA and protein. Furthermore, GT1-7 cells are NADPH-diaphorase-positive (a marker of NOS activity) and the histochemical reaction can be abolished by treatment with a competitive NOS blocker. The presence of citrulline in these cells provides further evidence for the biological activity of NOS. These observations indicate that an active NO synthesizing machinery is present in immortalized LHRH neurons. In addition, we show that LHRH secretion is enhanced by NO in a cGMP-dependent manner. Since pulsatile LHRH secretion from immortalized LHRH neurons in vitro is abolished by NOS blockers and NO scavengers, it appears that NO is a unique neurotransmitter that is necessary to set LHRH neurons in phase to establish synchronized pulsatile LHRH secretion.

The transforming growth factor alpha gene family is involved in the neuroendocrine control of mammalian puberty.

The concept is proposed that the central control of mammalian female puberty requires the interactive participation of neuronal networks and glial cells of the astrocytic lineage. According to this concept neurons and astrocytes control the pubertal process by regulating the secretory activity of those neurons that secrete luteinizing hormone-releasing hormone (LHRH). LHRH, in turn, governs sexual development by stimulating the secretion of pituitary gonadotropins. Astrocytes affect LHRH neuronal function via a cell-cell signaling mechanism involving several growth factors and their corresponding receptors. Our laboratory has identified two members of the epidermal growth factor/transforming growth factor (EGF/TGF alpha) family as components of the glial-neuronal interactive process that regulates LHRH secretion. Transforming growth factor alpha (TGF alpha) and its distant congener neu-differentiation factor, NDF, are produced in hypothalamic astrocytes and stimulate LHRH release via a glial intermediacy. The actions of TGF alpha and NDF on hypothalamic astrocytes involve the interactive activation of their cognate receptors and the synergistic effect of both ligands in stimulating the glial release of prostaglandin E2 (PGE2). In turn, PGE2 acts directly on LHRH neurons to stimulate LHRH release. A variety of experimental approaches has led to the conclusion that both TGF alpha and NDF are physiological components of the central mechanism controlling the initiation of female puberty.

Locus coeruleus lesions decrease norepinephrine input into the medial preoptic area and medial basal hypothalamus and block the LH, FSH and prolactin preovulatory surge

The aim of this work was to study the role of the dorsal noradrenergic ascending pathway (DNAP), which originates in the locus coeruleus (LC) on the preovulatory surge of luteinizing hormone (LH) follicle-stimulating hormone (FSH) and prolactin (PRL) by producing bilateral electrolytic lesions (cathodal or anodal) in this nucleus. LC lesions were placed at 11.00 h on proestrus in female rats with regular 4-day estrous cycles. Intact rats, sham-operated as well as animals with missed lesions served as controls. In Experiment I, anodal current was applied and hourly blood samples were withdrawn (from 13.00 to 17.00 h) via a jugular catheter from conscious, freely moving rats for determination of plasma LH, FSH and PRL concentrations. In Expt. II, Expt. I was repeated using cathodal current and collecting blood samples hourly from 13.00 to 18.00 h. In both experiments the animals were sacrificed on the next morning when the occurrence of ovulation was checked. The medial septal area (MSA), medial preoptic area (MPOA), and medial basal hypothalamus (MBH) were dissected and assayed for norepinephrine (NE), dopamine (DA) and 5-hydroxyindoleacetic acid (5-HIAA) content. Experiment III was performed in order to test if a hormonal discharge occurred immediately after lesion placement. Blood samples were collected immediately before and 15, 30, 60 and 90 min postoperatively (from 11.00 to 12.30 h). Either anodal or cathodal lesions blocked the proestrous surge of LH, FSH and PRL. The hypothesis that the lesions advanced or delayed these hormonal surges was rejected since we found no increases in the hormonal levels from 11.00 to 12.30 or from 13.00 to 18.00 h, and ovulation was not observed on the following morning in the lesioned animals. Since control, sham-operated and missed-lesion groups exhibited LH, FSH and PRL surges and ovulation, this blockage appears to be caused by the destruction of the LC neurons. Also, this blockade was correlated with a decrease in the NA content in the MPOA and MBH, but not in the MSA, whereas the DA and 5-HIAA content were not changed in all groups examined. The results lead us to suggest that the integrity of noradrenergic afferent input from the LC to luteinizing hormone-releasing hormone neurons in the MPOA and MBH is essential for triggering the preovulatory surge mechanisms for gonadotrophins and PRL.

Supportive Role of Cellular Bridge of Neurons Expressing a Highly Polysialylated Form of NCAM (NCAM-H) at the Initial Stage of Migration of LHRH Neurons

The early stage of cell migration from the olfactory placode to the forebrain was studied immunohistochemically in chick embryos to investigate the nature of early migrated cells and the role of these cells in the sebsequent migration of luteinizing hormone releasing hormone (LHRH) neurons. The initial cells migrating from the olfactory placode stained strongly positive for the highly polysialylated neural cell adhesion molecule (NCAM-H), but were negative for LHRH. These migrating NCAM-H immunoreactive cells were observed on embryonic day (ED) 2.5 as a bulge with a few cells from the base of the placode. By ED 3, they formed a wide cellular strand and developed into a cellular bridge between the olfactory placode and the ventro-rostral surface of the forebrain. These migrating cells are neurons because they stained positive for growth associated protein-43 (GAP-43), microtubule associated protein (MAP) 2, and MAP 5. Some of these cells seemed to migrate caudally along the ventro-lateral surface of the forebrain. LHRH-immunoreactive cells were not detected in the olfactory epithelium until after ED 3.5, which was one day after formation of the cellular bridge. Then, LHRH-immunoreactive cells appeared and began to cross the cellular bridge. The outgrowth of the olfactory nerve axon bundles from the olfactory epithelium was detected at around the same time when LHRH-immunoreactive cells first appeared. These olfactory nerve axons expressed NCAM-H, GAP-43, and MAP 5, as assessed by immunochemistry. After bundle formation, the olfactory nerve appeared to provide the migratory routes for LHRH neurons. These results suggest that the cellular bridge formed by the NCAM-H-immunoreactive neurons plays an important supportive role for LHRH neurons at the initial stage of their migration in chick embryos.

Two populations of luteinizing hormone‐releasing hormone neurons in the forebrain of the rhesus macaque during embryonic development

To investigate the possibility that a second luteinizing hormone-releasing hormone (LHRH) population appears during development in primates, embryos and fetal brains of rhesus monkeys were immunostained with antisera specific to different LHRH forms. Two LHRH cell populations were discernible by immunoreactivity to antisera LR-1 and GF-6. Because one LHRH cell type migrated out from the olfactory placode several days earlier than the other, they were referred to as “early” and “late” LHRH cells, respectively. Although late LHRH neurons were immunoreactive to all anti-mammalian LHRH antisera tested, early LHRH neurons were only detected by antiserum GF-6. Early LHRH neurons (∼10 × 7 μm) were smaller than late LHRH neurons (∼8 × 7 μm). Early LHRH neurons were first found around the olfactory placode, in the nasal mesenchyme, and in the rostroventral forebrain on embryonic day 30 (E30), whereas late LHRH neurons were first seen in the olfactory pit on E32. Early LHRH cells were located throughout the basal forebrain on E32–E42, whereas late LHRH cells were found in the olfactory pit and along the terminal nerve on E34–E36 and were not seen in the forebrain until E38. By E51–E62, late LHRH neurons reached into the basal hypothalamus in a distribution resembling that in the older brain, while early LHRH neurons were found in the septum, preoptic region, stria terminalis, medial amygdala, claustrum, internal capsule, and globus pallidus. Based on the distribution pattern of immunopositive cells with-antiserum LR-1, late LHRH cells are bona fide LHRH neurons that regulate the pituitary-gonadal axis. In contrast, the molecular form of early LHRH cells is unclear, although it is plausible that early LHRH cells may contain the molecule in which the C-terminal epitope of LHRH is modified or absent. It is concluded that in primates there is a second population of LHRH neurons that originates from the embryonic olfactory placode before the origin of mammalian LHRH-like neurons, and that these two populations of LHRH-immunopositive neurons have different morphologic features and different final distributions in the brain. J. Comp. Neurol. 380:293–309, 1997. © 1997 Wiley-Liss, Inc.

Two populations of luteinizing hormone-releasing hormone neurons in the forebrain of the rhesus macaque during embryonic development

To investigate the possibility that a second luteinizing hormone-releasing hormone (LHRH) population appears during development in primates, embryos and fetal brains of rhesus monkeys were immunostained with antisera specific to different LHRH forms. Two LHRH cell populations were discernible by immunoreactivity to antisera LR-1 and GF-6. Because one LHRH cell type migrated out from the olfactory placode several days earlier than the other, they were referred to as "early" and "late" LHRH cells, respectively. Although late LHRH neurons were immunoreactive to all anti-mammalian LHRH antisera tested, early LHRH neurons were only detected by antiserum GF-6. Early LHRH neurons (approximately 10 x 7 microm) were smaller than late LHRH neurons (approximately 18 x 7 microm). Early LHRH neurons were first found around the olfactory placode, in the nasal mesenchyme, and in the rostroventral forebrain on embryonic day 30 (E30), whereas late LHRH neurons were first seen in the olfactory pit on E32. Early LHRH cells were located throughout the basal forebrain on E32-E42, whereas late LHRH cells were found in the olfactory pit and along the terminal nerve on E34-E36 and were not seen in the forebrain until E38. By E51-E62, late LHRH neurons reached into the basal hypothalamus in a distribution resembling that in the older brain, while early LHRH neurons were found in the septum, preoptic region, stria terminalis, medial amygdala, claustrum, internal capsule, and globus pallidus. Based on the distribution pattern of immunopositive cells with antiserum LR-1, late LHRH cells are bona fide LHRH neurons that regulate the pituitary-gonadal axis. In contrast, the molecular form of early LHRH cells is unclear, although it is plausible that early LHRH cells may contain the molecule in which the C-terminal epitope of LHRH is modified or absent. It is concluded that in primates there is a second population of LHRH neurons that originates from the embryonic olfactory placode before the origin of mammalian LHRH-like neurons, and that these two populations of LHRH-immunopositive neurons have different morphologic features and different final distributions in the brain.

Effect of Alcohol on the Proestrous Surge of Luteinizing Hormone (LH) and the Activation of LH-Releasing Hormone (LHRH) Neurons in the Female Rat

Reproduction is adversely affected by alcohol abuse in humans and laboratory animals. In rats, alcohol exposure suppresses both luteinizing hormone (LH) and sex steroid secretion, although consensus is lacking as to which level of the hypothalamic-pituitary-gonadal (HPG) axis is primarily affected. We tested the hypothesis that acute alcohol treatment inhibits the HPG axis by blunting release of LH-releasing hormone (LHRH) in female rats, by examining the effect of this drug on the central reproductive endocrine event; i.e., the proestrous surge of gonadotropins, which triggers ovulation. In a first series of experiments, we injected alcohol at 8 A.M. and 12 P.M. on proestrus and measured plasma levels of LH, estradiol (E2), and progesterone during the afternoons of proestrus and estrus. Alcohol administration blocked the proestrous surge of LH and ovulation. In subsequent experiments, alcohol inhibited the surge of LHRH (measured by push-pull cannulation) and LHRH neuronal activation (measured by Fos labeling in LHRH neurons). Because alcohol also decreased E2 levels, we reasoned that it might have prevented positive feedback; however, alcohol retained its ability to inhibit the LH surge evoked by E2 implantation in ovariectomized females, disproving this hypothesis. Additionally, alcohol does not act via increased corticosteroid secretion, because alcohol also blocked the proestrous surge in adrenalectomized females. Last, exogenous administration of LHRH to alcohol-blocked animals evoked LH secretion and ovulation, indicating that pituitary and/or ovarian function could be restored by mimicking the hypothalamic signal. Collectively, these data indicate that in female rats, alcohol inhibits the gonadotropin surge primarily by decreasing LHRH secretion.

Dual label in situ hybridization studies provide evidence that luteinizing hormone-releasing hormone neurons do not synthesize messenger ribonucleic acid for mu, kappa, or delta opiate receptors.

Abundant evidence suggests that opiatergic neurons play an important intermediary role in the regulation of LHRH release by ovarian steroids; however, it is unclear whether opiates communicate directly or indirectly with LHRH neurons. To investigate this issue, we used dual label in situ hybridization histochemistry to determine whether LHRH neurons synthesize messenger RNA (mRNA) for mu, kappa, and/or delta opiate receptors. For these studies, we examined both intact (n = 3) and ovariectomized, steroid-treated rats. Ten of the ovariectomized rats were implanted 1 week later (day 0) with SILASTIC brand (Dow Corning) capsules of estradiol. On the morning of day 2, half of the estradiol-treated rats were injected with 5 mg progesterone. All animals were killed at approximately 1530 h on day 2. We found that cells expressing mu, kappa, and delta opiate receptor mRNAs were in all sections that also contained LHRH neurons. In every case, LHRH neurons were seen to be surrounded by or in close proximity to cells containing mu, kappa, or delta mRNAs. However, regardless of steroid treatment, we found no neurons containing both LHRH mRNA and mRNAs encoding any of the three receptor subtypes. These results support the hypothesis that LHRH neurons are regulated indirectly by opiatergic neurons.

Dual Label in Situ Hybridization Studies Provide Evidence that Luteinizing Hormone-Releasing Hormone Neurons Do Not Synthesize Messenger Ribonucleic Acid for , , or Opiate Receptors

Dual Label in Situ Hybridization Studies Provide Evidence that Luteinizing Hormone-Releasing Hormone Neurons Do Not Synthesize Messenger Ribonucleic Acid for , , or Opiate Receptors

Targeting transforming growth factor alpha expression to discrete loci of the neuroendocrine brain induces female sexual precocity.

Precocious puberty of cerebral origin is a poorly understood disorder of human sexual development, brought about by the premature activation of those neurons that produce luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation. An increased production of transforming growth factor alpha (TGF alpha) in the hypothalamus has been implicated in the mechanism underlying both normal and precocious puberty. We have now used two gene delivery systems to target TGF alpha overexpression near LHRH neurons in immature female rats. Fibroblasts infected with a retroviral construct in which expression of the human TGF alpha gene is constitutively driven by the phosphoglycerate kinase promoter, or transfected with a plasmid in which TGF alpha expression is controlled by an inducible metallothionein promoter, were transplanted into several regions of the hypothalamus. When the cells were in contact with LHRH nerve terminals or in the vicinity of LHRH perikarya, sexual maturation was accelerated. These results suggest that precocious puberty of cerebral origin may result from a focal disorder of TGF alpha production within the confines of the LHRH neuron microenvironment.

Sexually dimorphic processing of somatosensory and chemosensory inputs to forebrain luteinizing hormone-releasing hormone neurons in mated ferrets.

The ferret is a reflexively ovulating species in which mating induces a preovulatory LH surge in the estrous female but significantly decreases LH secretion in the breeding male. This sexually dimorphic hormonal response is reflected in a sex difference in Fos-like immunoreactivity (Fos-IR) in forebrain LHRH and non-LHRH neurons after mating. We used dual immunocytochemistry for Fos and LHRH to determine whether the sex dimorphism occurs in the initial detection and transmission or in the central processing of sensory stimuli associated with mating? We also assessed the ability of chemosensory cues alone to augment neuronal Fos-IR in the ferret forebrain. Breeding male and female ferrets were paired, whereupon the male partner achieved an intromission lasting for 16-90 min. Mated male and female subjects were always perfused 90 min after the onset of the male's intromission. Additional male and female subjects were placed alone in a cage in which an opposite sex ferret in breeding condition had been housed for 48 h. Other control ferrets were placed alone in a clean cage. Chemosensory-stimulated and unpaired control subjects were perfused 90 min after being placed in their respective cages. In both sexes mating augmented neuronal Fos-IR in the granular layer of the main olfactory bulb, the caudal thalamic central tegmental field, and the medial amygdala, regions situated early in the putative input pathway to mediobasal hypothalamic LHRH neurons. Neuronal Fos-IR was also increased in these same forebrain regions (the central tegmental field excluded) in both sexes after exposure to chemosensory cues alone. However, more central components of this input pathway, including the preoptic area, the bed nucleus of the stria terminalis, and the ventrolateral portion of the ventromedial hypothalamus as well as the mediobasal hypothalamic LHRH neurons themselves were activated by mating only in the female. In estrous females, exposure only to chemosensory stimuli from a breeding male augmented Fos-IR in the preoptic area and the ventrolateral portion of the ventromedial hypothalamus, but not in the bed nucleus of the stria terminalis or mediobasal hypothalamic LHRH neurons. In breeding males, exposure only to chemosensory cues from an estrous female failed to affect Fos-IR in any of these proximal components of the input pathway or in LHRH neurons themselves. These results suggest that the sex dimorphism in mating-induced LH secretion reflects a sex difference in the central processing of genital-somatosensory stimuli and possibly of chemosensory inputs as well.

Sexually Dimorphic Processing of Somatosensory and Chemosensory Inputs to Forebrain Luteinizing Hormone-Releasing Hormone Neurons in Mated Ferrets

The ferret is a reflexively ovulating species in which mating induces a preovulatory LH surge in the estrous female but significantly decreases LH secretion in the breeding male. This sexually dimorphic hormonal response is reflected in a sex difference in Fos-like immunoreactivity (Fos-IR) in forebrain LHRH and non-LHRH neurons after mating. We used dual immunocytochemistry for Fos and LHRH to determine whether the sex dimorphism occurs in the initial detection and transmission or in the central processing of sensory stimuli associated with mating? We also assessed the ability of chemosensory cues alone to augment neuronal Fos-IR in the ferret forebrain. Breeding male and female ferrets were paired, whereupon the male partner achieved an intromission lasting for 16–90 min. Mated male and female subjects were always perfused 90 min after the onset of the male’s intromission. Additional male and female subjects were placed alone in a cage in which an opposite sex ferret in breeding condition had been housed for 48 h. Other control ferrets were placed alone in a clean cage. Chemosensory-stimulated and unpaired control subjects were perfused 90 min after being placed in their respective cages. In both sexes mating augmented neuronal Fos-IR in the granular layer of the main olfactory bulb, the caudal thalamic central tegmental field, and the medial amygdala, regions situated early in the putative input pathway to mediobasal hypothalamic LHRH neurons. Neuronal Fos-IR was also increased in these same forebrain regions (the central tegmental field excluded) in both sexes after exposure to chemosensory cues alone. However, more central components of this input pathway, including the preoptic area, the bed nucleus of the stria terminalis, and the ventrolateral portion of the ventromedial hypothalamus as well as the mediobasal hypothalamic LHRH neurons themselves were activated by mating only in the female. In estrous females, exposure only to chemosensory stimuli from a breeding male augmented Fos-IR in the preoptic area and the ventrolateral portion of the ventromedial hypothalamus, but not in the bed nucleus of the stria terminalis or mediobasal hypothalamic LHRH neurons. In breeding males, exposure only to chemosensory cues from an estrous female failed to affect Fos-IR in any of these proximal components of the input pathway or in LHRH neurons themselves. These results suggest that the sex dimorphism in mating-induced LH secretion reflects a sex difference in the central processing of genital-somatosensory stimuli and possibly of chemosensory inputs as well.

Fos expression by naloxone in LHRH neurons of the mediobasal hypothalamus and effects of pentobarbital sodium in the proestrous rat.

Because Fos is thought to be induced in neurons that are activated, we examined whether luteinizing hormone-releasing hormone (LHRH) neurons expressed Fos protein when they were stimulated by an opioid receptor antagonist naloxone (NAL), expecting to identify LHRH neurons which are regulated by opioid neurons directly or indirectly. Further, we examined whether an ovulation-blocking dosage of pentobarbital sodium (PB) would affect the NAL-induced Fos expression. Female rats were infused with naloxone (5 mg/kg/h) for 90 min (10.00-11.30) in the morning of proestrus, during which infusion blood sampling was done, and were killed by i.v. injection with an overdose of PB at 11.30-12.00. Dual immunoperoxidase/immunofluorescence staining for both Fos and LHRH revealed that some LHRH immunoreactive (ir) neurons in the forebrain expressed Fos-ir, associated with an increase in serum LH concentrations, but little co-localization was found in rats in proestrus which were infused with saline as the control. The proportion of LHRH-ir neurons which expressed Fos-ir was about 35-62% in the caudal part of the forebrain including the mediobasal hypothalamus, and this was larger than that (10%) in the rostral part of the forebrain including the preoptic area. PB injection (32 mg/kg bw, i.p.) 15 min prior to the beginning of NAL infusion significantly enhanced the increase in LH secretion due to NAL, and also enhanced Fos-ir expression in LHRH-ir neurons. Together with the well-established fact that PB blocks the LHRH surge generator and our previous findings that NAL stimulates the LHRH pulse generator even in the PB-blocked proestrous rat, these results strongly suggest that the LHRH pulse generator exists in the mediobasal hypothalamus which contains LHRH neurons that are responsive to NAL and express Fos protein.

Granulocyte-macrophage colony stimulating factor suppresses LHRH release by inhibition of nitric oxide synthase and stimulation of gamma-aminobutyric acid release.

Several cytokines produced by immune cells act within the hypothalamus and/or on the pituitary to produce the pattern of pituitary hormone secretion that characterizes infection. Granulocyte-macrophage colony stimulating factor (GMCSF) was first described as a hematopoietic cytokine; however, its synthesis is also stimulated during infection, and it has been found in glia in the brain. Previous research indicates that interleukin-1 inhibits release of luteinizing hormone-releasing hormone (LHRH) both in vivo and in vitro. In the present study, we determined that GMCSF inhibited the release of LHRH in vitro and evaluated the mechanisms involved. After a 1-hour preincubation in Krebs-Ringer bicarbonate glucose buffer (KRB), medial basal hypothalamic explants were incubated in KRB together with recombinant murine GMCSF for 0.5 h in a Dubnoff metabolic shaker (50 cycles/min) in an atmosphere of 95% O2/5% CO2. LHRH release into the media was determined by radioimmunoassay. At concentrations of 10(-12) and 10(-11) M, GMCSF significantly inhibited LHRH release. There was a U-shaped dose-response curve and LHRH release was not inhibited at lower or higher cytokine concentrations. The inhibition was specific since it was completely blocked by GMCSF antiserum. Since sodium nitroprusside (NP; 300 microM), a releaser of nitric oxide (NO), stimulates LHRH, presumably by acting within the LHRH neurons, we examined the effect of GMCSF (10(-11) M) on NP-induced LHRH release. It completely suppressed NP-induced release of LHRH. Bicuculline (10(-5) M), a gamma-aminobutyric acid (GABA) receptor antagonist, partially reversed the inhibitory effects of GMCSF on LHRH release. This dose completely reversed the suppression of LHRH release induced by GABA. The present results indicate that the inhibitory effects of GMCSF on LHRH release are partially caused by blockade of NO-induced LHRH release by its activation of GMCSF receptors on GABAergic neurons. The stimulated release of GABA acts on the GABA-a receptors on the LHRH terminals to inhibit their response to NO. At the end of the experiment, NO synthase (NOS) activity was measured in the tissue homogenate by the citrulline method. NOS activity was highly significantly reduced by GMCSF (10(-11) M) indicating that part of its suppressive action on LHRH release is mediated by reduction in NOS activity in the medial basal hypothalamus.

GABAergic Neurons in the Embryonic Olfactory Pit/Vomeronasal Organ: Maintenance of Functional GABAergic Synapses in Olfactory Explants

In previous work, we showed a robust γ-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic micein vivoand study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures.In vivo,glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around −50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl−conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pitin vivo.