Luteinizing Hormone-releasing Hormone Neurons Research Articles

Topography and associations of luteinizing hormone-releasing hormone and neuropeptide Y-immunoreactive neuronal systems in the human diencephalon.

Neuropeptide Y (NPY) potentiates the effect of luteinizing hormone-releasing hormone (LHRH) on luteinizing hormone secretion in several species, including human. In addition to the pituitary sites, the interactions of the NPY and LHRH systems may involve diencephalic loci. However, the morphologic basis of this putative communication has not yet been elucidated in the human brain. To discover interaction sites, the distribution and connections of LHRH and NPY-immunoreactive (IR) neuronal elements in the human hypothalamus were investigated by means of light microscopic single- and double-label immunocytochemistry. NPY-IR perikarya and fibers were found to be widely distributed in the ventral diencephalon, with high densities in the preopticoseptal, periventricular, and tuberal regions. Small neuronal cell groups were infiltrated with a dense network of varicose NPY-IR fibers in the lateral preoptic area. The LHRH-IR perikarya were located mainly in the preopticoseptal region, diagonal band of Broca, lamina terminalis, and periventricular and infundibular nuclei. A few LHRH-IR neurons and fibers were scattered in the mamillary region. The overlap between the NPY and LHRH systems was apparent in the periventricular, paraventricular, and infundibular nuclei. Double-labeling immunohistochemistry showed NPY-IR axon varicosities in contact with LHRH-IR perikarya and main dendrites. The putative innervation of LHRH neurons by NPY-IR fibers was also seen in 1-microm-thick plastic sections and with confocal laser scanning microscope, thus further supporting the functional impact of NPY-IR terminals on LHRH-IR neurons. The present findings suggest that the hypophysiotropic LHRH-synthesizing neurons may be innervated by intrahypothalamic NPY-IR fibers. Confirmation by ultrastructural analysis would demonstrate that the LHRH system in the human hypothalamus is regulated by NPY, as has been demonstrated in nonhuman species.

Gender, neuroendocrine-immune interactions and neuron-glial plasticity. Role of luteinizing hormone-releasing hormone (LHRH).

Signals generated by the hypothalamic-pitutary-gonadal (HPG) axis powerfully modulate immune system function. This article summarizes some aspects of the impact of gender in neuroendocrine immunomodulation. Emphasis is given to the astroglial cell compartment, defined as a key actor in neuroendocrine immune communications. In the brain, the principal hormones of the HPG axis directly interact with astroglial cells. Thus, luteinizing hormone releasing hormone, LHRH, influences hypothalamic astrocyte development and growth, and hypothalamic astrocytes direct LHRH neuron differentiation. Hormonally induced changes in neuron-glial plasticity may dictate major changes in CNS output, and thus actively participate in sex dimorphic immune responses. The impact of gender in neuroimmunomodulation is further underlined by the sex dimorphism in the expression of genes encoding for neuroendocrine hormones and their receptors within the thymus, and by the potent modulation exerted by circulating sex steroids during development and immunization. The central role of glucocorticoids in the interactive communication between neuroendocrine and immune systems, and the impact of gender on hypothalamic-pituitary-adrenocortical (HPA) axis modulation is underscored in transgenic mice expressing a glucocorticoid receptor antisense RNA.

Some Hypothalamic Hamartomas Contain Transforming Growth Factorα , a Puberty-Inducing Growth Factor, But Not Luteinizing Hormone-Releasing Hormone Neurons1

Activation of LH-releasing hormone (LHRH) secretion, essential for the initiation of puberty, is brought about by the interaction of neurotransmitters and astroglia-derived substances. One of these substances, transforming growth factor alpha (TGFalpha), has been implicated as a facilitatory component of the glia-to-neuron signaling process controlling the onset of female puberty in rodents and nonhuman primates. Hypothalamic hamartomas (HH) are tumors frequently associated with precocious puberty in humans. The detection of LHRH-containing neurons in some hamartomas has led to the concept that hamartomas advance puberty because they contain an ectopic LHRH pulse generator. Examination of two HH associated with female sexual precocity revealed that neither tumor had LHRH neurons, but both contained astroglial cells expressing TGFalpha and its receptor. Thus, some HH may induce precocious puberty, not by secreting LHRH, but via the production of trophic factors--such as TGFalpha--able to activate the normal LHRH neuronal network in the patient's hypothalamus.

Sympathetic‐related neurons in the preoptic region of the rat identified by viral transneuronal labeling

The viral transneuronal labeling method was used to localize sympathetic-related neurons in the preoptic region following pseudorabies virus (PRV) injections into either the superior cervical ganglion, stellate ganglion, celiac ganglion, or adrenal gland of rats. A general pattern of infection was detected. First, neuronal labeling was found in the medial preoptic area, medial preoptic nucleus, median preoptic nucleus, and lateral preoptic area, and then it spread to the anteroventral periventricular, anteroventral preoptic, and parastrial nuclei. Finally, the forebrain circumventricular organs: organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO) became infected. Neuropeptide-containing preoptic neurons were analyzed following PRV injections in the stellate ganglion. Some thyrotropin-releasing hormone and neurotensin neurons were labeled, but none of the calcitonin gene-related peptide, cholecystokinin, corticotropin-releasing factor, galanin, luteinizing hormone-releasing hormone, enkephalin, substance P, or tyrosine hydroxylase neurons were PRV infected. Two major sympathetic networks appear to be represented in the preoptic region. One is linked to the OVLT, SFO, and anteroventral third ventricular (AV3V) region, sites previously implicated in fluid and electrolyte balance as well as cardiovascular control. The other descending sympathetic pathway appears to target the medial preoptic nucleus as its key nodal point, receiving inputs from infralimbic cortex and limbic regions, such as the lateral septum, medial nucleus of the amygdala, subiculum, and amygdalohippocampal area, and then, projecting caudally to the hypothalamus and brainstem. This second sympathetic network may subserve affiliative, defensive and sexual behaviors. J. Comp. Neurol. 414:361–378, 1999. © 1999 Wiley-Liss, Inc.

The role of nitric oxide in reproduction.

Nitric oxide (NO) plays a crucial role in reproduction at every level in the organism. In the brain, it activates the release of luteinizing hormone-releasing hormone (LHRH). The axons of the LHRH neurons project to the mating centers in the brain stem and by afferent pathways evoke the lordosis reflex in female rats. In males, there is activation of NOergic terminals that release NO in the corpora cavernosa penis to induce erection by generation of cyclic guanosine monophosphate (cGMP). NO also activates the release of LHRH which reaches the pituitary and activates the release of gonadotropins by activating neural NO synthase (nNOS) in the pituitary gland. In the gonad, NO plays an important role in inducing ovulation and in causing luteolysis, whereas in the reproductive tract, it relaxes uterine muscle via cGMP and constricts it via prostaglandins (PG).

Sympathetic-related neurons in the preoptic region of the rat identified by viral transneuronal labeling

The viral transneuronal labeling method was used to localize sympathetic-related neurons in the preoptic region following pseudorabies virus (PRV) injections into either the superior cervical ganglion, stellate ganglion, celiac ganglion, or adrenal gland of rats. A general pattern of infection was detected. First, neuronal labeling was found in the medial preoptic area, medial preoptic nucleus, median preoptic nucleus, and lateral preoptic area, and then it spread to the anteroventral periventricular, anteroventral preoptic, and parastrial nuclei. Finally, the forebrain circumventricular organs: organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO) became infected. Neuropeptide-containing preoptic neurons were analyzed following PRV injections in the stellate ganglion. Some thyrotropin-releasing hormone and neurotensin neurons were labeled, but none of the calcitonin gene-related peptide, cholecystokinin, corticotropin-releasing factor, galanin, luteinizing hormone-releasing hormone, enkephalin, substance P, or tyrosine hydroxylase neurons were PRV infected. Two major sympathetic networks appear to be represented in the preoptic region. One is linked to the OVLT, SFO, and anteroventral third ventricular (AV3V) region, sites previously implicated in fluid and electrolyte balance as well as cardiovascular control. The other descending sympathetic pathway appears to target the medial preoptic nucleus as its key nodal point, receiving inputs from infralimbic cortex and limbic regions, such as the lateral septum, medial nucleus of the amygdala, subiculum, and amygdalohippocampal area, and then, projecting caudally to the hypothalamus and brainstem. This second sympathetic network may subserve affiliative, defensive and sexual behaviors.

Glial-neuronal interactions in the neuroendocrine control of mammalian puberty: facilitatory effects of gonadal steroids.

It is now clear that astroglial cells actively contribute to both the generation and flow of information within the central nervous system. In the hypothalamus, astrocytes regulate the secretory activity of neuroendocrine neurons. A small subset of these neurons secrete luteinizing hormone-releasing hormone (LHRH), a neuropeptide essential for sexual development and adult reproductive function. Astrocytes stimulate LHRH secretion via cell-cell signaling mechanisms involving growth factors recognized by receptors with either serine/threonine or tyrosine kinase activity. Two members of the epidermal growth factor (EGF) family and their respective tyrosine kinase receptors appear to play key roles in this regulatory process. Transforming growth factor-alpha (TGFalpha) and its distant congeners, the neuregulins (NRGs), are produced in hypothalamic astrocytes. They stimulate LHRH secretion indirectly, via activation of erbB-1/erbB-2 and erbB-4/erbB-2 receptor complexes also located on astrocytes. Activation of these receptors leads to release of prostaglandin E(2) (PGE(2)), which then binds to specific receptors on LHRH neurons to elicit LHRH secretion. Gonadal steroids facilitate this glia-to-neuron communication process by acting at three different steps along the signaling pathway. They (a) increase astrocytic gene expression of at least one of the EGF-related ligands (TGFalpha), (b) increase expression of at least two of the receptors (erbB-4 and erbB-2), and (c) enhance the LHRH response to PGE(2) by up-regulating in LHRH neurons the expression of specific PGE(2) receptor isoforms. Focal overexpression of TGFalpha in either the median eminence or preoptic area of the hypothalamus accelerates puberty. Conversely, blockade of either TGFalpha or NRG hypothalamic actions delays the process. Thus, both TGFalpha and NRGs appear to be physiological components of the central neuroendocrine mechanism controlling the initiation of female puberty. By facilitating growth factor signaling pathways in the hypothalamus, ovarian steroids accelerate the pace and progression of the pubertal process.

Estrogen receptor immunoreactivity is present in the majority of central histaminergic neurons: evidence for a new neuroendocrine pathway associated with luteinizing hormone-releasing hormone-synthesizing neurons in rats and humans.

The central regulation of the preovulatory LH surge requires a complex sequence of interactions between neuronal systems that impinge on LH-releasing hormone (LHRH)-synthesizing neurons. The reported absence of estrogen receptors (ERs) in LHRH neurons indicates that estrogen-receptive neurons that are afferent to LHRH neurons are involved in mediating the effects of this steroid. We now present evidence indicating that central histaminergic neurons, exclusively located in the tuberomammillary complex of the caudal diencephalon, serve as an important relay in this system. Evaluation of this system revealed that 76% of histamine-synthesising neurons display ERalpha-immunoreactivity in their nucleus; furthermore histaminergic axons exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both the rodent and the human brain. Our in vivo studies show that the intracerebroventricular administration of the histamine-1 (H1) receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine, can block the LH surge in ovariectomized estrogen-treated rats. These data are consistent with the hypothesis that the positive feedback effect of estrogen in the induction of the LH surge involves estrogen-receptive histamine-containing neurons in the tuberomammillary nucleus that relay the steroid signal to LHRH neurons via H1 receptors.

Intracellular Ca2+Oscillations in Luteinizing Hormone-Releasing Hormone Neurons Derived from the Embryonic Olfactory Placode of the Rhesus Monkey

To understand the mechanism of pulsatile luteinizing hormone-releasing hormone (LHRH) release, we examined whether cultured LHRH neurons exhibit spontaneous intracellular Ca(2+) ([Ca(2+)](i)) signaling. The olfactory placode and the ventral migratory pathway of LHRH neurons from rhesus monkey embryos at embryonic ages 35-37 were dissected out and cultured on glass coverslips. Two to five weeks later, cultured cells were labeled with fura-2 and examined for [Ca(2+)](i) signaling by recording changes in [Ca(2+)](i) every 10 sec for 30-175 min. Cells were fixed and immunostained for LHRH and neuron-specific enolase. In 20 cultures, 572 LHRH-positive cells exhibited [Ca(2+)](i) oscillations at an interpulse interval (IPI) of 8.2 +/- 0.7 min and a duration of 88.8 +/- 2.9 sec. LHRH-negative neurons in culture exhibited only occasional [Ca(2+)](i) oscillations. In 17 of 20 cultures with LHRH-positive cells, [Ca(2+)](i) oscillations occurred synchronously in 50-100% of the individual cells, whereas [Ca(2+)](i) oscillations in cells in the remaining three cultures did not synchronize. Strikingly, in 12 of 17 cultures the synchronization of [Ca(2+)](i) oscillations repeatedly occurred in complete unison at 52.8 +/- 3.0 min intervals, which is similar to the period observed for LHRH release, whereas in 5 of 17 cultures the less tight synchronization of [Ca(2+)](i) oscillations repeatedly occurred at 23.4 +/- 4.6 min intervals. IPI of [Ca(2+)](i) oscillations in cells with tight synchronization and less tight synchronization did not differ from IPI in cells without synchronization. The results indicate that LHRH neurons derived from the monkey olfactory placode possess an endogenous mechanism for synchronization of [Ca(2+)](i) oscillations. Whether synchronization of [Ca(2+)](i) oscillations relates to neurosecretion remains to be investigated.

Advantage of double labeled in situ hybridization for detecting the effects of glucocorticoids on the mRNAs of protooncogenes and neural peptides (TRH) in cultured hypothalamic neurons

In order to study the effect of glucocorticoids on thyrotropin-releasing hormone (TRH) and protooncogenes, we describe a double labeled in situ hybridization method to explore this issue. The development of non-isotopic in situ hybridization histochemistry has proven to be an important tool for cellular and molecular studies in neurobiology [C.L.E. Moine, E. Normand, B. Bloch, Use of non-radioactive probes for mRNA detection by in situ hybridization: interests and applications in the central nervous system, Cell. Mol. Biol. 41 (1995) 917–923]. These methods involve the anatomic localization of labeled RNA or DNA molecules which hybridize with complementary target RNA or DNA sequences in the cell. With regard to gene expression, in situ hybridization allows the study of specific mRNA levels and the distribution between various cell types. It also allows the comparison of mRNA levels at various stages of development. Double labeled in situ hybridization is able to detect the colocalization of two different mRNAs simultaneously. Accordingly, this approach is utilized for specific studies involving the expression and distribution of TRH mRNA and the protooncogenes, c- fos/c- jun, in cultured rat hypothalamic neurons [L.G. Luo, I.M.D. Jackson, Glucocorticoids stimulate TRH and c- fos/c- jun gene co-expression in cultured hypothalamic neurons, Brain Research 791 (1998) 56–62]. Our protocol for double labeled in situ hybridization reflects a modification of a number of original protocols developed by others [H. Breitschopf, G. Suchanek, R.M. Gould, D.R. Colman, H. Lassmann, In situ hybridization with digoxigenin-labeled probes: sensitive and reliable detection method applied to myelinating rat brain, Acta Neurropathol. 84 (1992) 581–587; S. McQuaid, J. McMahon, G.M. Allan, A comparison of digoxigenin and biotin labeled DNA and RNA probes for in situ hybridization, Biotech. Histochem. 70 (1995) 147–154; E. Hrabovszky, M.E. Vrontakis, S.L. Petersen, Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats, J. Histochem. Cytochem. 43 (1995) 363–370]. This technique can be readily applied to various studies of cellular gene expression in the mammalian nervous system involving other neural peptides and transcription factors. Theme E: Endocrine and autonomic regulation Topic: Neuroendocrine regulation: other

Locus ceruleus lesions block pulsatile LH release in ovariectomized rats

Luteinizing hormone (LH) secretion during the reproductive cycle and in ovariectomized (OVX) rats is pulsatile and this pattern of secretion is determined by intermittent discharges of LH-releasing hormone (LHRH) into the hypophysial portal vessels. LHRH secretion is probably controlled by prior pulsatile norepinephrine (NE) release. The locus ceruleus (LC) is an important source of NE to the LHRH neurons. We have shown previously that LC lesions block the preovulatory LH surge and ovulation and also cause a decrease in plasma LH concentrations in OVX rats. The possible role of the LC in regulating pulsatile LH release has not been explored. Therefore, the aim of this work was to investigate, in OVX rats, the effects of LC lesions on pulsatile LH secretion. LC lesions were produced in adult female rats three weeks after OVX. On the next morning, the jugular vein was catheterized and, on the afternoon of the same day, blood samples (0.3 ml) were withdrawn every 5 min, during 90 min, from conscious freely moving rats. Plasma LH was measured by radioimmunoassay. LC lesions greatly suppressed pulsatile LH secretion by decreasing both LH pulse frequency and amplitude. The basal as well as total secretion of LH were also decreased. This inhibitory effect of the lesions was observed only when at least 50% of the nucleus was destroyed. Data from sham-operated animals as well as those with less than 50% destruction of the LC did not differ from those of the control rats without brain lesions. Since LC lesions induce a decrease in NE content in the preoptic area and median eminence, the inhibition of pulsatile LH release in ovariectomized rats with LC lesions occurs presumably as result of decreased pulsatile NE release into these areas of the brain that decreases both the frequency and the amount of LHRH released per pulse.

An increase in glutamate release follows a decrease in gamma aminobutyric acid and the pubertal increase in luteinizing hormone releasing hormone release in the female rhesus monkeys.

Previously we have shown that release of gamma-aminobutyric acid (GABA) in the stalk-median eminence (S-ME) is high in prepubertal monkeys and that a decrease in GABA release triggers the onset of puberty. However, it is still unclear how disinhibition of the luteinizing hormone releasing hormone (LHRH) neuronal system from GABA input is followed (or accompanied) by an increase in stimulatory signals, such as glutamatergic input to LHRH neurons. To clarify the temporal relationship between the reduction of the GABAergic inhibitory signal and the enhancement of the glutamatergic stimulatory signal in the control of LHRH release at the onset of puberty, we conducted two experiments using a push-pull perfusion method. In the first experiment, we measured developmental changes in release of LHRH, GABA, and glutamate in the S-ME. LHRH levels were very low in prepubertal monkeys, increased to higher levels in early pubertal monkeys, with the highest LHRH levels occurring in mid-pubertal monkeys. As we previously observed, GABA levels were high in prepubertal monkeys and then decreased in early- and mid-pubertal monkeys. In contrast, glutamate levels were very low in prepubertal monkeys, increased dramatically in early pubertal monkeys, and then slightly decreased in mid-pubertal monkeys, although mid-pubertal levels remained much higher than prepubertal levels. In the second experiment, we measured GABA, glutamate and LHRH in the same samples obtained from prepubertal monkeys which were infused with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD) 67 mRNA into the S-ME. GAD67 is a catalytic enzyme for GABA synthesis from glutamate, and AS GAD67 mRNA interferes with GAD67 synthesis. Infusion of the AS GAD67 induced a decrease in GABA release, which subsequently resulted in an increase in LHRH release. Surprisingly, glutamate release also increased several hours after the decrease in GABA release, and the increased LHRH release continued. These data are interpreted to mean that a decrease in GABA synthesis by interference with GAD67 synthesis and the reduction of GABA release in the S-ME trigger an increase in LHRH release, but that a subsequent increase in glutamate release in the S-ME further contributes to the pubertal increase in LHRH release at the onset of puberty. The data further support our hypothesis that GAD plays an important role in the mechanism of the onset of puberty.

Pulsatile release of luteinizing hormone-releasing hormone (LHRH) in cultured LHRH neurons derived from the embryonic olfactory placode of the rhesus monkey.

To study the mechanism of LH-releasing hormone (LHRH) pulse generation, the olfactory pit/placode and the migratory pathway of LHRH neurons from monkey embryos at embryonic age 35-37 were dissected out, under the microscope, and cultured on plastic coverslips coated with collagen in a defined medium for 2-5 weeks. First, we examined whether cultured neurons release the decapeptide into media. It was found that LHRH cells release LHRH in a pulsatile manner at approximately 50-min intervals. Further, LHRH release was stimulated by depolarization with high K+ and the Na+ channel opener, veratridine. However, whereas the Na+ channel blocker, tetrodotoxin suppressed the effects of veratridine, tetrodotoxin did not alter the effects of high K+. Subsequently, the role of extracellular and intracellular Ca2+ in LHRH release was examined. The results are summarized as follows: 1) exposing the cells to a low Ca2+ (20 nM) buffer solution suppressed LHRH release, whereas exposure to a normal Ca2+ solution (1.25 mM) maintained pulsatile LHRH release; 2) LHRH release from cultured LHRH cells was stimulated by the voltage-sensitive L-type Ca2+ channel agonist, Bay K 8644 (10 microM), whereas it was suppressed by the L-type Ca2+ channel blocker, nifedipine (1 microM), but not by the N-type channel blocker, omega-conotoxin GVIA (1 microM); 3) the intracellular Ca2+ stimulant, ryanodine (1 microM), stimulated LHRH release, whereas the intracellular Ca2+ transporting adenosine triphosphatase antagonist, thapsigargin (1 and 10 microM), did not yield consistent results; and 4) carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (1 microM), a mitochondrial Ca2+ mobilizer, stimulated LHRH release, whereas ruthenium red, a mitochondrial Ca2+ uptake inhibitor, did not induce consistent results. These results indicate that: 1) the presence of extracellular Ca2+ is essential for LHRH neurosecretion; 2) Ca2+ enters the cell via L-type channels but not N-type channels; and 3) mobilization of intracellular Ca2+ from inositol 1,4,5-triphosphate-sensitive stores, as well as mitochondrial stores, seem to contribute to LHRH release in these cells.

Pulsatile Release of Luteinizing Hormone-Releasing Hormone (LHRH) in Cultured LHRH Neurons Derived from the Embryonic Olfactory Placode of the Rhesus Monkey

To study the mechanism of LH-releasing hormone (LHRH) pulse generation, the olfactory pit/placode and the migratory pathway of LHRH neurons from monkey embryos at embryonic age 35–37 were dissected out, under the microscope, and cultured on plastic coverslips coated with collagen in a defined medium for 2–5 weeks. First, we examined whether cultured neurons release the decapeptide into media. It was found that LHRH cells release LHRH in a pulsatile manner at approximately 50-min intervals. Further, LHRH release was stimulated by depolarization with high K+ and the Na+ channel opener, veratridine. However, whereas the Na+ channel blocker, tetrodotoxin suppressed the effects of veratridine, tetrodotoxin did not alter the effects of high K+. Subsequently, the role of extracellular and intracellular Ca2+ in LHRH release was examined. The results are summarized as follows: 1) exposing the cells to a low Ca2+ (20 nm) buffer solution suppressed LHRH release, whereas exposure to a normal Ca2+ solution (1.25 mm) maintained pulsatile LHRH release; 2) LHRH release from cultured LHRH cells was stimulated by the voltage-sensitive L-type Ca2+ channel agonist, Bay K 8644 (10 μm), whereas it was suppressed by the L-type Ca2+ channel blocker, nifedipine (1 μm), but not by the N-type channel blocker, ω-conotoxin GVIA (1μ m); 3) the intracellular Ca2+ stimulant, ryanodine (1 μm), stimulated LHRH release, whereas the intracellular Ca2+ transporting adenosine triphosphatase antagonist, thapsigargin (1 and 10 μm), did not yield consistent results; and 4) carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (1 μm), a mitochondrial Ca2+ mobilizer, stimulated LHRH release, whereas ruthenium red, a mitochondrial Ca2+ uptake inhibitor, did not induce consistent results. These results indicate that: 1) the presence of extracellular Ca2+ is essential for LHRH neurosecretion; 2) Ca2+ enters the cell via L-type channels but not N-type channels; and 3) mobilization of intracellular Ca2+ from inositol 1,4,5-triphosphate-sensitive stores, as well as mitochondrial stores, seem to contribute to LHRH release in these cells.

Luteinizing hormone-releasing hormone quantified in tissues and slice explant cultures of postnatal rat hypothalami.

LH-releasing hormone (LHRH) peptide from postnatal rat preoptic area (POA)/hypothalamic tissues in vivo and slice explant cultures maintained in vitro was quantitated using an enzyme-linked immunosorbant assay. Moreover, messenger RNA (mRNA) copy number was calculated in LHRH neurons maintained in culture using in situ hybridization histochemistry with autoradiographic film analysis. POA/hypothalami from postnatal day 5-6 pups averaged 1250 pg of LHRH, with approximately 28% of peptide residing within rostral tissues where most LHRH perikarya reside. Explant cultures maintained 18 days in vitro contained 30.4-92.0 pg/slice with a whole animal total of 244.8 pg. Considering cell numbers in vivo and in vitro, LHRH neurons in whole animal produce 1.0 pg of LHRH/cell, whereas those in culture average 2.0 pg/cell. Furthermore, LHRH mRNA copies/cell in organotypic culture was estimated conservatively at 1410 copies/cell, a relatively high number. This work shows that, compared with whole animal, cultures have substantial LHRH stores, indicating maturation of synthetic activity and/or formation of new terminals in vitro. High LHRH mRNA copy number also suggests a high rate of peptide biosynthesis. Our analysis, demonstrating the dynamic potential of LHRH neurons, suggests that subtle changes in LHRH mRNA expression in all cells or a subpopulation can dramatically alter the LHRH system biosynthetic capacity.

Polysialic Acid Facilitates Migration of Luteinizing Hormone-Releasing Hormone Neurons on Vomeronasal Axons

Luteinizing hormone-releasing hormone (LHRH) neurons migrate from the olfactory placode to the forebrain in association with vomeronasal nerves (VNN) that express the polysialic acid-rich form of the neural cell adhesion molecule (PSA-NCAM). Two approaches were used to investigate the role of PSA-NCAM: injection of mouse embryos with endoneuraminidase N, followed by the analysis of LHRH cell positions, and examination of LHRH cell positions in mutant mice deficient in the expression of NCAM or the NCAM-180 isoform, which carries nearly all PSA in the brain. The enzymatic removal of PSA at embryonic day 12 significantly inhibited the migration of nearly half of the LHRH neuron population, without affecting the VNN tract itself. Surprisingly, the absence of NCAM or NCAM-180 did not produce this effect. However, a shift in the route of migration, resulting in an excess number of LHRH cells in the accessory olfactory bulb, was observed in the NCAM-180 mutant. Furthermore, it was found that PSA expressed by the proximal VNN and its distal branch leading to the accessory bulb, but not the branch leading to the forebrain, was associated with the NCAM-140 isoform and thus was retained in the NCAM-180 mutant. These results provide two types of evidence that PSA-NCAM plays a role in LHRH cell migration: promotion of cell movement along the VNN tract that is sensitive to acute (enzymatic), but not chronic (genetic), removal of PSA-NCAM, and a preference of a subset of migrating LHRH cells for a PSA-positive axon branch over a PSA-negative branch in the NCAM-180 mutant.

Periventricular preoptic area neurons coactivated with luteinizing hormone (LH)-releasing hormone (LHRH) neurons at the time of the LH surge are LHRH afferents.

Earlier studies demonstrated coactivation of the periventricular preoptic area (pePOA) with LHRH neurons at the time of an induced or spontaneous LH surge, suggesting that the pePOA might regulate LHRH neurons. To investigate this hypothesis, studies were conducted to determine the temporal pattern of pePOA Fos activation during the rat estrous cycle and establish the connections of the pePOA neurons with LHRH neurons. Fos activation within LHRH and pePOA neurons showed the same temporal pattern. Both were absent during diestrous I, diestrous II, and the morning of proestrus. Fos was induced in the pePOA and LHRH neurons beginning on the afternoon of proestrus (4 h before lights off), with a decline 8 h later on proestrous evening. Tract-tracing studies then established the relationship between LHRH and pePOA neurons. Retrograde labeling with fluorogold determined that a portion of the Fos-positive pePOA neurons present at the time of the LH surge sent a projection to regions that contain LHRH cells. Anterograde tracer (neurobiotin) injections established that the pePOA neurons sent axons to the LHRH cells. Taken together, these data indicate that the pePOA provides direct input to LHRH neurons that is likely to stimulate LHRH neurons at the time of the LH surge.

Periventricular Preoptic Area Neurons Coactivated with Luteinizing Hormone (LH)-Releasing Hormone (LHRH) Neurons at the Time of the LH Surge Are LHRH Afferents

Earlier studies demonstrated coactivation of the periventricular preoptic area (pePOA) with LHRH neurons at the time of an induced or spontaneous LH surge, suggesting that the pePOA might regulate LHRH neurons. To investigate this hypothesis, studies were conducted to determine the temporal pattern of pePOA Fos activation during the rat estrous cycle and establish the connections of the pePOA neurons with LHRH neurons. Fos activation within LHRH and pePOA neurons showed the same temporal pattern. Both were absent during diestrous I, diestrous II, and the morning of proestrus. Fos was induced in the pePOA and LHRH neurons beginning on the afternoon of proestrus (4 h before lights off), with a decline 8 h later on proestrous evening. Tract-tracing studies then established the relationship between LHRH and pePOA neurons. Retrograde labeling with fluorogold determined that a portion of the Fos-positive pePOA neurons present at the time of the LH surge sent a projection to regions that contain LHRH cells. Anterograde tracer (neurobiotin) injections established that the pePOA neurons sent axons to the LHRH cells. Taken together, these data indicate that the pePOA provides direct input to LHRH neurons that is likely to stimulate LHRH neurons at the time of the LH surge.

Origin and migration of luteinizing hormone-releasing hormone neurons in mammals.

Luteinizing hormone-releasing hormone (LHRH) neurons are unique among hypothalamic neurons in that they originate outside of the central nervous system. In most vertebrates, LHRH-immunoreactive (-ir) neurons are detected in the epithelium of the medial olfactory pit soon after its formation. The LHRH-ir neurons migrate out of the placodal epithelium and into the brain along a migration route that consists of the central processes of the terminal, olfactory, and vomeronasal nerves. LHRH-ir cell migration follows a highly ordered course from the initial appearance of the LHRH-ir cells in the epithelium of the medial olfactory pit, to the crossing of these cells in cords on the nasal septum, to their entrance into the forebrain. Here they separate and follow an arching trajectory to their final destinations in the septal and preoptic areas and in the hypothalamus. Examination of the molecular makeup of the developing migration route reveals the presence of neural cell adhesion molecule (N-CAM) in non-LHRH-ir cells. The N-CAM-ir cells migrate into the nasal mesenchyme, trailed by N-CAM-ir axons of the olfactory, vomeronasal, and terminal nerves. These N-CAM cells and axons link the olfactory epithelia with the developing forebrain and together form scaffolding along which the LHRH-ir cells migrate into the brain. The focus of this review is on the origin and migration of LHRH-ir neurons in mammals, including humans. A discussion of Kallmann's syndrome (hypogonadotropic hypogonadism with anosmia) is included, in which there is an absence of LHRH-ir in the brain but clusters of LHRH-ir cells in the nasal cavity. This "experiment of nature" lends support to the hypothesis that all LHRH-ir cells in humans originate in the olfactory placode.

Olfactory origin of luteinizing hormone-releasing hormone (LHRH) neurons

1.性腺刺激ホルモン放出ホルモン 性腺刺激ホルモン放出ホルモンは,黄体形成ホルモ ン放出ホルモン(LHRH)とも,ゴナドトロピン(LH と FSH)のいずれの放出にも関与するという意味をこ めてゴナドトロピン放出ホルモン(GnRH)ともよば れ,LH と FSH の合成も刺激する.雌動物では LHRH の間欠分泌により LH の間欠分泌が起こり,卵巣から の排卵を促し,妊娠可能の状態が用意される(排卵分 泌).LHRH 細胞の分泌は,発生学的研究から,基本 的には一定の周期をもつ自発的分泌であることがわ かってきたが,生体内では,卵巣ホルモンや神経性の 制御をうけ,一定周期の律動分泌,パルス分泌をおこ なっている(基礎分泌).そのうえに,生体時計による 制御をうけて,周期的に排卵分泌が加わる.一方, LHRH の脳内,あるいは皮下投与によって,排卵分泌 とともに,ロルドーシスなど生殖行動を惹き起こすの で, LHRH が生殖現象の要であることが示唆される. LHRH は 10 個のアミノ酸からなり,TRH より早く 抗体が作られ,免疫組織化学的に脳内局在が調べられ た.その後,系統発生学的にも研究がすすみ,最初に 構造決定された哺乳類の mLHRH 以外に,非脊椎動物 の 2 種を含めて 10 種の LHRH が同定され,LHRH ファミリーを構成している.しかし,mLHRH 以外の LHRH に関しては,その生産細胞の起源や機能の詳細 はまだ明らかではない. Barry による研究は,哺乳類の視床下部 LHRH 細胞 の局在と下垂体への LHRH 放出の場の同定であった. 免疫陽性細胞体は視索前野,中隔,視床下部前核,弓 状核,視交叉上核などに認められた.細胞体からでる 軸索突起は第三脳室前壁(終板)や視床下部正中隆起 (ME)に投射された.線維終末には免疫陽性の分泌顆 粒がふくまれた.細胞の局在は動物によりことなるが, ME と終板での線維の集積は動物間で差はない.特に, ME には下垂体門脈系一次毛細血管が分布しており, 線維終末から放出された LHRH は門脈血管により下 垂体に運ばれゴナドトロプスを刺激することが理解さ れ,Harris が唱えた仮設が形態学的に立証された.Silverman ら はモルモットを主にして,いくつかの哺乳 類をもちいて,LHRH 細胞分布の動物種差を研究し て,LHRH 線維を前脳胞腹側面にある前有孔質に発見 した.この部には前大脳動脈からでる細血管が前脳底 部にはいるためにできた多数の小孔があり,線維はこ の細血管にともない,LHRH 細胞体も認められた.こ れは LHRH 細胞の脳内侵入を示唆する.Silverman は翌年,Schwanzel-Fukuda とともにモルモット胎児 で研究し,これら線維や細胞の分布が終神経のそれに 一致することに着目して,終神経に属する New LHRH neuron system の存在を提唱した.このことにより,鼻 プラコード,鋤鼻器,終神経,鋤鼻神経がにわかに脚 光を浴びることになる. 日医大誌 1999 第 66 巻 第 2 号