Rat Median Eminence Research Articles

Effects of Chronic NMDA‐NR2b Inhibition in the Median Eminence of the Reproductive Senescent Female Rat

Gonadotrophin-releasing hormone (GnRH) neurones of the hypothalamic-pituitary-gonadal (HPG) axis drive reproductive function and undergo age-related decreases in activation during the transition to reproductive senescence. Decreased GnRH secretion from the median eminence (ME) partially arises from attenuated glutamatergic signalling via the NMDA receptor (NMDAR) and may be a result of changing NMDAR stoichiometry to favour NR2b over NR2a subunit expression with ageing. We have previously shown that the systemic inhibition of NR2b-containing receptors with ifenprodil, an NR2b-specific antagonist, stimulates parameters of luteinising hormone (used as a proxy for GnRH) release in both young and middle-aged females. In the present study, we chronically administered ifenprodil, an NR2b-specific antagonist, at the site of GnRH terminals in the ME or at GnRH perikarya in the preoptic area, in reproductively senescent middle-aged female rats, aiming to determine whether NR2b antagonism could restore aspects of reproductive functionality. Effects on oestrous cyclicity, serum hormones, and protein expression of GnRH, NR2b and phosphorylated NR2b (Tyr-1472) in the ME were measured. Chronic ifenprodil treatment in the ME (but not the preoptic area) altered oestrous cyclicity by increasing the percentage of days spent in pro-oestrus. This was accompanied by increased GnRH fluorescence intensity in the external ME zone and a greater proportion of GnRH terminals that co-labelled with pNR2b with treatment. We also observed changes in the relationships between protein immunofluorescence, serum hormone levels and other aspects of reproductive physiology in acyclic females, as revealed by bionetwork analysis. Together, these data support the hypothesis that NMDAR-NR2b expression and phosphorylation state play a role in reproductive senescence and highlight the ME as a major player in reproductive ageing.

A Novel Pathway Regulates Thyroid Hormone Availability in Rat and Human Hypothalamic Neurosecretory Neurons

Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.

Differential co-localization with choline acetyltransferase in nervus terminalis suggests functional differences for GnRH isoforms in bonnethead sharks ( Sphyrna tiburo)

The nervus terminalis (NT) is a vertebrate cranial nerve whose function in adults is unknown. In bonnethead sharks, the nerve is anatomically independent of the olfactory system, with two major cell populations within one or more ganglia along its exposed length. Most cells are immunoreactive for either gonadotropin-releasing hormone (GnRH) or RF-amide-like peptides. To define further the cell populations and connectivity, we used double-label immunocytochemistry with antisera to different isoforms of GnRH and to choline acetyltransferase (ChAT). The labeling patterns of two GnRH antisera revealed different populations of GnRH-immunoreactive (ir) cell profiles in the NT ganglion. One antiserum labeled a large group of cells and fibers, which likely contain mammalian GnRH (GnRH-I) as described in previous studies and which were ChAT immunoreactive. The other antiserum labeled large club-like structures, which were anuclear, and a sparse number of fibers, but with no clear labeling of cell bodies in the ganglion. These club structures were choline acetyltrasferase (ChAT)-negative, and preabsorption control tests suggest they may contain chicken-GnRH-II (GnRH-II) or dogfish GnRH. The second major NT ganglion cell-type was immunoreactive for RF-amides, which regulate GnRH release in other vertebrates, and may provide an intraganglionic influence on GnRH release. The immunocytochemical and anatomical differences between the two GnRH-immunoreactive profile types indicate possible functional differences for these isoforms in the NT. The club-like structures may be sites of GnRH release into the general circulation since these structures were observed near blood vessels and resembled structures seen in the median eminence of rats.

Alteration by Thyroid Hormones of TRH in the Rat Median Eminence: Role of the Hypothalamic Paraventricular Nucleus

We have shown that hypothyroidism produced a progressive decrease in the TRH content of the hypothalamic median eminence. In the present study, we explored whether thyroid hormones might alter TRH content in the median eminence by acting on the hypothalamic paraventricular nucleus (PVN) in the rat. After thyroidectomy, adult male rats received a hypothalamic deafferentation or electrolytic lesions in the bilateral PVNs. These rats were i.p. administered daily with T4 in a dose of 1.5 micrograms per 100 g body weight. Two weeks later, these rats were decapitated, and TRH in the median eminence and TSH in the peripheral blood were determined by RIAs. Thyroidectomy caused a significant reduction in TRH content of the median eminence. The hypothalamic deafferentation and PVN-lesions further reduced the TRH content. T4-replacement restored the decreased content of median eminence TRH to the normal level. The hypothalamic deafferentation and PVN-lesions prevented T4-induced restoration of TRH content. The present data imply that thyroid hormones regulate the amount of THR in the median eminence through acting on the hypothalamic PVN.

Hypocretin/Orexin: A Molecular Link Between Sleep, Energy Regulation, and Pleasure

Hypocretin (Hcrt) is a neurotransmitter of the dorsal and lateral hypothalamus that regulates sleep, appetite, and energy consumption. Recent evidence indicates that it is also involved in pleasure/reward-seeking. Mutation of the Hcrt-receptor gene causes narcolepsy in canines, and Hcrt knockout mice exhibit narcolepsy-like symptoms. Human narcoleptics do not commonly have mutations in the ligand or receptor but do have degeneration of Hcrt-containing neurons, possibly through an autoimmune mechanism. When Hcrt neurons degenerate in mice, hypophagia and obesity are observed, symptoms that are also present in some human narcoleptics. This article reviews the recent literature with regard to the many functions of this single molecule. The authors suggest that eating habits and impulsivity may be topics worth exploring in the evaluation of narcoleptic patients.

Multiple signaling pathways involved in the effect of endothelin type b receptor in rat median eminence

We assessed the possible link between endothelin receptor mediated phosphoinositide breakdown and NO/cGMP signaling pathways in rat arcuate nucleus-median eminence fragments (AN-ME), brain structures known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers, together with densely arranged endothelin ETB-receptors-like immunoreactive fibres. Our data show that ET-1, ET-3 and the ETB-receptors agonist, IRL 1620, increased inositol monophosphate (InsP1) accumulation, NOS activity and cGMP formation, in a similar degree. The stimulatory effect of ETs on InsP1 accumulation and cGMP formation was inhibited by the phospholipase C (PLC) inhibitor, neomycin, and the absence of extracellular calcium, suggesting that calcium is involved in endothelin receptor-induced PLC activation. The L-arginine analog, L-NAME, inhibited ET-1 or IRL1620-stimulated cGMP formation. The ETA receptor antagonists BQ 123, did not alter, while the ETB receptor antagonists BQ788 inhibited ETs-induced increase in the PI metabolism, NOS activity and cGMP generation. Our data indicate that in AN-ME, ETB receptor signals through receptor-mediated calcium dependent-stimulation of phosphoinositide breakdown and activation of NOS/cGMP signaling pathway.

Heterogeneous occurrence of aquaporin-4 in the ependyma and in the circumventricular organs in rat and chicken

Aquaporins are selective water channel proteins critical in volume homeostasis. In the CNS AQP4 predominates, localized mainly in the glia limitans, the perivascular endfeet and ependyma. The present immunofluorescent study reveals the distribution of aquaporin-4 in the circumventricular organs in rat and chicken brains. The ventricular ependyma (especially in the third one), the subfornical organ, the area postrema, the rat pineal body (in part), and the vascular organ of lamina terminalis were marked by intense immunopositivity. Several areas, however, proved to be immunonegative: the central canal, the subcommissural organ, the ependymal zone of the median eminence in rat but its whole thickness in chicken, the subtrochlear organ, and the paraventricular organ. The immunostaining of the lateral septal and subseptal organs were similar to their environment. Results on developing rats suggested that the aquaporin-4 immunonegativity is a secondary phenomenon. Surveying other structural and functional features, no clear explanation of the heterogeneous occurrence of aquaporin-4 was found. The absence of aquaporin-4 seems to correlate with some features of the "ependymal organs" (thickened, pseudostratified ependyma, presence of blood-brain barrier) and with the avoidance of GFAP. On the other hand, the organs rich in aquaporin-4 have features of the "hypendymal organs" (glial and vascular plexus but no blood-brain barrier). There are organs, however, which do not fit into either group completely, i.e. the lateral septal and subseptal organs. Presence of tight junctions coincides with the absence of aquaporin-4 in the ependyma of spinal cord, the subcommissural organ and the ependyma of median eminence.

Morphologic evidence that neurokinin B modulates gonadotropin‐releasing hormone secretion via neurokinin 3 receptors in the rat median eminence

Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual-label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin-releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB-immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH-ir somata exhibited NK3R staining. ProNKB and NK3R-ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB-ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis.

Changes of melanin-concentrating hormone related to LHRH release in the median eminence of rats

The present research was carried out to study the distribution of melanin-concentrating hormone (MCH) fibers in the median eminence of rats and to evaluate if changes in the MCH content of the median eminence could be related to the release of LHRH. Immunocytochemical studies in the median eminence of males and estrous females showed the presence of MCH fibers, mainly in its internal layer. Diestrous and proestrous animals displayed MCH immunoreactivity in both the internal and external layers of the median eminence. Longitudinal sections of the median eminence in proestrous animals showed that MCH-immunoreactive (ir) density is higher at 12 than at 9 h in both layers of the median eminence. MCH was assayed by radioimmunoassay in median eminences of males and in females in all stages of the estrous cycle at 10 h. It was observed that the content of MCH at diestrus-1 and -2 was higher than in estrus and in male rats. In the day of proestrus, MCH and LHRH were assayed at 10, 12, 13, 14, 15 and 17 h. At 12 h, the content of MCH and LHRH showed the maximal values. At 13 h, MCH content showed a decline, while LHRH was still high. At 14 h, the LHRH content started to decrease. The present results suggest that MCH is involved in the regulation of LHRH release in the female rat.

Autoantibodies against alpha -MSH, ACTH, and LHRH in anorexia and bulimia nervosa patients.

The hypothalamic arcuate nucleus is involved in the control of energy intake and expenditure and may participate in the pathogenesis of eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN). Two systems are of particular interest in this respect, synthesizing alpha-melanocyte-stimulating hormone (alpha-MSH) and synthesizing neuropeptide Y, respectively. We report here that 42 of 57 (74%) AN andor BN patients studied had in their plasma Abs that bind to melanotropes andor corticotropes in the rat pituitary. Among these sera, 8 were found to bind selectively to alpha-MSH-positive neurons and their hypothalamic and extrahypothalamic projections as revealed with immunostaining on rat brain sections. Adsorption of these sera with alpha-MSH peptide abolished this immunostaining. In the pituitary, the immunostaining was blocked by adsorption with alpha-MSH or adrenocorticotropic hormone. Additionally, 3 ANBN sera bound to luteinizing hormone-releasing hormone (LHRH)-positive terminals in the rat median eminence, but only 2 of them were adsorbed with LHRH. In the control subjects, 2 of 13 sera (16%) displayed similar to ANBN staining. These data provide evidence that a significant subpopulation of ANBN patients have autoantibodies that bind to alpha-MSH or adrenocorticotropic hormone, a finding pointing also to involvement of the stress axis. It remains to be established whether these Abs interfere with normal signal transduction in the brain melanocortin circuitryLHRH system andor in other central and peripheral sites relevant to food intake regulation, to what extent such effects are related to andor could be involved in the pathophysiology or clinical presentation of ANBN, and to what extent increased stress is an important factor for production of these autoantibodies.

Antibodies obtained by xenotransplantation of organ-cultured median eminence specifically recognize hypothalamic tanycytes.

Tanycytes are specialized ependymal cells lining the infundibular recess of the third ventricle of the cerebrum. Early and recent investigations involve tanycytes in the mechanism of gonadotropin-releasing hormone (GnRH) release to the portal blood. The present investigation was performed to obtain a specific immunological marker of tanycytes and to identify the compound(s) responsible for this labeling. After 30 days of organ culture, explants of bovine median eminence formed spherical structures mostly constituted by tanycytes. These tanycyte spheres were xenotransplanted to rats, and the antibodies raised by the host animals against the transplanted living tanycytes were used for immunochemical studies of the bovine and rat median eminence. This antiserum immunoreacted with two compounds of 60 kDa and 85 kDa present in extracts of bovine and rat median eminence. The individual immunoblotting analysis of rat medial basal hypothalami showed a decrease in the amount of the 85-kDa compound in castrated rats as compared to control rats processed at oestrus and dioestrus. The antiserum, labeled as anti-P85, when used for immunostaining of sections throughout the rat central nervous system, immunoreacted specifically with the hypothalamic tanycytes. Within tanycytes, P-85 immunoreactivity was exclusively present in the basal processes. It is suggested that the 85-kDa and 60-kDa compounds correspond to two novel proteins selectively expressed by tanycytes. The possibility that they are secretory proteins involved in GnRH release is discussed. Anti-P85 appears to be the first specific marker of hypothalamic tanycytes.

Role of endothelin type B receptor in NO/cGMP signaling pathway in rat median eminence.

We studied the effect of endothelins (ETs) on receptor-mediated NO/cGMP signaling in rat arcuate nucleus-median eminence (AN-ME) fragments, an hypothalamic structure known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers together with densely arranged ET(B)-receptor-like immunoreactive fibers. NOS activity was determined measuring the conversion of [3H] arginine to [3H] citrulline, as an index of NO produced. cGMP production was determined by radio immunoassay. ET-1, ET-3, and the selective ET(B) receptor agonist, IRL1620, significantly increased cGMP formation and NOS activity. Preincubation of AN-ME fragment with L-arginine analog, N-nitro-L-arginine (L-NAME), inhibited ET-1 or IRL1620-stimulated cGMP formation. The addition of theselective ET(B) receptor antagonist, BQ788, blocked ET-1-, ET-3-, or IRL1620-induced increase in NOS activity and cGMP generation, while BQ123, a selective ET(A) receptor antagonist, was ineffective. Our results demonstrate that in whole rat AN-ME fragments, ETs stimulate NO/cGMP signaling pathway through the interaction with the ET(B) receptor subtype, supporting the concept that ETs may represent an important regulator of reproductive and neuroendocrine function.

Involvement of NPY Y2 receptor subtype in the control of the spontaneous NO/GnRH release at the rat median eminence

The role of nitric oxide (NO) from vascular endothelium in the control of GnRH release at the median eminence (ME) level is well established. Interactions between NPY receptor/endothelium/nitric oxide are clearly demonstrated. While several studies implicate NPY Y1 receptor in the control of GnRH/LH at the time of the preovulatory LH surge, our results also demonstrate the importance of NPY Y2 receptor in the control of GnRH release via endothelial NO. We conclude that NPY may be one of the elements implicated in the generation of the spontaneous NO/GnRH via Y2 receptor located on endothelium.

Characterization of GFAP expression and cell proliferation in the rat median eminence following hypophysectomy.

To analyze whether the reorganization of the rat median eminence after hypophysectomy might be related to changes in glial fibrillary acidic protein (GFAP)- and cellular proliferation, the distribution of cells immunoreactive for GFAP and the proliferation rate of such cells were analyzed at 20, 40 and 60 days posthypophysectomy. For this study, four rostro-caudal regions of the median eminence were differentiated: the retrochiasmatic, preinfundibular, infundibular and postinfundibular regions. In each of these regions, three layers were studied: the ependymal, the internal and the external. At 20 and 40 days after hypophysectomy, significant increases in cellular proliferation affecting all three layers studied in the preinfundibular and infundibular regions were found. At the same time points, increases in GFAP expression were also observed. However, after 60 days, GFAP and proliferative cellular nuclear antigen (PCNA) expression decreased. Although variations of PCNA and GFAP levels were evident, no colocalisation of PCNA and GFAP was found in the cells of the median eminence in untreated or hypophysectomized rats when sections were analyzed by double immunohistochemical staining. Our results suggest that reorganization of median eminence involves alterations (or modulation) of GFAP-immunoreactive cells together with a proliferation of cells that are not GFAP-immunoreactive. This study also demonstrates that this reorganization is completed within the first two months after hypophysectomy.

Variation of Endothelial Nitric Oxide Synthase Synthesis in the Median Eminence during the Rat Estrous Cycle: An Additional Argument for the Implication of Vascular Blood Vessel in the Control of GnRH Release

Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.

Variation of endothelial nitric oxide synthase synthesis in the median eminence during the rat estrous cycle: an additional argument for the implication of vascular blood vessel in the control of GnRH release.

Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.

Axonal projections from the hypothalamus to the median eminence in rats during ontogenesis: DiI tracing study

This study has determined the ontogenetic schedule of the arrival of the axons from the hypothalamus and the diagonal band in the median eminence in rats by using the fluorescent lipophilic carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as a retrograde tracer. After fixation of the brain, the crystals of the dye were implanted in the median eminence on the 13th, 14th, 15th, 16th, 17th, 20th embryonic days, and on the 2nd postnatal day. This was followed by fluorescent staining of the neuronal cell bodies in the hypothalamus. According to our data, the axons of rare hypothalamic neurons first reached the primordium of the median eminence on the 14th embryonic day. For two subsequent days, the number of neurons projecting the axons to the median eminence appeared to increase considerably. They were widely distributed through the hypothalamus and in the ventromedial region of the more rostral forebrain. Till the 20th embryonic day, the majority of the fluorescent neurons were concentrated mainly in the paraventricular nucleus (dorsal and medial parts) and the arcuate nucleus, and to a lesser extent in the medial preoptic nucleus, the supraoptic nucleus, the diagonal band, and the retrochiasmatic nucleus. In neonates, DiI-labelled neurons appeared additionally in the accessory dorsolateral nucleus, medial preoptic area lateral to the diagonal band, anterior hypothalamic area, and in the anterior periventricular nucleus. Thus, the axons of differentiating neurons arrive in the median eminence from the 14th embryonic day till the neonatal period, providing the pathway for the neurohormone transfer to the hypophysial portal circulation.

Presence of sst2(a) receptor immunoreactivity in rat ependyma and tanycytes.

Somatostatin sst2(a) receptor was observed, by immunofluorescence, in ependymal cells and in tanycytes of the wall of the ventricle and the hypothalamic recess of the male rat median eminence. Strong immunoreactivity for the receptor protein was observed in lateral tanycytes (alpha-type) while a moderate signal was seen in medial tanycytes (beta-type). In high magnification the immunoreactive material, of moderate intensity, had a coarse granular appearance. Only few of the alpha-tanycytes also displayed immunoreactive GFAP. The apical portion of the ependymal cells as well as of tanycytes contained immunoreactive S-100 (alphabeta). Since rather high levels of somatostatin are demonstrated to occur in the cerebrospinal fluid of the third ventricle, it is suggested that somatostatin via the sst2(a) receptor may regulate the physiology of tanycytes.

Evidence for a spontaneous nitric oxide release from the rat median eminence: influence on gonadotropin-releasing hormone release.

The involvement of nitric oxide (NO) as a gaseous neurotransmitter in the hypothalamic control of pituitary LH secretion has been demonstrated. NO, as a diffusible signaling gas, has the ability to control and synchronize the activity of the neighboring cells. NO is secreted at the median eminence (ME), the common termination field for the antehypophysiotropic neurons, under the stimulation of other signaling substances. At the ME, NO stimulates GnRH release from neuroendocrine terminals. The present studies were undertaken to determine whether NO is secreted spontaneously from ME fragments ex vivo and whether its secretion is correlated to GnRH release. To accomplish this, female rats were killed at different time points of the day and/or of the estrous cycle. The spontaneous NO release was monitored in real time, with an amperometric probe, during 4 periods of 30 min, from individual ME fragments (for each time point, n = 4). GnRH levels were measured in parallel for each incubation-period by RIA. The results revealed that NO was released in a pulsatile manner from female ME fragments and, unambiguously, that the amplitude of NO secretion varied markedly across the estrous cycle. Indeed, though the NO pulse period (32 +/- 1 min, n = 36) and duration (21 +/- 2 min, n = 36) did not vary significantly across the estrous cycle, the amplitude of this secretion pulse was significantly higher on proestrus (Pro; 39 +/- 3 nM, n = 20), compared with diestrus (16 +/- 1 nM, n = 8) or estrus (23 +/- 3 nM, n = 8, P < 0.05). The GnRH levels in the incubation medium were positively correlated to NO secretion across the estrous cycle (r = 0.86, P < 0.003, n = 9), confirming that NO and GnRH release are coupled. Furthermore, 5 x 10(-7) M L-N(5)-(1-iminoethyl)ornithine (L-NIO), a NO synthase inhibitor, succeeded in inhibiting the strong NO-GnRH secretory coupling and GnRH release on PRO: Because at this concentration, L-NIO selectively inhibits endothelial NO synthase, the results further demonstrate that the major source of NO involved in GnRH release at the ME is endothelial in origin. Additionally, the induction of a massive NO/GnRH release in 15-day ovariectomized rat treated with estradiol benzoate strongly suggested that estradiol is participating in the stimulation of NO release activity between diestrus II and PRO: The present study is the first demonstrating that ME can spontaneously release NO and that NO's rhythm of secretion varies markedly across the estrous cycle. This pulsatile/cyclic ME NO release may constitute the synchronizing link to anatomically scattered GnRH neurons.

Evidence for a Spontaneous Nitric Oxide Release from the Rat Median Eminence: Influence on Gonadotropin-Releasing Hormone Release

Evidence for a Spontaneous Nitric Oxide Release from the Rat Median Eminence: Influence on Gonadotropin-Releasing Hormone Release