Dibutyryl cyclic adenosine monophosphate stimulates in vitro luteinizing hormone-releasing hormone release only from median eminence derived from ovariectomized, estradiol-primed rats

The present study examined the effect of intermittent infusion of dibutyryl cyclic AMP (dbcAMP; 10(-7) M; 10 min on, 20 min off) on in vitro luteinizing hormone-releasing hormone (LH-RH) release from the rat median eminence (ME) derived from immature rats: intact females, intact males, ovariectomized (OVX) females, castrated (CAST) males, ovariectomized, estradiol primed (OVX + E2) females and castrated, estradiol primed (CAST + E2) males. In intact, OVX and CAST conditions, spontaneous LH-RH release from MEs was not modified by dbcAMP infusion. However, E2 implants in OVX and CAST rats selectively affected the responsiveness of MEs to dbcAMP: ME from OVX + E2 were highly responsive to dbcAMP; contrarily, MEs from CAST + E2 were unresponsive to this nucleotide. Therefore, these differences in MEs responsiveness to dbcAMP-induced LH-RH release appear to be dependent upon a critical effect of E2 priming on this tissue in female but not in male rats.     

Orphanin FQ stimulates prolactin and growth hormone release in male and female rats

Intracerebroventricular administration of Orphanin FQ (5.5, 55 or 550 pmol) caused a dose-related increase in prolactin secretion in both male and female rats and stimulated GH secretion in males. The magnitude of the prolactin secretory response was greater in females than in males. These effects of OFQ on prolactin and growth hormone release are the same as the stimulatory effects of the endogenous opioid peptides.     

Noradrenergic afferents to median eminence: Inhibitory role in rhythmic growth hormone secretion

The involvement of noradrenergic (NA) afferents to the median eminence (ME) in the regulation of growth hormone (GH) secretion was investigated in chronically cannulated, unanaesthetized male rats by using systematically administered 6-hydroxy-dopamine (6-OHDA).One day after 6-OHDA treatment (50 mg/kg, i.v.) GH peak frequency was substantially increased and examination of catecholamine (CA) fluorescence indicated disruption of CA innervation of the ME, but not other central nervous system (CNS) structures. Prolactin secretion at this time was normal and the administration of butaclamol (1.0 mg/kg, i.v.), a dopamine (DA) antagonist, was effective in increasing secretion, indicating that ME DA mechanisms were functionally intact following 6-OHDA treatment. GH secretory patterns returned to normal by 21 days following 6-OHDA treatment and this corresponded with re-emergence of a normal pattern of CA-fluorescence in the ME.In an additional study, the administration of the α-NA agonist clonidine (130 μg/kg, i.v.) increased GH secretion in previously untreated animals. Administration of the α-NA agonist oxymetazoline (45 μg/kg, i.v.), which does not readily pass the blood-brain barrier (BBB), suppressed GH secretion.These findings indicate that ME NA afferents are inhibitory to GH secretion and are a major determinant of the 3 h pattern of GH release in the rat. This inhibitory input is subsidiary to a NA stimulatory input to an as yet unidentified site inside the BBB.     

Involvement of central catecholamines in the feedback actions of 17beta-estradiolbenzoate on luteinizing hormone secretion in the ovariectomized female rat.

(1) Evidence has been presented, based on quantitative microfluorimetric estimations of dopamine (DA) and noradrenaline (NA) levels and turnover, and on radioimmunological measurements of serum luteinizing hormone (LH), and follicle-stimulating hormone (FSH) and prolactin levels, that the central inhibitory feedback action of estradiol on LH secretion mainly involves a marked increase in DA turnover of the lateral palisade zone (LPZ) of the median eminence and also involves a reduction of NA turnover, mainly located in the medial preoptic area (MPOA) and in the subependymal layer (SEL). (2) The mechanism for these changes in catecholamine (CA) turnover is discussed. The possibility is favored that they involve the stimulation of central cytosol estrogen receptors which may even be located in the arcuate DA and possibly reticular NA cell bodies themselves. (3) The marked and long-lasting hypersecretion of prolactin caused by estrogen could be involved in mediating the sustained increase of DA turnover in the median eminence and the sustained reduction of NA turnover. (4) The central facilitatory feedback action of estrogen, on the other hand, may be mainly responsible for the sharp increase of NA turnover in the MPOA and SEL and also the associated reduction of DA turnover in the LPZ in the critical period of both adult cyclic rats and of immature female rats treated with PMS. (5) We take the view that these latter NA and DA turnover changes take precedence over cytosol estrogen receptors, which previous evidence indicates (see Sawyer, 1975) are located in preoptic and amygdaloid regions. (These estradiol concentrating neurons then directly and/or indirectly make connections with the NA and DA pathways). (6) FSH secretion is not controlled by DA and NA pathways. (7) The hypothesis given above is based on the assumption that different estradiol concentrating neurons are involved in the central inhibitory and facilitatory feedback action of estradiol and that the estrogen receptors of these respective neurons have differences which allow their differential activation, the inhibitory feedback leading mainly to a marked increase in the ratio DA activity/NA activity in the median eminence, whereas the facilitatory feedback causes a marked reduction of this ratio.     

孤啡肽对脑缺血损伤后N-甲基-D-天门冬氨酸受体表达的影响

目的研究孤啡肽(OFQ)对脑缺血损伤后N-甲基-D-天门冬氨酸受体(NMDAR1)表达的影响。方法成年健康雄性Sprague-Dawley大鼠40只,应用Longa线栓法建立大鼠大脑中动脉闭塞(MCAO)模型,侧脑室微量注射OFQ干预,原位TUNEL检测细胞凋亡,免疫组织化学法检测NMDAR1受体表达。结果脑缺血后脑组织凋亡细胞增多,NMDAR1表达增强(P<0.05)。应用OFQ后凋亡细胞增多,NMDAR1表达较缺血组显著增强(P<0.05),而且与剂量呈正比例关系。结论OFQ可能通过上调NMDAR1表达,加重脑缺血引起的氧化损伤。     

Autoradiography of GABA in the rat hypothalamic median eminence

Light microscopy autoradiographs of the rat hypothalamic median eminence were prepared after injection of high specific activity tritiated GABA and GABA structural analogs.Following intracardiac injection of labeled GABA with short (15 min) survival time, a dense accumulation of silver grains was observed over the external layer of the median eminence. The silver grains appeared much less numerous and randomly scattered over the internal layer. No conspicuously labeled cells could be detected in the median eminence.A similar pattern of labeling was observed after 10 min in vitro incubation of the median eminence with a low concentration(2.5 × 10⁻⁷M) of labeled GABA. Clusters of silver grains were also visible over the external layer following intraventicular injection of labeled GABA. In this latter case, however, other sites of labeling were revealed over the internal and ependymal layers.The dense labeling over the external layer with tritiated GABA was partially reduced by a simultaneous intracardiac injection of a 50-fold excess of non-radioactive cis-aminocyclohexane car☐ylic acid — a reported preferential substrate for GABA neuronal uptake — but it was not displaced by a 2000-fold excess of non-radioactive β-alanine — a reported specific substrate for GABA glial uptake.Intracardiac injection of triated β-alanine led to a faint and even labeling over the entire median eminence with no preferential accumulation of silver grains over the various layers. Following intraventricular injection of labeled β-alanine the tanycytes and their processes as well as numerous glial cells appeared heavily labeled.These results suggested that there exist cell elements in the external layer of the hypothalamic median eminence which are capable of accumulating exogenous GABA according to its neuronal uptake characteristics. Although the exact nature of these cells is not readily apparent at this stage of our investigations, these findings led us to speculate that there might be a subpopulation of GABAergic nerve endings in the vicinity of the primary plexus capillaries.     

Neurohemal contact in the internal zone of the rabbit median eminence

The concept of neurosecretion as the mechanism by which neural control of adenohypophyseal function is accomplished was based on the observation that long capillary loops penetrate deeply into the supra-opticohypophyseal tract as it passes through the median eminence internal zone. However, neural contact upon these capillary loops has not been demonstrated in the mammalian median eminence. The present transmission electron microscopic investigation of the rabbit median eminence demonstrates neurohemal contact in the median eminence internal zone. Axons containing small lucent vesicles 53.3 ± 3.28 nm in diameter (mean ± SEM) or small lucent and large granular vesicles with a mean diameter of 122.4 (±3.28 nm) in their terminals make neurohemal contact with capillary loops in the internal zone and form a cuff about them. These terminals resemble terminals found in the external zone. Intravenous injection of the false neurotransmitter 5-hydroxydopamine (5-OH-DA) renders small lucent vesicles granular in both the external and internal zone. The effect of 5-OH-DA injecting is abolished by concurrent reserpine administration. Whereas large granular vesicles in many terminals become lucent after reserpine administration, in others they remained electron dense. Viewed in the light of previous studies our findings suggest that the internal plexus arises from the external plexus and invaginates the neuropil carrying connective tissue and parvicellular axon terminals of aminergic and peptidergic systems from the external zone into the internal zone, that some elements making neurohemal contact with long capillary loops are terminals of the noradrenergic reticular infundibular tract arising outside the hypothalamus in the brainstem, and that long capillary loops form a system of repeating microvascular modules which markedly increase the surface available for neurohemal contact.     

Intravenous administration of tumor necrosis factor-alpha stimulates corticotropin releasing hormone secretion in the push-pull cannulated median eminence of freely moving rats.

Utilizing push-pull perfusion, we examined the effects of intravenous (iv) administration of human recombinant tumor necrosis factor (TNF)-alpha on the levels of plasma adrenocorticotropin (ACTH) and corticotropin releasing hormone (CRH) in the median eminence (ME) of freely moving male rats. The ME was perfused with artificial cerebrospinal fluid between 11:00 and 14:00 h, and perfusates and blood samples were collected every 20 min. TNF-alpha (1.0 microgram), but not vehicle only, given as an iv bolus at 12:00 h significantly stimulated both plasma ACTH and ME-CRH. The increase in ME-CRH clearly preceded that of plasma ACTH. This is the first to characterize the temporal profile of CRH secretion in the ME after iv administration of TNF-alpha to freely moving rats. These in vivo data strongly suggest that TNF-alpha stimulates ACTH secretion, at least in part, by triggering hypothalamic CRH release. In addition, combined with our previous data obtained by iv administration of human recombinant interleukin-1 under the same experimental condition, the present study also suggests that iv injected TNF-alpha and interleukin-1 may share a common site of action in the brain, such as the ME, to stimulate CRH secretion.     

Activity of monoamine oxidase in the medial preoptic area and median eminence of female rats of different ages

Total monoamine oxidase activity in the medial preoptic area and median eminence (with surrounding tissue) has been studied in female rats of three age groups, viz., those aged 1.5–2 months (peripubertal), 4–5 months (mature), and over 12 months (aging). Monoamine oxidase activity was measured using kynuramine as a substrate and changes in the concentration of product (4-hydroxyquinoline) were recorded at 327 nm. In the medial preoptic area, the lowest activity (nmole kynuramine/min/mg protein, M ± m) was found during the peripubertal period (1.55 ± 0.11), while in mature and aging rats the activities were similar (1.93 ± 0.12 and 2.01 ± 0.15, respectively). In the median eminence, the greatest activity of monoamine oxidase was found in the aging rats (6.61± 0.56), whereas in the rats of peripubertal and mature age the activities were similar (4.79 ± 0.57 and 4.36 ± 0.25, respectively). In animals aged 4–5 months, we found a tendency toward a negative correlation between the activity of monoamine oxidase in the medial preoptic area and the activity in the median eminence. Our results suggest that opposing changes in enzyme activity are necessary for the coordinated work of the monoaminergic systems in the areas studied.     

The influence of hGRF, CRF, TRH and LHRH on SRIF release from median eminence fragments

The influence of 4 releasing factors on the release of somatostatin (SRIF) from the median eminence of the hypothalamus in rats was studied using an in vitro system. Synthetic growth hormone-releasing factor (hGRF-40) and corticotropin releasing factor (CRF) stimulated SRIF release, whereas thyrotrophin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH) did not stimulate its release. CRF and GRF may be physiologically involved in the regulation of SRIF release.     

Endopeptidase EC 3.4.24.15 Presence in the Rat Median Eminence and Hypophysial Portal Blood and its Modulation of the Luteinizing Hormone Surge

The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. Among its numerous roles in metabolizing and processing biologically-active peptides, the enzyme degrades gonadotropin-releasing hormone (GnRH) by cleaving the central Tyr⁵-Gly⁶ bond. The aim of the present studies was to determine whether EP24.15 can modulate the concentrations of GnRH within the hypothalamo-hypophysial portal blood and thereby play a physiological role in reproduction. Our data suggest the presence of immunoreactive EP24.15 in the perivascular space of the median eminence and that this enzyme is secreted into portal blood. We have also shown a physiological role for this enzyme in that an inhibition of its activity with a specific inhibitor augmented the steroid-induced LH increase in ovariectomized rats. The present results suggest that secretory and post-secretory mechanisms are important in shaping the GnRH signal from the central nervous system; GnRH metabolism by EP24.15 may be one such mechanism.     

Data on the absence of axon terminals of medial preoptic area neurons in the surface zone of the median eminence

Summary In order to investigate whether neurons of the medial preoptic area (MPOA) project to the surface zone (zona palisadica) of the median eminence (ME) and proximal part of the pituitary stalk, an electrolytic slesion or a frontal cut was placed in the pre- and suprachiasmatic region of the rat hypothalamus and the mentioned zone examined under the electron microscope. Degenerated nerve profiles were not observed in the zona palisadica following a lesion restricted to the MPOA or after a frontal cut at the posterior border of the MPOA. Altered elements were seen only in those cases in which the posterior part of the suprachiasmatic region was destroyed. The present data indicate that neurons of the MPOA do not terminate in the zona palisadica of the ME but presumably end on the nerve cells of the medial basal hypothalamus.     

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.     

Identification of gonadotropin releasing hormone (GnRH) neurons projecting to the median eminence from third ventricular preoptic area grafts in hypogonadal mice

Functional gonadotropin releasing hormone (GnRH) neurosecretory activity can be restored in genetically hypogonadal (hpg) adult mice with grafts of GnRH-containing fetal or neonatal septal-preoptic area (S/POA) tissue. Neurons implanted into the third ventricle of the host brain survive and send out axons which innervate one of the normal targets of these neurons, the median eminence (ME). Fibers terminate near primary portal vessels where GnRH is available for release into the vasculature, and this axonal outgrowth is essential for the stimulation of gonadotropin secretion, gonadal and accessory sex structure growth, gametogenesis, and fertility. Although it is known that GnRH axons reach their target, it is not known if all such neurons in a graft contribute to the projection. Taking advantage of the fact that axons in the ME, the sole host target, are outside the blood-brain barrier (BBB), long-term grafted animals were injected intraperitoneally with a retrograde tracer, Fluorogold (FG). Normal male mice were injected for comparison. Animals were sacrificed 5 days after injection and brain sections in the area of the graft were stained immunocytochemically for GnRH. In the normal male mice, two-thirds of the GnRH neurons were double-labeled with FG. In grafted individuals which showed increased gonadal growth, the percentage of labeled cells ranged from 17 to 75%. The results indicate that despite tissue injury, ectopic location, and a vastly reduced population, transplanted fetal GnRH neurons recapitulate a pattern seen in normal intact mice where some but not all neurons were capable of capturing a peripherally delivered tracer.     

Plasticity of vasopressin- and oxytocin-containing fibers in the median eminence in hypophysectomized young and old mice

Rearrangements of vasopressin- and oxytocin-containing fibers in the external layer of the median eminence after hypophysectomy were compared between young and old mice. In 3-month-old hypophysectomized mice, the increase in the number of fibers containing vasopressin was greater than that observed in 19-month-old hypophysectomized ones, suggesting a decrease in axonal plasticity in old mice. No difference with age was detected for the plasticity of fibers containing oxytocin.     

Characterization of Membrane-Associated Peptidase Activities Expressed by Endothelial Cells of the Ovine Median Eminence

The capillary endothelial cells of the median eminence represent a potential site for the degradation/modification of both circulating and hypothalamic peptides passing through the hypophysial portal system toward the pituitary. This study examines endothelial cell peptidase expression in vitro by monitoring the metabolism of gonadotropin-releasing hormone (GnRH) by cultured endothelial cells from sheep median eminence. Cleavage of GnRH by median eminence endothelial cell membranes generated GnRH_1–5 as the primary stable product, which was then degraded to GnRH_1–3 and free amino acids. Degradation of GnRH was completely inhibited by TPCK, ZnCI₂ and N-ethylmaleimide, and partially inhibited by EDTA and by a specific inhibitor of the metalloendopeptidase EC 3.4.24.15, CFP-AAY-pAB. Interestingly, an increase in GnRH_1–9 production was seen with the latter inhibitors, suggesting a two-step mechanism of GnRH degradation involving a primary cleavage at the Pro⁹-Gly¹⁰-NH₂ bond, inhibitable by TPCK, ZnCI₂, and NEM, followed by cleavage by EC 3.4.24.15 to generate GnRH_1–5. Phosphoramidon and angiotensin converting enzyme inhibitors (as well as other non-specific inhibitors) were without effect, indicating that endopeptidase EC 3.4.24.11 and angiotensin converting enzyme are not involved. Neither bovine aortic endothelial cell nor AtT-20 cell membranes exhibited this pattern of peptidase activity. Degradation of GnRH by intact median eminence endothelial cells in culture was also observed, suggesting an extracellular orientation for these enzymes; the potential role of such peptidases in the fine regulation of both pituitary function and local blood flow is currently under investigation.     

Age‐related changes in the morphology of tanycytes in the human female infundibular nucleus/median eminence

Tanycytes are emerging as key players in the neuroendocrine control of gonadotrophin‐releasing hormone (GnRH) release. Rodent studies have demonstrated that the structural relationship between tanycytes and GnRH terminals in the median eminence is highly dynamic, regulated by gonadal steroids and undergoes age‐related changes. The present study aimed to determine whether the number and organisation of tanycytes changes throughout life in the female infundibular nucleus/median eminence (INF/ME) region. Post‐mortem hypothalamic tissues were collected at the Netherlands Brain Bank and were stained for vimentin by immunohistochemistry. Hypothalami of 22 control female subjects were categorised into three periods: infant/prepubertal, adult and elderly. We measured the fractional area covered by vimentin immunoreactivity in the INF. Qualitative analysis demonstrated a remarkable parallel organisation of vimentin‐immunoreactive processes during the infant/prepubertal and adult periods. During the elderly period, this organisation was largely lost. Semi‐quantitatively, the fractional area covered in vimentin immunoreactivity was significantly higher at the infant/prepubertal compared to the adult period and almost reached statistical significance compared to the elderly period. By contrast, the number of tanycyte cell bodies did not appear to change throughout life. The results of the present study thus demonstrate that the number and structure of tanycytic processes are altered during ageing, suggesting that tanycytes might be involved in the age‐related changes observed in GnRH release.