Luteinizing Hormone-releasing Hormone Cells Research Articles

Spatiotemporal appearance of developing LHRH neurons in the rat brain.

To obtain insight into the development of the heterogeneous intracerebral populations of luteinizing hormone-releasing hormone (LHRH) neurons, their spatiotemporal appearance was examined at different stages in normal rat embryos, in nasal epithelial explants in vitro, and in intrauterine nasal-operated embryos. Following the appearance of nerve cell adhesion molecule in the nasal placode at embryonic day (E) 12.5, LHRH neurons, generated in the nasal placode at E13.5, penetrated the forebrain vesicle (FV) by E14.5-15.5. After E16.5, as the FV elongated to form the olfactory bulb, the migrating neurons traversed posteriorly through the interhemispheric space to penetrate the septopreoptic (S-P) area. By E18.5, LHRH neurons were detected in the preoptic-diagonal band (P-D) area as well as in the S-P region, along with some scattered extrahypothalamic LHRH neurons. To determine the source of these neurons, we separately cultured dissected parts of E12.5 nasal pit epithelium. Neuronal generation was predominantly from the medial wall epithelium (NAP), but some LHRH neurons originated in the roof epithelium. Cocultures of the NAP (E12.5) with the FV, median eminence-arcuate complex, Rathke's pouch, mesencephalon, or medulla oblongata from E14.5 embryos revealed the ability of LHRH cells to penetrate all of these tissues. Uni- or bilateral nasal destruction was conducted at E16.5 or E15.5, respectively, and examined at E18.5 and E21.5. In the operated embryos, most LHRH neurons were present in the P-D system and some in the S-P area. This finding suggests that the neurons generated before E15.5 are primarily predisposed to form the P-D system, whereas those derived afterward form the S-P system.

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.

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.

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.

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.

Migration of luteinizing hormone-releasing hormone (LHRH) neurons in early human embryos.

Luteinizing hormone-releasing hormone (LHRH) neurons originate in the epithelium of the medial olfactory pit and migrate from the nose into the forebrain along nerve fibers rich in neural cell adhesion molecule (N-CAM). The present study examined the ontogenesis of LHRH neurons in early human embryos and found a similar pattern of development of these cells. Luteinizing hormone-releasing hormone immunoreactivity was detected in the epithelium of the medial olfactory pit and in cells associated with the terminal-vomeronasal nerves at 42 (but not 28-32) days of gestation. The migration route of these cells was examined with antibodies to N-CAM and antibodies to polysialic acid (PSA-N-CAM), which is present on N-CAM at certain stages of development. Neural cell adhesion molecule immunoreactivity was present in a population of cells in the olfactory placode of the earliest embryos examined (28-32 days) and later (42 and 46 days) throughout the migration route. The PSA-N-CAM immunoreactivity was not detected until 42 days and was present in a more limited distribution in nerve fibers streaming from the olfactory placode and along the caudal part of the migration route below the forebrain. Previous studies have indicated that the highly sialated form of N-CAM is less adhesive. The PSA-N-CAM may therefore facilitate the migration of these cells by lessening the adhesion between the fascicles that make up the migration route, expediting the passage of cords of LHRH cells between the nerve fibers as these cells move toward the brain.

2418 Effect of abdominal vagotomy on interleukin-1β induced noradrenaline release in the paraventricular nucleus in conscious rats

Seasonal breeding mammals exhibit a reversible annual cycle of fertility, and thus represent valuable models for investigating the organization of luteinizing hormone-releasing hormone (LHRH) neurons which mediate the neuroendocrine control of reproduction. Electron microscopic immunocytochemistry was used to examine the ultrastructure of LHRH neurons and their projections in a seasonal breeder, the golden hamster, housed under photoperiodic conditions which are reproductively stimulatory. LHRH perikarya in the diagonal band, medial septum, and preoptic area were bipolar or unipolar cells which possessed nuclei with prominent indentations and multiple nucleoli. LHRH reaction product within these cells was associated with neurosecretory granules and the rough endoplasmic reticulum (RER), particularly those portions of RER adjacent to the outer nuclear envelope. LHRH cell bodies and dendrites in the hamster received an extremely limited amount of synaptic input; of a total of twenty-five cells analyzed, we found only five instances of morphologically identified synapses onto immunoreactive dendrites. Occasionally close somatic appositions were seen between LHRH cells and non-immunoreactive neurons, and in one instance between two LHRH somas. In the organum vasculosum of the lamina terminalis (OVLT), LHRH fibers appeared as isolated strings of varicosities. In contrast, within the median eminence immunoreactive axonal profiles were frequently bundled together in fascicles although synaptic specializations were not observed between individual axons. The existence of contacts between LHRH axons in the median eminence, coupled with the extreme paucity of synaptic inputs onto LHRH neurons in this species, suggests that the median eminence may be a site where neural or hormonal signals could influence the coordinated release of LHRH from cells of dispersed origin.

The LHRH-astroglial network of signals as a model to study neuroimmune interactions: assessment of messenger systems and transduction mechanisms at cellular and molecular levels.

Neurons and astrocytes have a close anatomic and functional relationship that plays a crucial role during development and in the adult brain. Astrocytes in the central nervous system (CNS) express receptors for a variety of growth factors (GFs), neurotransmitters and/or neuromodulators; in turn, neuronal cells can respond to astrocyte-derived GFs and control astrocyte function via a common set of signaling molecules and intracellular transducing pathways. There is also increasing evidence that soluble factors from lymphoid/mononuclear cells are able to modulate the growth and function of cells found in the CNS, specifically macroglial and microglial cells. Furthermore, glial cells can secrete immunoregulatory molecules that influence immune cells as well as the glial cells themselves. As neuronal and immune cells share common signaling systems, the potential exists for bidirectional communication not only between lymphoid and glial cells, but also between neuronal cells and immune and glial cells. In the present work, interactions of luteinizing-hormone-releasing hormone (LHRH) and the astroglial cell are proposed as a prototype for the study of neuroimmune communication within the CNS in the light of (1) the commonality of signal molecules (hormones, neurotransmitters and cytokines) and transduction mechanisms shared by glia LHRH neurons and lymphoid cells; (2) the central role of glia in the developmental organization and pattern of LHRH neuronal migration during embryogenesis, and (3) the strong modulatory role played by sex steroids in mechanisms involved in synaptic and interneuronal organization, as well as in the sexual dimorphisms of neuroendocrine-immune functions. During their maturation and differentiation in vitro, astroglial cells release factors able to accelerate markedly the LHRH neuronal phenotypic differentiation as well as the acquisition of mature LHRH secretory potential, with a potency depending on both the 'age' and the specific brain localization of the astroglia, as well as the degree of LHRH neuronal differentiation in vitro. Regional differences in astroglial sensitivity to estrogens were also measured. Different experimental paradigms such as coculture and mixed-culture models between the immortalized LHRH (GT1-1) neuronal cell line and astroglial cells in primary culture, disclosed the presence of a bidirectional flow of informational molecules regulating both proliferative and secretory capacities of each cell type. The importance of adhesive mechanisms in such cross-talk is underscored by the complete abolition of GT1-1 LHRH production and cell proliferation following the counteraction of neuronal-neuronal/neuronal-glial interactions through addition of neural-cell adhesion molecule antiserum. Other information came from pharmacological experiments manipulating the astroglia-derived cytokines and/or nitric oxide, which revealed cross-talk between the neuronal and astroglial compartments. From the bulk of this information, it seems likely that interactions between astroglia and LHRH neurons play a major role in the integration of the multiplicity of brain signals converging on the LHRH neurons that govern reproduction. Another important facet of neuronal-glial interactions is that concerning neuron-guided migration, and unraveling astroglial/LHRH-neuronal networks might then constitute an additional effort in the comprehension of defective LHRH-neuronal migration in Kallman's syndrome.

Cross-talk between luteinizing hormone-releasing hormone (LHRH) neurons and astroglial cells: developing glia release factors that accelerate neuronal differentiation and stimulate LHRH release from GT1-1 neuronal cell line and LHRH neurons induce astroglia proliferation

Recent evidences indicate that the bidirectional flow of informations governing neuron-astrocyte interactions plays a crucial role during the development and in the adult brain. In the present study, we have used the immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neuronal cell line (GT(1-1), subclone) to investigate LHRH-astroglial cell interactions and addressed the following questions: (a) does the astroglial cell compartment influence GT(1-1) neuron morphology, LHRH secretion and/or proliferation?; (b) does the bidirectional flow of informational molecules released during neuron-astroglia interactions influence one or both cell compartments?; (c) are receptor-mediated cell-cell interactions between neurons and astroglia involved in such crosstalk? In this experimental design, GT(1-1) neuronal cells were grown either: (1) in Dulbecco's modified eagle's medium (DMEM); (2) in the presence of conditioned medium from astroglial cell (ACM) cultures at different stages of glia differentiation and maturationin vitro; 93) in the presence of astroglial cells, in co-cultures or mixed-cultures; and (4) in the absence or the presence of antibodies (Abs) for neural cell adhesion molecule, (N-CAM) receptor. This work shows that during its maturation and differentiationin vitro (8-40 days, DIV), astroglial cells in primary culture release factors able to markedly influence GT(1-1) cell morphology and accelerate LHRH cell secretory potential, with a potency depending on both the 'age' of astroglia and the degree of GT(1-1) neuron differentiationin vitro. Regional differences in glial-derived factors that promote LHRH neuronal differentiation and secretion were observed, with hypothalamic astroglia being the most potent neurotrophic stimulus. Such effects were specific for astroglia conditioned medium (CM), since oligodendrocyte CM was without effect. Boiling of the ACM for 10 min completely abolished stimulatory activity on neuronal cells. When immature astroglial cells (12 DIV) were co-cultured with GT(1-1) neurons, LHRH release increased by about 2- to 3-fold over basal levels and GT(1-1) neuron proliferation was doubled. Astroglial cells responded to GT(1-1) neuronal signals with an almost doubling of the [(3)H]-thymidine incorporation and DNA synthesis. Extensive neurite outgrowth and establishment of cell-cell contacts between the two cell compartments were observed in the mixed culture preparation, accompanied by a marked stimulatory effect on both cell proliferation and LHRH secretion. Addition of N-CAM-Ab in the GT(1-1)-astroglial cell mixed cultures resulted in a dramatic disruption of GT(1-1)-astroglia morphology and a 95% suppression of the stimulatory effect on both cell proliferation and LHRH release suggesting the local adhesive mechanisms are importantly involved in the crosstalk between GT(1-1) neurons and astroglial cellsin vitro. This work shows for the first time the presence of a bidirectional interaction between the LHRH neurons and astroglial cells and suggest a potential interplay between the two compartments in the regulation of LHRH neuronal physiology.

Signal Requirements for Production of Luteinizing Hormone Releasing-Hormone by Human T Cells

We have previously demonstrated that both human CD4+ and CD8+ T lymphocytes produce enhanced levels of luteinizing hormone-releasing hormone (LHRH) mRNA and peptide upon stimulation with monoclonal antibody directed at the CD3 component of the T cell receptor for antigen (TCR) or mitogenic lectin. In the current study, we define the signaling pathways that control TCR-mediated LHRH production by using agents known to affect distinct signals, and compare the messenger systems required for LHRH response to other T-cell-associated activation responses, such as expression of CD69 and interleukin-2 receptor (IL-2R) molecules and production of interleukin-2 (IL-2). Results indicate that the activation of protein kinase C (PKC) is essential for LHRH production by previously nonstimulated T cells, not increased concentration of cytosolic-free calcium ([Ca]2+]1). Phorbol ester (PMA), a direct activator of PKC, provoked LHRH production and cell surface expression of CD69 and IL-2R molecules by T cells, but not IL-2 synthesis. The synthesis of IL-2 by T cells required costimulation with PMA and ionomycin, a Ca2+ ionophore. Consistent with these observations, H7, an inhibitor of PKC, prevented T cells from producing LHRH upon activation with mitogen. However, LHRH production was not suppressed by HA 1004, which inhibits all cyclic nucleotide-dependent protein kinases except for PKC. Genistein, a selective inhibitor of protein tyrosine kinase, blocked PMA-induced increase in LHRH production, but not CD69 and IL-2R expression, suggesting that protein tyrosine phosphorylation events distal from PKC activation may play a role in regulating LHRH response.

LHRH Cells Migrate on Peripherin Fibers in Embryonic Olfactory Explant Cultures: An in Vitro Model for Neurophilic Neuronal Migration

Luteinizing hormone releasing hormone (LHRH) neurons arise from progenitor cells in the olfactory placode. During prenatal development, these cells migrate via neurophilic interactions, in track-like arrangements along axons of the olfactory complex. The mechanisms by which these cells attain an adult-like distribution are unknown. In this study, we established an in vitro, embryonic mouse olfactory explant model to examine the factors directing LHRH cell migration. Cultures were generated from E11.5 embryos and maintained for up to 3 weeks. Typically 20-50% (160-400 LHRH cells) of the total LHRH neuronal population survived and maintained gene expression in these explants. Fibronectin and laminin staining delineated substratum producing cells which concentrically spread, from Days 1-7, from the periphery of the entire explanted tissue. In contrast, LHRH cells emigrated exclusively from inside olfactory pit areas to the surface of the culture after 3 days. The relationship between groups of LHRH cells emerging from bilateral olfactory pits was not random, but highly organized; in 93% of the cultures examined, the angular relationship between these groups was 180-270°. After 5 or more days in vitro, LHRH cells were found in directional tracks similar to those observed in vivo forming a continuum from the olfactory pit out onto the substratum, where many bipolar LHRH cells were discretely located. The maximum distance away from the olfactory pit that LHRH cells were detected was 0.9 mm, which is compatible with the distance traversed by LHRH cells through the nasal region in vivo. The reproducible spatiotemporal appearance of LHRH cells was unrelated to the concentric spread of the fibronectin and laminin producing cells of the explant. Taken together, these data indicate that LHRH cells migrated directionally in these explants and that the molecular cues governing the initial migration of these neurons remained intact in this system in the absence of brain tissue. Double-label immunocytochemistry indicated that at least three populations of neuronal fibers existed in the explants: N-CAM-positive, peripherin-positive, and N-CAM/peripherin-positive. Although all three fibers groups showed highly organized spatiotemporal distribution patterns, only peripherin-positive fibers correlated with the location of LHRH cells. LHRH cells were always preceded by, and in close association with, peripherin-positive fibers. We hypothesize that signals arising from the peripherin-positive axons provide the appropriate guidance cues to LHRH cells as they emigrate from the olfactory pit.

A Subset of Peripherin Positive Olfactory Axons Delineates the Luteinizing Hormone Releasing Hormone Neuronal Migratory Pathway in Developing Mouse

Luteinizing hormone releasing hormone (LHRH) neurons in the CNS are derived from cells of the olfactory placode and thereafter migrate from the olfactory pit into the diencephalon. In this study, we examined embryonic LHRH neurons and the LHRH migratory pathway for several markers. During development, N-CAM and peripherin mRNA were expressed by olfactory epithelia, but not by LHRH cells. In nasal regions, olfactory axons were not immunostained by laminin or fibronectin antibodies, but were robustly peripherin and N-CAM immunoreactive. Although the majority of these axonal tracks entered the developing olfactory bulbs, a small population of peripherin positive but N-CAM negative axons turned caudally into the developing forebrain. LHRH cells were consistently juxtaposed to these axons. We propose that this peripherin positive/N-CAM negative fiber track is the anatomical pathway upon which LHRH cells migrate from the olfactory pit into the diencephalon.

Luteinizing hormone-releasing hormone (LHRH)-expressing cells in the nasal septum of human fetuses

In 12–19 weeks old human fetuses, LHRH-immunoreactive (LHRH-Ir) cells were detected in the nerve fascicles arising from the vomeronasal organ (VNO), from their origin to their end point at the medial aspect of the olfactory bulb (vomeronasal and/or terminal nerves). In one 22-weeks-old fetus, LHRH-Ir cells were present only in the upper part of the nasal septum, in the nerve fascicles crossing the cribriform plate. No LHRH cells could be detected in two fetuses, 23 and 36 weeks old. Our results are discussed with regard to the migratory hypothesis of LHRH cells from the medial olfactory placode to the brain.

Gene expression in a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons prior to the preovulatory gonadotropin surge

Gene expression in luteinizing hormone-releasing hormone (LHRH) neurons was analyzed during the periovulatory period to (1) characterize temporal patterns of LHRH gene expression and their relationship(s) to gonadotropin surges, and (2) determine if any such changes are uniform or dissimilar at different rostrocaudal levels of the basal forebrain. The number of neurons expressing mRNA for the decapeptide, and the relative degree of expression per cell were analyzed using in situ hybridization and quantitative image analysis. Rats were killed at 1800 hr on metestrus (Met), 0800 hr, 1200 hr, 1800 hr, and 2200 hr on proestrus (Pro), or 0200 hr, 0800 hr, and 1800 hr on estrus (E; n = 5-6 rats/group). All sections were processed for LHRH mRNA in a single in situ hybridization assay. Sections were atlas matched and divided into four rostrocaudal groups for analysis: vertical limb of the diagonal band of Broca (DBB), rostral preoptic area/organum vasculosum of the lamina terminalis (rPOA/OVLT), medial preoptic area (mPOA), and suprachiasmatic/anterior hypothalamic area (SCN/AHA). Plasma LH and FSH levels from all animals were analyzed by RIA. The labeling intensity per cell was similar among all time points at all four rostrocaudal levels. The number of cells expressing LHRH mRNA, however, varied as a function of time of death during the estrous cycle, and this temporal pattern varied among the four anatomical regions. At the level of the mPOA, the number of cells was highest at 1200 hr on Pro, and then declined and remained low throughout the morning of E. At the level of the rPOA/OVLT, the greatest number of LHRH neurons was noted later in Pro, at 1800 hr, dropping rapidly to lowest numbers at 2200 hr. No significant changes in LHRH cell number occurred at the DBB or SCN/AHA levels. At all anatomical levels, the secondary surge of FSH was unaccompanied by any change in the number of neurons expressing LHRH mRNA. These data demonstrate that (1) the number of detectable LHRH mRNA-expressing cells fluctuates during the periovulatory period and (2) peak numbers of LHRH-expressing cells are attained in the mPOA before the onset of the LH surge and before peak LHRH cell numbers are seen at more rostral levels. A model is proposed in which gene expression in this subpopulation of LHRH neurons may be activated by preovulatory estrogen secretion and acutely reduced following the proestrous surge of progesterone.

Vomeronasal organ-mediated induction of fos in the central accessory olfactory pathways in repetitively mated female rats

Removal of the VNO significantly reduced the enhancement of lordosis and the induction of fos immunoreactivity in luteinizing hormone-releasing hormone (LHRH) neurons in ovariectomized estrogen-primed rats. There was a significant positive correlation between the two variables. In the accessory olfactory bulb (AOB) of the repetitively mated rats, the number of fos-positive cells in the granule (G) cell layer was significantly lower in the VNO-removed rats, whereas that in the mitral (M) cell layer was not significantly different between VNO-removed and VNO-sham females. The G/M ratio (calculated by dividing the mean number of fos-positive cells in the G cell layer by that in the M cell layer), taken as an estimate of the output of the AOB, was relatively larger in the VN-sham as compared with the VNO-removed rats. There were significant positive correlations between G/M ratio and the increase in LQ and between the G/M ratio and the percentage of fos-positive LHRH cells. The positive correlation between the number of fos-positive cells in the posterodorsal medial amygdala (PDMA) and the increase in LQ and that between the number of fos-positive cells in the PDMA and the percentage of fos-positive LHRH cells were significant, supporting the role of the medial nucleus of amygdala in lordosis. However, the correlation between G/M ratio and the number of fos-positive cells in the PDMA was not significant, indicating that fos immunoreactivity in the PDMA is not directly related to that in the AOB. In conclusion, the results support the involvement of the accessory olfactory system in mediating the facilitatory effects of repeated mating on lordosis in female rats and suggest that the influence of the accessory olfactory system is mediated likely through the LHRH neuronal system. Integration and filtering of sensory information may take place at various levels of the brain, such as the AOB and the medial amygdala, before being transmitted to higher brain centers controlling lordosis behavior in female rats.

Nitric oxide regulates luteinizing hormone-releasing hormone secretion

We have analyzed the role of nitric oxide (NO), an unorthodox and novel neuromodulator, on luteinizing hormone-releasing hormone (LHRH) secretion. Sodium nitroprusside (SNP), an NO donor, was used to challenge LHRH neurons using both hypothalamic explants and an immortalized neuronal cell line (GT1 cells) in vitro. In both paradigms, SNP was able to stimulate LHRH release in a dose-dependent manner. This action of SNP was accompanied by an elevation in both extra- and intra-cellular cGMP levels. In addition, exposure of LHRH cells (GT1-7 cells) to increasing concentrations of a soluble analog of cGMP (8-Br-cGMP) enhanced LHRH release in a dose-dependent manner, indicating that LHRH neurons have the intrinsic ability to respond to the intracellular messenger elicited by NO, i.e., cGMP. Furthermore, sodium nitroprusside-induced LHRH secretion from GT1-7 cells was blocked, in a dose-dependent manner, by Rp-8-Br-cGMPS, a cGMP analog which blocks cGMP-dependent protein kinase. These data clearly demonstrate that NO stimulates LHRH secretion by activating guanylate cyclase, and support a potential role of NO as a neuroactive agent involved in the control of LHRH secretion and, thereby, reproductive functions.

Nitric oxide regulates luteinizing hormone-releasing hormone secretion.

We have analyzed the role of nitric oxide (NO), an unorthodox and novel neuromodulator, on luteinizing hormone-releasing hormone (LHRH) secretion. Sodium nitroprusside (SNP), an NO donor, was used to challenge LHRH neurons using both hypothalamic explants and an immortalized neuronal cell line (GT1 cells) in vitro. In both paradigms, SNP was able to stimulate LHRH release in a dose-dependent manner. This action of SNP was accompanied by an elevation in both extra- and intra-cellular cGMP levels. In addition, exposure of LHRH cells (GT1-7 cells) to increasing concentrations of a soluble analog of cGMP (8-Br-cGMP) enhanced LHRH release in a dose-dependent manner, indicating that LHRH neurons have the intrinsic ability to respond to the intracellular messenger elicited by NO, i.e., cGMP. Furthermore, sodium nitroprusside-induced LHRH secretion from GT1-7 cells was blocked, in a dose-dependent manner, by Rp-8-Br-cGMPS, a cGMP analog which blocks cGMP-dependent protein kinase. These data clearly demonstrate that NO stimulates LHRH secretion by activating guanylate cyclase, and support a potential role of NO as a neuroactive agent involved in the control of LHRH secretion and, thereby, reproductive functions.

Characterization of neuronal cell varieties migrating from the olfactory epithelium during prenatal development in the rat. Immunocytochemical study using antibodies against olfactory marker protein (OMP) and luteinizing hormone-releasing hormone (LH-RH)

The development of neurons located outside the olfactory epithelium was studied by using antisera against olfactory marker protein (OMP) and luteinizing hormone-releasing hormone (LH-RH) in the rat. The study was restricted to the localization of these cells in the nasal cavity and in the region of the olfactory bulb during development. We describe groups of cells that stain positively for OMP located principally on the ventro-lateral aspect of the olfactory bulbs. A comparison is made with the LH-RH-immunoreactive system of cells which predominate on the medial aspect following the known trajectory of the nervus terminalis. OMP-immunoreactive cells appeared along the course of the olfactory fibers when they were first detected at embryonic day 16. These cells became restricted to a small group above the cribriform plate, ventral to the olfactory bulbs that seemed to disappear shortly after birth. It is concluded that these cells, which like the LH-RH cells have most probably migrated from the olfactory placode, represent a group of intervening neurons between the olfactory receptor cells and the olfactory bulb, serving as hints for olfactory axons to reach their targets during prenatal development.

Influence of male rats on the luteinizing hormone-releasing hormone neuronal system in female rats: role of the vomeronasal organ.

Olfactory information processed by the vomeronasal system is reported to influence reproductive functions in a variety of mammals. The present studies were designed to determine if male-associated cues affect the luteinizing hormone-releasing hormone (LHRH) neuronal system, and, if so, to determine the extent to which these cues are processed by the vomeronasal organ (VNO). Ovariectomized rats underwent VNO removal (VNX) or sham surgery (VN-Sham). Forty-eight hours after estrogen priming (5 micrograms), they were subjected to one of the following treatments: repeated mating, repeated exposure to male-soiled bedding or repeated exposure to clean bedding. In animals treated for 180 min, coronal brain sections were double labelled for Fos protein and LHRH. An intense Fos immunoreactivity was induced following mating in the majority of LHRH neurons in the VN-Sham females, whereas removal of the VNO significantly suppressed the mating-induced Fos staining. Exposure of female rats to male-soiled bedding or clean bedding did not induce appreciable Fos immunoreactivity in LHRH neurons. Following 90 min of mating or exposure to bedding, blood samples were assayed for luteinizing hormone (LH). Mating stimulated the release of LH in VN-Sham females, while the removal of the VNO significantly suppressed the mating-induced LH release. Exposure of the females to male-soiled bedding or clean bedding did not induce an LH surge. The present results demonstrate that male-originating sensory cues (i.e. repeated mating) can influence the LHRH neuronal system, as evidenced by the presence of Fos immunoreactivity in LHRH cell bodies, and indicate that this effect is mediated through the VNO to a certain extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing Hormone‐Releasing Hormone Neurons which are R‐etrogradely Labeled After Peripheral Fluoro‐Gold Administration in the Male Ferret

This study identified luteinizing hormone-releasing hormone (LHRH)-producing neurons which have access to fenestrated capillaries in prepubertal male European ferrets. Fluoro-Gold was injected intraperitoneally to retrogradely label neurons with terminals outside the blood-brain barrier. LHRH neurons were identified by immunofluorescence using a secondary antibody tagged with tetramethylrhodamine isothiocyanate. Cell bodies which demonstrated both tetramethylrhodamine isothiocyanate and Fluoro-Gold fluorescence were defined as LHRH-producing neurons with axon terminals in regions containing fenestrated capillaries. The total number and neuroanatomical distribution of immunopositive (LHRH +) cells concurred with previous studies in the ferret in which cell bodies were diffusely distributed from rostral forebrain through caudal diencephalon, with approximately 70% of the LHRH + cell bodies located in retrochiasmatic hypothalamus. In the present study, an average of 59.8% of all LHRH+ neuronal perikarya also contained Fluoro-Gold. The majority of Fluoro-Gold filled LHRH+ neurons demonstrated only faint to moderate amounts of Fluoro-Gold when compared to other Fluoro-Gold filled neurosecretory neurons. This limited uptake of Fluoro-Gold may be due to a relative inactivity of LHRH neurons projecting outside the blood-brain barrier. Double-labeled LHRH + neurons were dispersed throughout the entire population of LHRH+ cell bodies and no apparent nuclear groups of double-labeled neurons were found. This observation suggests that the LHRH+ neurons responsible for neurosecretion into the median eminence coexist with the LHRH+ neurons responsible for intracerebral neurotransmission or neuromodulation. One distinguishable population of LHRH + neurons was consistently observed in all the brains. Only 26% of total LHRH+ perikarya within the caudal arcuate nucleus contained Fluoro-Gold, while at least 50% of LHRH+ neurons in other structures, including the rostral arcuate nucleus, contained Fluoro-Gold. Thus, in the prepubertal male ferret, the majority of LHRH cell bodies located in the caudal arcuate nucleus may be differentially regulated and/or involved in non-neuroendocrine functions.