Juxtapositions between the somatostatinergic and growth hormone-releasing hormone (GHRH) neurons in the human hypothalamus.

Somatostatin is a 14-28 amino acid peptide that is located not only in the gastrointestinal system but also in multiple sites of the human brain. The inhibitory effect of somatostatin on the growth hormone (GH) secretion of the pituitary gland is a well-established phenomenon. There is a general consensus that somatostatin is released into the hypophysial portal blood and modulates GH secretion by hormonal action. In the present study, we explored the possibility that in addition to the hormonal route, somatostatin may also influence GH secretion via influencing the growth hormone-releasing hormone (GHRH) secretion by direct contacts that may be functional synapses. Since the verification of these putative synapses by electron microscopy is virtually impossible in humans due to the long post mortem time, in order to reveal the putative somatostatinergic-GHRH juxtapositions, light microscopic double-label immunohistochemistry was utilized. By examining the slides with high magnification, we observed that the vast majority of the GHRH perikarya received contacting somatostatinergic axonal varicosities in the arcuate nucleus. In contrast, GHRH axonal varicosities rarely contacted somatostatinergic perikarya. The morphology and the abundance of somatostatin to GHRH juxtapositions indicate that these associations are functional synapses, and they represent, at least partially, the morphological substrate of the somatostatin-influenced GHRH secretion. Thus, in addition to influencing the GH secretion directly via the hypophysial portal system, somatostatin may also modulate GH release from the anterior pituitary by regulating the hypothalamic GHRH secretion via direct contacts. The rare GHRH to somatostatin juxtapositions indicate that the negative feedback effect of GH targets the somatostatinergic system directly and not via the GHRH system.

Intimate associations between the endogenous opiate systems and the growth hormone-releasing hormone system in the human hypothalamus.

Although it is a general consensus that opioids modulate growth, the mechanism of this phenomenon is largely unknown. Since endogenous opiates use the same receptor family as morphine, these peptides may be one of the key regulators of growth in humans by impacting growth hormone (GH) secretion, either directly, or indirectly, via growth hormone-releasing hormone (GHRH) release. However, the exact mechanism of this regulation has not been elucidated yet. In the present study we identified close juxtapositions between the enkephalinergic/endorphinergic/dynorphinergic axonal varicosities and GHRH-immunoreactive (IR) perikarya in the human hypothalamus. Due to the long post mortem period electron microscopy could not be utilized to detect the presence of synapses between the enkephalinergic/endorphinergic/dynorphinergic and GHRH neurons. Therefore, we used light microscopic double-label immunocytochemistry to identify putative juxtapositions between these systems. Our findings revealed that the majority of the GHRH-IR perikarya formed intimate associations with enkephalinergic axonal varicosities in the infundibular nucleus/median eminence, while endorphinergic-GHRH juxtapositions were much less frequent. In contrast, no significant dynorphinergic-GHRH associations were detected. The density of the abutting enkephalinergic fibers on the surface of the GHRH perikarya suggests that these juxtapositions may be functional synapses and may represent the morphological substrate of the impact of enkephalin on growth. The small number of GHRH neurons innervated by the endorphin and dynorphin systems indicates significant differences between the regulatory roles of endogenous opiates on growth in humans.

Distribution and morphology of the catecholaminergic neural elements in the human hypothalamus.

Previous studies have demonstrated that catecholaminergic, tyrosine hydroxylase (TH)-immunoreactive (IR) perikarya and fibers are widely distributed in the human hypothalamus. Since TH is the key and rate-limiting enzyme for catecholaminergic synthesis, these IR neurons may represent dopaminergic, noradrenergic or adrenergic neural elements. However, the distribution and morphology of these neurotransmitter systems in the human hypothalamus is not entirely known. Since the different catecholaminergic systems can be detected by identifying the neurons containing the specific key enzymes of catecholaminergic synthesis, in the present study we mapped the catecholaminergic elements in the human hypothalamus using immunohistochemistry against the catecholaminergic enzymes, TH, dopamine beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT). Only a few, PNMT-IR, adrenergic neuronal elements were found mainly in the infundibulum and the periventricular zone. DBH-IR structures were more widely distributed in the human hypothalamus occupying chiefly the infundibulum/infundibular nucleus, periventricular area, supraoptic and paraventricular nuclei. Dopaminergic elements were detected by utilizing double label immunohistochemistry. First, the DBH-IR elements were visualized; then the TH-IR structures, that lack DBH, were detected with a different chromogen. In our study, we conclude that all of the catecholaminergic perikarya and the majority of the catecholaminergic fibers represent dopaminergic neurons in the human hypothalamus. Due to the extremely small number of PNMT-IR, adrenergic structures in the human hypothalamus, the DBH-IR fibers represent almost exclusively noradrenergic neuronal processes. These findings suggest that the juxtapositions between the TH-IR and numerous peptidergic systems revealed by previous reports indicate mostly dopaminergic synapses.

Catecholaminergic input to the oxytocin neurosecretory system in the human hypothalamus.

Previous studies revealed that oxytocin release is increased by various forms of stress. Hypertonic saline injection, immobilization, and several other stressors elevated the blood level of oxytocin in rats. However, the mechanism of the stress-induced oxytocin release in human is not elucidated yet. Although numerous studies indicate that catecholamines play a pivotal role in modulating the release of oxytocin, there is a lack of data regarding the morphological substrate of this phenomenon. In order to reveal putative juxtapositions between tyrosine hydroxylase-immunoreactive (TH-IR) catecholaminergic and the oxytocinergic systems in the human hypothalamus, we utilized double-label immunohistochemistry in the present study. Numerous TH-IR axon varicosities abutted on oxytocin-IR neurons in the supraoptic and paraventricular nuclei, forming synapse-like associations. Close examination of these juxtapositions with high magnification failed to reveal any gaps between the contacting elements. In summary, the intimate associations between the TH-IR and oxytocin-IR elements may be functional synapses and may represent the morphological substrate of stress-influenced oxytocin release. The finding that several oxytocin-IR perikarya did not receive apparent TH innervation suggests that additional mechanisms may play significant roles in the oxytocin modulation by stressors.

Associations between the human growth hormone-releasing hormone- and neuropeptide-Y-immunoreactive systems in the human diencephalon: a possible morphological substrate of the impact of stress on growth.

Previous studies revealed that stress is a pivotal factor in the regulation of growth. Psychological harassment may result in psychosocial dwarfism with delayed puberty, short stature and depression. Growth hormone (GH) secretion is suppressed by stress, possibly via the attenuation of growth hormone-releasing hormone (GHRH) secretion. However, the morphological substrate of this phenomenon has not been elucidated yet. Since neuropeptide Y (NPY) levels in the plasma is increased by administration of various stressors, the common consensus is that NPY plays a crucial role in the stress response. In the present study, we examined the putative juxtapositions between the NPY- and GHRH-immunoreactive (IR) systems in the human hypothalamus using double-label immunohistochemistry. Our findings revealed that the majority of the GHRH-IR perikarya formed intimate associations with NPY-IR fiber varicosities. The majority of these juxtapositions were found in the infundibular nucleus/median eminence where NPY-IR fiber varicosities often covered a significant surface area of the GHRH neurons. Since the juxtapositions between the GHRH-IR perikarya and NPY-IR fiber varicosities may be functional synapses, they may represent the morphological substrate of stress-suppressed GH secretion. The large number of contacting elements indicates that NPY plays a pivotal role in GH release, and may be considered as a major factor in the attenuation of growth by stress in humans.

Gonadotropin-releasing hormone neurons express estrogen receptor-beta.

CONTEXT: Recent identification of the second estrogen receptor (ER) isoform (ER-beta) within GnRH neurons of the rodent brain has generated much enthusiasm in the field of neuroendocrine research by questioning the dogma that GnRH cells do not directly sense changes in circulating estrogens. OBJECTIVE: To address the issue of whether GnRH neurons of the human hypothalamus also contain ER-beta, we have performed dual-label immunocytochemical studies. DESIGN: Tissue sections were prepared from autopsy samples of male human individuals (n = 8; age < 50 yr), with sudden causes of death. Technical efforts were made to minimize postmortem interval (<24 h), optimize tissue fixation (use of a mixture of 2% paraformaldehyde and 4% acrolein for four tissue samples), and sensitize the immunocytochemical detection of ER-beta (application of silver-intensified nickel-diaminobenzidine chromogen). MAIN OUTCOME MEASURE: Distribution and percent ratio of GnRH neurons that also contained ER-beta immunoreactivity were analyzed under the light microscope. RESULTS: With acrolein in tissue fixative, nuclear ER-beta immunoreactivity was observed in 10.8-28.0% of GnRH neurons of the four different individuals. ER-beta-containing GnRH neurons were widely distributed in the hypothalamus, without showing a noticeable preference in regional location. CONCLUSIONS: The demonstration of ER-beta and the previous lack of detection of ER-alpha in human GnRH cells indicate that estrogens may exert direct actions upon GnRH neurons exclusively through ER-beta. In the light of differing ligand-binding characteristics of ER-beta from those of ER-alpha, this discovery offers a potential new approach to influence estrogen feedback to GnRH neurons through ER-beta-selective receptor ligands.

Morphological substrate of the catecholaminergic input of the vasopressin neuronal system in humans.

It has been postulated that the stress response is associated with water balance via regulating vasopressin release. Nausea, surgical stress and insulin-induced hypoglycaemia were shown to stimulate vasopressin secretion in humans. Increased vasopressin release in turn induces water resorption through the kidneys. Although the mechanism of the stress-mediated vasopressin release is not entirely understood, it is generally accepted that catecholamines play a crucial role in influencing water balance by modulating the secretion of vasopressin. However, the morphological substrate of this modulation has not yet been established. The present study utilised double-label immunohistochemistry to reveal putative juxtapositions between tyrosine hydroxylase (TH)-immunoreactive (IR) catecholaminergic system and the vasopressin systems in the human hypothalamus. In the paraventricular and supraoptic nuclei, numerous vasopressin-IR neurones received TH-IR axon varicosities. Analysis of these juxtapositions with high magnification combined with oil immersion did not reveal any gaps between the contacted elements. In conclusion, the intimate associations between the TH-IR and vasopressin-IR elements may be functional synapses and may represent the morphological basis of vasopressin release modulated by stressors. Because certain vasopressin-IR perikarya receive no detectable TH innervations, it is possible that additional mechanisms may participate in the stress-influenced vasopressin release.

Three-dimensional representation of the neurotransmitter systems of the human hypothalamus: inputs of the gonadotrophin hormone-releasing hormone neuronal system.

The gonadotrophin-releasing hormone (GnRH) represents the final common pathway of a neuronal network that integrates multiple external and internal factors to control fertility. Among the many inputs GnRH neurones receive, oestrogens play the most important role. In females, oestrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurones to generate the preovulatory luteinising hormone surge and ovulation. Until recently, the belief has been that the GnRH neurones do not contain oestrogen receptors and that the action of oestrogen upon GnRH neurones is indirect, involving several, oestrogen-sensitive neurotransmitter and neuromodulator systems that trans-synaptically regulate the activity of the GnRH neurones. Although this concept still holds for humans, recent studies indicate that oestrogen receptor-beta is expressed in GnRH neurones of the rat. This review provides three dimensional stereoscopic images of GnRH-immunoreactive (IR) and some peptidergic (neuropeptide Y-, substance P-, beta-endorphin-, leu-enkaphalin-, corticotrophin hormone-releasing- and galanin-IR) and catecholaminergic neurones and the communication of these potential oestrogen-sensitive neuronal systems with GnRH neurones in the human hypothalamus. Because the post-mortem human tissue does not allow the electron microscopic identification of synapses on GnRH neurones, the data presented here are based on light microscopic immunocytochemical experiments using high magnification with oil immersion, semithin sections or confocal microscopy.

Bi-directional associations between galanin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon.

Evidence suggests that galanin plays an important role in the regulation of reproduction in the rat. Galanin is colocalized with luteinizing hormone (LH)-releasing hormone (LHRH) in a subset of LHRH neurons in female rats and galanin-immunoreactive (galanin-IR) nerve terminals innervate LHRH neurons. Recent studies indicate that galanin may control gonadal functions in rats at two different levels: (i) via direct modulation of pituitary LH secretion and/or (ii) indirectly via the regulation of the hypothalamic LHRH release. However, the morphological substrate of any similar modulation is not known in human. In the present series of experiments we first mapped the galanin-IR and LHRH-IR neural elements in human brain, utilizing single label immunohistochemistry. Then, following the superimposition of the maps of these systems, the overlapping sites were identified with double labeling immunocytochemistry and examined in order to verify the putative juxtapositions between galanin-IR and LHRH-IR structures. LHRH and galanin immunoreactivity were detected mainly in the medial basal hypothalamus, in the medial preoptic area and along the diagonal band of Broca. Careful examination of the IR elements in the overlapping areas revealed close, bi-directional contacts between galanin-IR and LHRH-IR structures, which have been verified in semithin plastic sections. These galanin-LHRH and LHRH-galanin juxtapositions were most numerous in the medial preoptic area and in the infundibulum/median eminence of the human diencephalon. In conclusion, the present study is the first to reveal bi-directional juxtapositions between galanin- and LHRH-IR neural elements in the human diencephalon. These galanin-LHRH and LHRH-galanin contacts may be functional synapses, and they may be the morphological substrate of the galanin-controlled gonadal functions in humans.

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.

The effects of progesterone on oxytocin mRNA levels in the paraventricular nucleus of the female rat can be altered by the administration of diazepam or RU486.

Oxytocin (OT) facilitates the onset of maternal behaviour in the late pregnant rat, enhances uterine contractility at parturition, and elicits milk ejection during lactation. If the rising estradiol (E2 and declining progesterone (P) of late pregnancy is reproduced in a virgin ovariectomized rat by implanting E2- and P-filled capsules for 2 weeks followed by removal of P-containing implants 36-48 h prior to death, OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of the rat. Both E2 administration and P withdrawal are necessary to increase OT mRNA, but the mechanisms of these effects are not understood. P may work within the PVN although P receptors are reported to be sparse or non-existent in the PVN or outside the PVN on PR-containing neurones that project to OT-containing neurones or via membrane bound receptors that are known to bind neurosteroids and gamma aminobutyric acid (GABA). To determine the mechanism through which P may inhibit or P withdrawal may increase OT mRNA levels, virgin ovariectomized (OVX) rats received sequential E2 and P via Silastic implants for 14 days. On day 13, prior to removal of P capsules on day 14, the rats were given the benzodiazepine agonist, diazepam, or saline injections subcutaneously (s.c.) twice daily until death on day 16. OT mRNA levels were increased in the steroid-treated group that received saline but not diazepam. In experiment 2, P capsules were removed on day 14 or pharmacological P withdrawal was induced by injecting RU486 injections s.c. twice daily until death 48 h later. OT mRNA levels were increased in the steroid-treated group that received RU486. Subsequent studies demonstrated the expression of PR mRNA within the rat PVN. The data suggest that gonadal steroids may influence PVN OT mRNA levels by modulating the GABA(A) receptor or by directly altering gene transcription via the PR.

Expression of growth differentiation factor-9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues.

Growth differentiation factor-9 (GDF-9) is a member of the transforming growth factor-beta family that is reported to be expressed exclusively in the ovary, specifically in the oocyte. Female mice deficient in GDF-9 are infertile, suggesting that GDF-9 receptor agonists and antagonists may specifically modulate fertility. We now report that GDF-9 messenger RNA (mRNA) is expressed in nonovarian tissues in mice, rats, and humans. GDF-9 mRNA was detected in mouse and rat ovary, testis, and hypothalamus by Northern blot and RT-PCR analyses. The localization of GDF-9 mRNA specifically in oocytes of the mouse ovary was confirmed by in situ hybridization histochemistry. In mouse testis, although localization in Sertoli cell cytoplasm could not be ruled out, mRNA expression was observed in large pachytene spermatocytes and round spermatids. The expression of GDF-9 mRNA in human tissues was assessed by Northern blot and RT-PCR analyses. GDF-9 mRNA was observed in ovary and testis and, surprisingly, in diverse nongonadal tissues, including pituitary, uterus, and bone marrow. Therefore, GDF-9 mRNA expression in rodents is not exclusive to the ovary, but includes the testis and hypothalamus. Furthermore, human GDF-9 mRNA is expressed not only in the gonads, but also in several extragonadal tissues. The function and relevance of nongonadal GDF-9 mRNA are not known, but may affect strategies for contraception and fertility that are based on GDF-9 activity.

The distribution of estrogen receptor-beta mRNA in the rat hypothalamus.

The recent cloning of a second form of the estrogen receptor (ER-beta) has made it possible to map the distribution of ER-beta mRNA-containing perikarya in the rat hypothalamus. The present in situ hybridization histochemical studies have detected ER-beta mRNA in the medial preoptic area; the anterior periventricular, paraventricular, supraoptic, arcuate, medial tuberal and medial mammillary nuclei; the bed nucleus of the stria terminals, and zona incerta. As previously described for the classical ER (ER-alpha) mRNA, a dense accumulation of ER-beta mRNA-expressing perikarya is present in the medial preoptic area and bed nucleus of the stria terminalis. In contrast, ER-beta mRNA was also concentrated in the paraventricular and supraoptic nuclei, brain regions which contain little or no ER-alpha mRNA. Moreover, the arcuate and ventromedial nuclei, areas with abundant ER-alpha. contain only a weak level of ER-beta hybridization signal. The description of ER-beta mRNA-containing perikarya in the rat hypothalamus provides a foundation for further morphological and physiological studies aimed at elucidating the role of ER-beta in the hypothalamus.

Glucagon-like peptide-1 receptor (GLP1-R) mRNA in the rat hypothalamus.

GLP-1 has been shown to dramatically reduce food intake in fasted rats and is thought to exert its effects by modulating neuronal function in the hypothalamus. To date, little is known about the distribution of GLP1-R and its mRNA in the rodent hypothalamus. The purpose of the present study was to utilize in situ hybridization histochemistry to determine the anatomical distribution of GLP1-R mRNA in the rat hypothalamus. The results of these studies revealed an extensive distribution of GLP1-R mRNA throughout the rostral-caudal extent of the hypothalamus; with a dense accumulation of labeled cells in the supraoptic, paraventricular, and arcuate nuclei. Additional labeled cells were also detected in medial and lateral preoptic areas, periventricular nucleus, ventral division of the bed nucleus of the stria terminalis, lateral hypothalamus, and dorsomedial nucleus. The results of these in situ hybridization histochemical studies have provided detailed and novel information about the distribution of GLP1-R mRNA in the rat hypothalamus. In addition, this morphological data provides important information about the neuronal systems modulated by GLP-1 and their potential role in feeding behavior.

Induction of proenkephalin in tuberoinfundibular dopaminergic neurons by hyperprolactinemia: the role of sex steroids.

The observation that tuberoinfundibular dopaminergic (TIDA) neurons of pregnant, pseudopregnant, lactating, and aged rats express enkephalins suggested that chronically elevated PRL levels, which are characteristic for these animals, are essential for the induction of proenkephalin gene expression in TIDA neurons. The present studies investigated further the role of PRL in this phenomenon. Elevated PRL levels were achieved either experimentally by implanting anterior pituitaries under the kidney capsule of intact or hypophysectomized female rats or by using lactating rats. For controls, the elevated PRL levels were reduced with bromocryptine, a dopamine receptor agonist. The role of sex steroids in PRL-induced enkephalin gene expression was also studied in cycling, sex hormone-treated, hypophysectomized or ovariectomized rats, pituitary-implanted/sex hormone-treated rats, and ovariectomized mothers. Enkephalin immunoreactivity was detected by immunocytochemistry and enkephalin messenger RNA with in situ hybridization histochemistry using 35S- or digoxigenin-labeled riboprobes. Enkephalin or its messenger RNA was present in TIDA neurons in all experimental animals with elevated PRL levels. Although estradiol had no or only a minor effect on PRL-induced enkephalin gene expression, progesterone supported the effect of PRL. The present observations suggest that the expression of enkephalin in TIDA neurons is PRL dependent and supported by sex steroids, primarily progesterone.

Induction of enkephalin in tuberoinfundibular dopaminergic neurons of pregnant, pseudopregnant, lactating and aged female rats.

The tuberoinfundibular dopaminergic (TIDA) neurons projecting to the external zone of the median eminence arise in the dorsomedial and ventrolateral subdivisions of the arcuate nucleus. In cycling female rats these regoins contain only scattered enkephalin-immunoreactive (ENK-i) neurons some of which coexpress dopamine, detected by immunostaining for tyrosine hydroxylase (TH). The present immunocytochemical, in situ hybridization and retrograde-labeling studies show that each TIDA neuron of pregnant, pseudopregnant, lactating, and aged female rats contains ENK-like immunoreactivity and pro-ENK mRNA and projects to the hypophysial portal circulation. Ovariectomy of lactating and aged rats did not change ENK staining within TIDA neurons, suggesting that ovarian steroids do not play a critical role in the colocalization of ENK and dopamine. Since prolactin levels are elevated in each of these experimental animals, a possible role for prolactin in the induction of the ENK gene in TIDA neurons is suggested. Prolactin stimulates dopamine and its own secretion via a short-loop feedback mechanism. The sensitivity of this regulatory mechanism is altered in these experimental animals, resulting in elevated prolactin secretion. ENK, which has prolactin-releasing activity and is colocalized with dopamine, could mediate the positive short-loop feedback regulation and sustain elevated levels of prolactin in pregnant, pseudopregnant, lactating, and aged animals.

Mapping of thyrotropin-releasing hormone (TRH) neuronal systems of rat forebrain projecting to the median eminence and the OVLT. Immunocytochemistry combined with retrograde labeling at the light and electron microscopic levels.

The thyrotropin-releasing hormone (TRH)-containing neurons that project to portal capillaries of the external zone of the median eminence (ME) and fenestrated capillaries of the organum vasculosum of the lamina terminalis (OVLT) were identified on thin paraffin and thick vibratome sections using a combination of retrograde labeling with peripherally administered Fluoro-Gold and fluorescence immunocytochemistry. The results indicate that the vast majority of those TRH neurons that project to the ME and the OVLT is located in the paraventricular nucleus (PVN), and most abundantly, in its medial parvicellular subdivision. Although numerous TRH-immunoreactive (TRH-i) neurons are present in other hypothalamic areas of the brain, only a few of them in the dorsal hypothalamic area behind the PVN and the periventricular preoptic nucleus could be retrogradely labeled. Since only a few Fluoro-Gold-accumulating and TRH-i perikarya were seen in other nuclei than the PVN, it is likely that the majority of nerve terminals in the OVLT also originates from TRH-i perikarya in the PVN. Fluoro-Gold, an electron-dense substance, is stored in the lysosomes of hypophysiotropic TRH-i perikarya and thus, it provides an excellent model for electron microscopic characterization of hypophysiotropic neurons at both the light and electron microscopic levels. The data together provides additional morphological evidence for the key role of the PVN in the regulation of TSH secretion.

Induction of enkephalin in tuberoinfundibular dopaminergic neurons during lactation.

Single- and double-labeling immunocytochemistry was used to demonstrate that the tuberoinfundibular dopaminergic (TIDA) neurons of lactating rats, in contrast to those of male or female rats on any day of the estrous cycle, synthesize enkephalin which is colocalized with dopamine. Each enkephalin-immunopositive perikaryon in the dorsomedial and ventrolateral subdivisions of the arcuate nucleus contains dopamine; therefore, the median eminence of lactating rats contains high levels of enkephalin compared to male or female rats. It has been shown that endogenous opiates, including enkephalin, stimulate PRL secretion by reversing the inhibitory action of dopamine at the level of the TIDA neurons. The present findings suggest that enkephalin, coproduced with dopamine in TIDA neurons of lactating rats, may be an endogenous source for this action and maintain elevated PRL and milk secretion during the nonsuckling periods of lactation.

Corticotropin-releasing hormone neurons in the paraventricular nucleus project to the external zone of the median eminence: a study combining retrograde labeling with immunocytochemistry.

Corticotropin-releasing hormone (CRH) is the major regulator of the pituitary-adrenal axis. CRH-immunoreactive perikarya are widely distributed in the central nervous system; however, only those which participate directly in the regulation of adrenocorticotropin are connected to the portal circulation in the external zone of the median eminence. The present study describes the identification of these hypophysiotropic neurons using retrograde labeling and CRH immunocytochemistry. Fluoro-Gold was injected peripherally then, 5 days later, the animals were treated with colchicine. Twenty-four hours later the animals were sacrificed, and their brains were immunostained for CRH with the indirect immunofluorescence technique. The results indicate that the vast majority of the Fluoro-Gold-accumulating and CRH-immunopositive perikarya (hypophysiotropic neurons) are located in the medial parvicellular subdivision of the paraventricular nucleus (PVN). However, not each CRH-immunoreactive neuron contains Fluoro-Gold, i.e. a small portion of these neurons project to areas of the brain other than the median eminence. The anterior, lateral and periventricular subdivisions of the PVN also contain hypophysiotropic CRH-immunoreactive perikarya, however, their number is much less than in the medial parvicellular subdivision. Scattered double-labeled cells are also present in the medial preoptic area and the dorsal hypothalamus, just behind the PVN. These results support previous observations that the PVN, particularly the medial parvicellular subdivision, is the predominant source of the hypophysiotropic CRH neurons.

Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons.

Mammalian reproduction is dependent upon intermittent delivery of luteinizing hormone-releasing hormone (LHRH) to the anterior pituitary. This mode of secretion is required to sensitize maximally the gonadotrophs to LHRH stimulation and to regulate gonadotropin gene expression. While LHRH secretion is pulsatile in nature, the origin of the pulse generator is unknown. In this report, we show that this oscillator could be located within the LHRH neuronal network. When immortalized LHRH neurons are placed into a perifusion system, LHRH is secreted into the medium in a pulsatile fashion under basal conditions. LHRH secretion and the number of LHRH pulses are reduced when calcium is removed from the medium. Perifusion also influences pro-LHRH processing, since the molar ratio of its processed products varies dramatically when the cells are transferred from a static system. Several different cellular mechanisms may underlie these changes in secretion and processing. Lucifer yellow experiments reveal that some cells are dye-coupled. Hence, these cells could be electrically coupled through gap junctions such that secretion from individual cells could be coordinated. Secretion could also be synchronized through the observed synapse-like contacts. These contacts could perform a negative-feedback role to regulate not only the amount of LHRH released but also the molecular forms secreted. The organization of LHRH neurons into interconnected clusters could serve to coordinate LHRH secretion from individual cells and, thereby, orchestrate functions in vivo as diverse as the onset of puberty, the timing of ovulation, and the duration of lactational infertility.