Median Eminence Region Research Articles

Ketogenic diet promotes the proliferation of oligodendrocyte precursor cells in ME region by activating fatty acid oxidation.

Hypothalamic median eminence (ME) is a potential niche for neurons and oligodendrocytes, and trophic factors may regulate hypothalamic function by inducing cellular changes in the ME region. To determine whether diet-induced plasticity exists in hypothalamic stem cells dormant under physiological conditions, we used a combination of a normal diet, a high-fat diet, and a ketogenic diet (a low-carb, high-fat diet) to compare the proliferation of tanycytes (TCs) and oligodendrocyte precursor cells (OPCs) in the ME area of mice under the different diets. The results showed that the ketogenic diet could induce and promote the proliferation of OPCs in the ME area, and blocking the fatty acid oxidation program could inhibit the proliferation of OPCs induced by a ketogenic diet. This study preliminarily revealed the diet-induced effect on OPCs in the ME region and provided enlightenment for further study on the function of OPCs in the ME region.

IMMUNOHISTOCHEMICAL EXPRESSION OF MONOCARBOXYLATE TRANSPORTER 1&4 IN TANYCYTE–LIKE CELLS OF THE SULCUS MEDIANUS ORGANUM

Background: Circumventricular organs (CVOs) are specialized structures border the brain ventricles and lack the blood-brain barrier. These CVOs are lined by specialized ependymal cells (ECs) called tanycyte. The sulcus medianus organum (SMO) locates at the floor of the 4th ventricle at the rostral part of the sulcus medianus (SM). Objective: To explore the expression of the monocarboxylate transporter 1 and monocarboxylate transporter 4 (MCT1 & MCT4) in the tanycytes of the median eminence (ME) and tanycyte-like cells of the SMO to add a functional evidence for describing the SMO as another CVOs and to start a roadmap for the citing the SMO specifically as a sensory or a secretory CVO. Methods: Ten adult male rats (Rattus norvegicus albinus), aged 3-6 months with 300±50 g, were used to study the histological characteristics of ECs in ME and SMO with Hematoxylin & Eosin and to explore the immunohistochemical expression of MCT1 & MCT4 in ME and SMO. Results: The ependymal cells were arranged in in 2-3 layers in the depth of SMO region and single layer in the ME region as seen with H&E stains. Immunohistochemical expression of MCT1 & MCT4 using Aperioscope image analysis in tanycytes of the ME is higher than that in tanycyte-like cells of the SMO with significant differences between the two regions as proved by t-test. Conclusion: the SMO has different structural and functional properties compared to ME suggesting that the SMO may be a sensory CVO. Keywords: Circumventricular organs, tanycytes, sulcus medianus organum, median eminence Citation: Jawad HF, Al-Kaabi MA, Al-Salihi AR. Immunohistochemical expression of monocarboxylate transporter 1&4 in tanycyte–like cells of the sulcus medianus organum. Iraqi JMS. 2019; 17(1): 83-99. doi: 10.22578/IJMS.17.1.12

A simple strategy for culturing morphologically-conserved rat hypothalamic tanycytes.

Hypothalamic tanycytes are specialized bipolar ependymal cells that line the floor of the third ventricle. Given their strategic location, tanycytes are believed to play several key functions including being a selective barrier and controlling the amount of hypothalamic-derived factors reaching the anterior pituitary. The in vitro culture of these cells has proved to be difficult. Here, we report an improved method for the generation of primary cultures of rat hypothalamic tanycytes. Ependymal cultures were derived from tissue dissected out of the median eminence region of 10-day-old rats and cultured in a chemically defined medium containing DMEM:F12, serum albumin, insulin, transferrin and the antibiotic gentamycin. After 7days in vitro, ∼30% of the cultured cells exhibited morphological features of tanycytes as observed by phase contrast or scanning electron microscopy. Tanycyte-like cells were strongly immuno-reactive for vimentin and dopamine-cAMP-regulated phospho-protein (DARPP-32) and weakly immune-reactive for glial fibrillary acidic protein. Tanycyte-like cells displayed a stable negative resting plasma membrane potential and failed to show spiking properties in response to current injections. When exposed to fluorescent beads in the culture medium, tanycyte-like cells exhibited a robust endocytosis. Thus, the present method effectively yields cultures containing tanycyte-like cells that resemble in vivo tanycytes in terms of morphologic features and molecular markers as well as electrical and endocytic activity. To our knowledge, this is the first protocol that allows the culturing of tanycyte-like cells that can be individually identified and that conserve the morphology of tanycytes in their natural physiological environment.

Second generation anti-epileptic drugs adversely affect reproductive functions in young non-epileptic female rats

Reproductive endocrine disturbances are a major health concern in women with epilepsy due to their long term use of antiepileptic drugs (AEDs). Second generation AEDs such as topiramate (TPM) and gabapentin are frequently used for the treatment of epilepsy as well as migraine, bipolar disorder etc. Despite the widespread clinical complications, however the definitive mechanism(s) mediating the side effects of TPM and gabapentin remain obscure. The present study was aimed to evaluate the long term effects of TPM and gabapentin on reproductive functions in young female Wistar rats. Estrous cyclicity, ovarian histology as well as estradiol, LH, leptin and insulin hormones level were studied to elucidate the long-term effect of these AEDs monotherapy on reproductive functions in non-epileptic animals. Further to explore the effects on gonadotropin releasing hormone (GnRH) neuroendocrine plasticity, the expression of GnRH, gamma-amino butyric acid (GABA), glutamic acid decarboxylase (GAD), glial fibrilliary acidic protein (GFAP) and polysialylated form of neural cell adhesion molecule (PSA-NCAM) was studied in median eminence (ME) region of these animals by immunohistochemistry, Western blot hybridization and RT-PCR. Our results demonstrate that TPM and gabapentin treatment for 8 weeks cause reproductive dysfunction as ascertained by disturbed hormonal levels and estrous cyclicity as well as alterations in GABAergic system and GnRH neuronal–glial plasticity. Our findings suggest that treatment with TPM and gabapentin disrupts the complete hypothalamo-hypophyseal-gonadal axis (HPG) through GnRH pulse generator in hypothalamus.

Neuroestradiol in the Hypothalamus Contributes to the Regulation of Gonadotropin Releasing Hormone Release

Release of gonadotropin releasing hormone (GnRH) from the medial basal hypothalamus (MBH)/median eminence region (S-ME) is essential for normal reproductive function. GnRH release is profoundly regulated by the negative and positive feedback effects of ovarian estradiol (E2). Here we report that neuroestradiol, released in the S-ME, also directly influences GnRH release in ovariectomized female monkeys, in which the ovarian source of E2 is removed. We found that (1) brief infusion of E2 benzoate (EB) to the S-ME rapidly stimulated release of GnRH and E2 in the S-ME of ovariectomized monkeys, (2) electrical stimulation of the MBH resulted in GnRH release as well as E2 release, and (3) direct infusion of an aromatase inhibitor to the S-ME suppressed spontaneous GnRH release as well as the EB-induced release of GnRH and E2. These findings reveal the importance of neuroestradiol as a neurotransmitter in regulation of GnRH release. How circulating ovarian E2 interacts with hypothalamic neuroestrogens in the control of GnRH release remains to be investigated.

Intermittent Fasting Dietary Restriction Regimen Negatively Influences Reproduction in Young Rats: A Study of Hypothalamo-Hypophysial-Gonadal Axis

Nutritional infertility is very common in societies where women fail to eat enough to match their energy expenditure and such females often present as clinical cases of anorexia nervosa. The cellular and molecular mechanisms that link energy balance and central regulation of reproduction are still not well understood. Peripheral hormones such as estradiol, testosterone and leptin, as well as neuropeptides like kisspeptin and neuropeptides Y (NPY) play a potential role in regulation of reproduction and energy balance with their primary target converging on the hypothalamic median eminence-arcuate region. The present study was aimed to explore the effects of negative energy state resulting from intermittent fasting dietary restriction (IF-DR) regimen on complete hypothalamo-hypophysial-gonadal axis in Wistar strain young female and male rats. Significant changes in body weight, blood glucose, estrous cyclicity and serum estradiol, testosterone and LH level indicated the negative role of IF-DR regimen on reproduction in these young animals. Further, it was elucidated whether serum level of metabolic hormone, leptin plays a mechanistic role in suppressing hypothalamo-hypophysial-gonadal (HPG) axis via energy regulators, kisspeptin and NPY in rats on IF-DR regimen. We also studied the effect of IF-DR regimen on structural remodeling of GnRH axon terminals in median eminence region of hypothalamus along with the glial cell marker, GFAP and neuronal plasticity marker, PSA-NCAM using immunostaining, Western blotting and RT-PCR. Together these data suggest that IF-DR regimen negatively influences reproduction in young animals due to its adverse effects on complete hypothalamus-hypophysial-gonadal axis and may explain underlying mechanism(s) to understand the clinical basis of nutritional infertility.

Enzymatic removal of polysialic acid from neural cell adhesion molecule interrupts gonadotropin releasing hormone (GnRH) neuron–glial remodeling

There is abundant evidence to prove that the astrocytes are highly dynamic cell type in CNS and under physiological conditions such as reproduction, these cells display a remarkable structural plasticity especially at the level of their distal processes ensheathing the gonadotropin releasing hormone (GnRH) axon terminals. The morphology of GnRH axon terminals and astrocytes in the median eminence region of hypothalamus show activity dependent structural plasticity during different phases of estrous cycle. In the current study, we have assessed the functional contribution of ∞-2,8-linked polysialic acid (PSA) on neural cell adhesion molecule (PSA-NCAM) in this neuronal–glial plasticity using both in vitro and in vivo model systems. In vivo experiments were carried out after stereotaxic injection of endoneuraminidase enzyme (endo-N) near median eminence region of hypothalamus to specifically remove PSA residues on NCAM followed by localization of GnRH, PSA-NCAM and glial fibrillary acidic protein (GFAP) by immunostaining. Using in vitro model, structural remodeling of GnV-3 cells, (a conditionally immortalized GnRH cell line) co-cultured with primary astrocytes was studied after treating the cells with endo-N. Marked morphological changes were observed in GnRH axon terminals in proestrous phase rats and control GnV-3 cells as compared to endo-N treatment i.e. after removal of PSA. The specificity of endo-N treatment was also confirmed by studying the expression of PSA-NCAM by Western blotting in cultures treated with and without endo-N. Removal of PSA from surfaces with endo-N prevented stimulation associated remodeling of GnRH axon terminals as well as their associated glial cells under both in vivo and in vitro conditions. The current data confirms the permissive role of PSA to promote dynamic remodeling of GnRH axon terminals and their associated glia during reproductive cycle in rats.

Early Postnatal Administration of Growth Hormone Increases Tuberoinfundibular Dopaminergic Neuron Numbers in Ames Dwarf Mice

Hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons secrete dopamine, which inhibits pituitary prolactin (PRL) secretion. PRL has demonstrated neurotrophic effects on TIDA neuron development in PRL-, GH-, and TSH-deficient Ames (df/df) and Snell (dw/dw) dwarf mice. However, both PRL and PRL receptor knockout mice exhibit normal-sized TIDA neuron numbers, implying GH and/or TSH influence TIDA neuron development. The current study investigated the effect of porcine (p) GH on TIDA neuron development in Ames dwarf hypothalamus. Normal (DF/df) and dwarf mice were treated daily with pGH or saline beginning at 3 d of age for a period of 42 d. After treatment, brains were analyzed using catecholamine histofluorescence, tyrosine hydroxylase immunocytochemistry, and bromodeoxyuridine (BrdU) immunocytochemistry to detect BrdU incorporation. DF/df males and df/df treated with pGH experienced increased (P </= 0.01) weight gain compared with those treated with saline. DF/df had greater (P </= 0.01) TIDA neuron numbers than df/df, regardless of treatment. TIDA neuron number in pGH-treated df/df was greater (P </= 0.01) than in saline-treated df/df. Zona incerta and periventricular dopamine neurons were not affected by treatment or genotype. There was no effect of genotype or treatment on BrdU incorporation in the arcuate nucleus, median eminence, or periventricular region surrounding the third ventricle. Saline-treated df/df experienced decreased (P </= 0.05) dentate gyrus BrdU incorporation compared with saline-treated DF/df. In the lateral ventricle, pGH-treated males had greater BrdU immunoreactivity than pGH-treated females. The results show an effect of pGH on TIDA neuron development, although this effect is less potent than that of PRL, and likely GH-induced preservation of TIDA neurons rather than generation of new TIDA neurons via neurogenesis.

GluR5-mediated glutamate signaling regulates hypothalamo–pituitary–adrenocortical stress responses at the paraventricular nucleus and median eminence

Glutamate is the main excitatory neurotransmitter in the central nervous system, and plays an excitatory role in generation of hypothalamic-pituitary-adrenocortical (HPA) axis responses to stress. The current study assesses the role of kainate-preferring receptors in glutamatergic excitation of the HPA axis. In situ hybridization and immunohistochemical analyses confirmed the existence of the GluR5 kainate subunit in the paraventricular nucleus of the hypothalamus (PVN). Importantly, GluR5 immunoreactivity was enriched in the external lamina of the median eminence, where it is co-localized with corticotropin releasing hormone (CRH). Intra-PVN infusion of LY382884 increased plasma adrenocorticotropin (ACTH), corticosterone and PVN c-Fos immunoreactivity. Infusions of LY382884 into the median eminence region, on the other hand, reduced restraint induced ACTH release without altering c-Fos expression. Together, these findings provide evidence for glutamate-mediated signaling in control of CRH release at the PVN and median eminence, mediated by way of kainate-preferring receptor complexes.

Gonadotropin-Releasing Hormone in Third Ventricular Cerebrospinal Fluid: Endogenous Distribution and Exogenous Uptake

GnRH is detectable in the cerebrospinal fluid (CSF), but its source remains unidentified. Previous studies have harvested CSF for GnRH analysis from the median eminence region, but it is unknown whether GnRH in CSF is restricted to this region. If CSF-GnRH plays a physiological role, through volume transmission, to communicate with brain regions that express GnRH receptors but are not evidently innervated by GnRH neurons, then it is essential to establish whether GnRH is more pervasive throughout the cerebroventricular system. Three cannulae were placed in the supraoptic, infundibular, and pineal recesses of the third ventricle. GnRH was undetectable in lateral ventricle CSF. GnRH pulses were detected in all ewes in infundibular recess CSF, but at sites more rostral (supraoptic) and caudal (pineal), GnRH pulse frequency and amplitude significantly (P<0.05) decreased. A GnRH surge was evident in CSF collected simultaneously from all cannulae, but the amplitude was greatest (P<0.05) at the infundibular recess. A final study established whether iv administered GnRH enters the CSF. A 250-ng GnRH dose did not affect CSF-GnRH concentrations (1.6+/-0.3 pg/ml), but 2.5 microg (2.7+/-0.2 pg/ml; P<0.001) and 1 mg (38.5+/-10.6 pg/ml; P<0.05) significantly increased CSF-GnRH concentrations. The present study shows: 1) the median eminence region is likely to be the major, if not only, source of GnRH entering the cerebroventricular system; and 2) exogenous GnRH crosses the blood-brain barrier, but extremely high doses are required to elevate CSF concentrations to physiological levels. Thus, CSF-GnRH may affect sites that are closer in proximity to the infundibular recess region than previously thought.

Inhibition of Metastin (Kisspeptin-54)-GPR54 Signaling in the Arcuate Nucleus-Median Eminence Region during Lactation in Rats

Follicular development and ovulation are suppressed during lactation in various mammalian species, mainly due to the suppression of pulsatile GnRH/LH secretion. Metastin (kisspeptin-54), a KiSS-1 gene product, is an endogenous ligand for GPR54, a G-protein-coupled receptor, and suggested to play a critical role in regulating the gonadal axis. The present study therefore aims to determine whether metastin (kisspeptin-54)-GPR54 signaling in discrete brain areas is inhibited by the suckling stimulus that causes suppression of LH secretion in lactating rats. Quantitative RT-PCR revealed that the KiSS-1 mRNA level was significantly lower in the arcuate nucleus (ARC)-median eminence region in lactating ovariectomized (OVX) and estrogen-treated OVX rats than in nonlactating controls. KiSS-1 mRNA in the anteroventral periventricular nucleus was kept at a low level in both lactating and nonlactating rats despite estrogen treatment. GPR54 mRNA levels were significantly lower in lactating than nonlactating rats in the anteroventral periventricular nucleus, but the levels in lactating mothers of the preoptic area and ARC-median eminence were comparable with nonlactating controls. Although KiSS-1 mRNA-expressing cells or metastin (kisspeptin-54) immunoreactivities were densely located in the ARC of nonlactating controls, few were found in the ARC of lactating OVX animals. Various doses of metastin (kisspeptin-54) (0.02, 0.2, and 2 nmol) injected into the third ventricle caused a significant increase in LH secretion in both lactating and nonlactating OVX rats, suggesting that lactating rats are responsive to metastin (kisspeptin-54) stimulus. Thus, the present study demonstrated that KiSS-1 mRNA/metastin (kisspeptin-54) expression is inhibited in the ARC by the suckling stimulus, suggesting that the inhibition is most probably involved in suppressing LH secretion in lactating rats.

Electrical Activity of the Pulse Generator of Gonadotropin-Releasing Hormone in 26-Month-Old Female Rats

To know whether age-related changes occur in the activity of the pulse generator of gonadotropin-releasing hormone (GnRH), old (26 months) and young (3 months) female rats were examined by recording multiunit activity (MUA) in the median eminence region of the hypothalamus, concurrently with blood samplings through an intra-atrial cannula at 6-min intervals to determine serum luteinizing hormone (LH) concentrations. We have regarded the MUA showing intermittent increases (volleys) at 20–30 min intervals, followed by LH pulses, as the electrical activity of the GnRH pulse generator. We were successful in recording MUA in 18 (26%) of 69 old ovariectomized rats and in 8 (32%) of 25 young ovariectomized rats. The overall mean (±SE) of the interval between MUA volleys in old ovariectomized rats was 35.1 ± 2.0 min (n = 18), which was significantly longer than that of 22.5 ± 1.5 min (n = 8) in young ovariectomized rats. The mean interval between LH pulses in old ovariectomized rats was 32.2 ± 3.6 (n = 10), also being significantly longer than that of 23.3 ± 1.0 (n = 8) in young ovariectomized rats. Further, the LH pulse amplitude in old rats (0.95 ± 0.07 ng/ml) was significantly smaller than in young rats (3.40 ± 0.36 ng/ml). The present study also confirmed that the increase in serum LH after intravenous injection of 50 ng GnRH was much smaller in old ovariectomized rats. These results show that the electrical activity of the GnRH pulse generator is certainly reduced with age. Taken together with findings suggesting an age-dependent decrease in stimulated transmitter release, attenuation in both frequency and amplitude of GnRH pulses as well as in pituitary responsiveness to GnRH pulses may account for the decreased pulsatile LH secretion observed in aging rats.

Suckling-Induced Change in Oxytocin but not in α-Msh Concentrations of the Median Eminence, The Neural-, Intermediate- and Anterior Lobes of the Pituitary Gland

Previous reports have implicated that pituitary-derived prolactin (PRL) is secreted from two distinct zones of mammotropes within the anterior lobe (AL). The inner zone (AL-IZ), located adjacent to the neuro-intermediate lobe (NIL), is supposed to be involved in the rapid and massive discharge of PRL from the pituitary gland due to suckling stimulus. Whereas the outer-zone (AL-OZ) gives the basal secretion and it does not play a role in the acute secretory response during nursing. Anatomically, the AL-IZ has an intimate contact with the NIL because the blood passing through the short portal vessels (SPV) bathes it first. Based on this fact it would be hypothesized that locally released and/or produced compounds, like OXY and a-MSH, can be delivered to the AL-IZ. In conjunction, OXY and a-MSH have already been implicated to play a role in the regulation of PRL release during suckling. Therefore, the purpose of this study was to examine the possible local transportation of these hormones into the median eminence and various regions of the pituitary gland of lactating rats. We have measured the concentrations of OXY and α-MSH from tissue samples of nonsuckled (NS) and 10 or 30 min after suckling (S) was initiated using specific RIAs. It has been shown that there are no changes in the concentration of OXY and α-MSH in the AL-IZ and AL-OZ due to suckling stimulus. In contrast, our data provide compelling evidence that OXY is transported into the IL, which can be further increased by suckling stimulus. These data suggest that blood transfusing NL passes through the IL before it is drained into the cavernous sinus, which opens the road for OXY into the general circulation. In addition, our data have unequivocally shown a lack of local delivery of either α MSH or OXY into the AL that raises serious doubt about their possible role in PRL secretion during suckling stimulus.

Multiple Forms of GnRH Are Released from Perifused Medial Basal Hypothalamic/Preoptic Area (MBH/POA) Explants in Birds

Both chicken gonadotropin-releasing hormones I and II (cGnRH I and II) were detected in abundant quantity by radioimmunoassay of extracts of Japanese quail medial basal hypothalamic/preoptic area (MBH/POA) fragments that included the median eminence (ME) region. However, in radioimmunoassayed extracts of Japanese quail ME alone, the concentration of cGnRH I greatly exceeded that of cGnRH II (≈450 pg/ME vs <10 pg/ME). Likewise, cGnRH I and II were released into perifusates from quail and turkey MBH/POA explants maintained in short-term perifusion. Release of both forms occurred whether or not explants included the ME region, i.e., from quail POA explants that did not include the ME or from turkey MBH/POA explants from which the ME region had been dissected out. This indicates that neuropeptides released from areas other than the ME can be a major source of neuropeptides detected in perifusates. Further, release of cGnRH I was altered following the addition of norepinephrine to perfusion media, whereas cGnRH II release was unaffected, again, whether or not explants included the ME. These results demonstrate that the release of neurohemoral substances from perifused explants cannot be assumed to represent regulated secretion from the ME.

Preoptic Area Grafts Implanted in Mammillary Bodies of Hypogonadal Mice: Patterns of GNRH Neuronal Projections

Gonadotropin-releasing hormone (GnRH) axons project to the median eminence, where the peptide is released to stimulate pituitary gonadotrophs. Hypogonadal mice (hpg) do not synthesize GnRH due to a deletion in the gene. When neonatal preoptic area (POA) tissue from normal mice containing GnRH neurons is transplanted into the third ventricle of hpg mice, GnRH axons exit the graft and specifically project to the median eminence, where the release of GnRH in the portal circulation induces the stimulation of the pituitary-gonadal axis. To test the hypothesis that the median eminence region is critical to targeting, we placed POA grafts in the region of the mammillary bodies, which never contains GnRH cell bodies, but is nevertheless close to the median eminence. Control mice received bilateral grafts into the anterior hypothalamus. GnRH axons innervated the median eminence in animals with grafts in the mammillary bodies and posterior hypothalamus. Mice with such grafts for 4–5 months had gonadal development, while those with grafts for shorter periods did not. Anterior hypothalamic grafts merged into the third ventricle and, consistent with previous studies, this resulted in GnRH innervation of the median eminence and gonadal development. However, when grafts were located within dorsal regions such as the thalamus, no median eminence innervation was seen. In these cases, GnRH axons borrowed other bundles of fibers to travel within the host brain. The pattern of innervation from grafts within ventro-caudal regions of the hypothalamus vs. that from dorsal regions supported the hypothesis that the median eminence releases diffusible substances directing GnRH outgrowth.

Effects of Treadmill Training on Plasma ACTH and Brain CRF Responses to Heterotypic Stress 648

We examined whether six wk (45min/d) of treadmill running (TT) moderates pituitary-adrenal cortical (PAC) responses and corticotropin-releasing factor[CRF] in microdissected nuclei (central nucleus of amygdala (CNA), paraventricular nucleus (PVN), median eminence (ME) to acute (10 min) heterotypic (novel) stress and no stress. Male Sprague-Dawley rats (N=24) were randomly assigned in a 2 GROUP (sedentary vs TT) *TEST(heterotypic-immobilization (IM) vs no stress (NS) *TIME (0,15,30,60 min) mixed factorial design with TEST and TIME repeated. After TEST, animals were returned to their home cages where blood was drawn (0.6ml) from a chronic indwelling jugular catheter. Plasma [ACTH] (pg/ml),[Corticosterone](CRT)(ug/dl) and [CRF] (pg/mg protein) were determined by radioimmunoassay. ANOVA and post hoc contrasts (p <.05) indicated a GROUP*TEST*TIME effect for ACTH. TT at 15 min (P=.04)(M=83.7, SD=14.2) and 30 min (P=.0038)(42.56,11.35) exhibited an attenuated ACTH effect compared with the sedentary controls min 15 (127.38, 28.5), min 30 (71.91,9.1) following heterotypic IM. A TEST effect was found for [CRT] and [ACTH].IM resulted in a greater PAC response compared with NS. ANOVA results indicated no difference (P>.4) in[CRF] between TT and sedentary in the CNA, PVN and ME regions. These data suggest a cross-stressor tolerance indicated by PAC hormones in male rats compared with previous findings of sensitization in estradiol-treated female rats. The failure of TT to moderate [CRF] in brain regions recognized to influence PAC responsiveness indicates the potential uniqueness of TT as a stressor and warrants further study.

17β-estradiol inhibits the production of dopamine by the tuberoinfundibular dopaminergic neurons of the male rat

Previous work in our laboratory suggests that the indirect stimulation of prolactin gene transcription observed in male rats between 3 and 6 h following estrogen injection results from an inhibition of dopamine release from the tuberoinfundibular dopaminergic neurons. To evaluate further the role of dopamine in the regulation of prolactin gene transcription by estrogens, we have examined the acute effects of 17β-estradiol (E 2) on dihydroxyphenylalanine accumulation in the arcuate median eminence region and the amount of dopamine associated with the arcuate median eminence region. Dihydroxyphenylalanine accumulation in the arcuate median eminence region was unaffected when examined 2 h following a single injection of E 2 (10 μg), and was reduced by 60% when examined 4 h following E 2 treatment. When examined 4 h after E 2 injection, dopamine content in the arcuate median eminence region was significantly increased. These data suggest that estrogens exert acute inhibitory effects on the tuberoinfundibular dopaminergic neurons of the male rat, and further support the assertion that the indirect stimulatory actions of estrogens on prolactin gene transcription result from inhibition of hypothalamic dopamine production.

Localization of [125I]IGF‐I Binding on the Ovine Pars Tuberalis

In the sheep, it has been shown that the pars tuberalis of the pituitary may mediate the photoperiodic control of seasonal changes in prolactin secretion. High concentrations of melatonin receptors are present on the ovine pars tuberalis and melatonin is known to inhibit forskolin-stimulated cyclic AMP production in this tissue. Other hormonal inputs to the ovine pars tuberalis have not yet been identified. In the rat mRNA for the IGF-I receptor has been identified in the pars tuberalis using in situ hybridization. In order to define whether IGF-I may influence the function of the ovine pars tuberalis the presence of receptors for IGF-I has been investigated. Using in vitro autoradiography specific [125I]IGF-I binding was found in high concentrations over the ovine pars tuberalis particularly associated with certain of the capillaries. Homogenate receptor assays showed saturable specific binding of [125I]IGF-I with a mean dissociation constant (Kd) of 0.5 +/- 0.1 nM (n = 4). Competition studies revealed a rank order of potency of IGF-I > IGF-II > > > insulin, in displacing [125I]IGF-I binding, indicative of a mixed population of IGF-I and IGF-II/mannose-6-phosphate receptors and insulin-like growth factor binding proteins (IGFBPs). Cross-linking of [125I]IGF-I to pars tuberalis membrane homogenates and analysis by SDS-PAGE under reducing conditions confirmed the presence of both IGF-I receptors and binding proteins. Autophosphorylation of a 97 kDa substrate, compatible with the beta-sub-unit of the IGF-I receptor, was increased in the presence of IGF-I, indicating the existence of functional IGF-I receptors on the ovine pars tuberalis. In contrast in the rat [125I]IGF-I binding was restricted to the median eminence region of the brain and was not detectable over the pars tuberalis.

Injection of bicuculline elicits firing of luteinizing hormone releasing hormone pulse generator in muscimol-treated ovariectomized rats

We have already demonstrated that, although exogenous γ-aminobutyric acid A (GABA A) receptor agonist is capable of inhibiting the activity of luteinizing hormone releasing hormone (LHRH) pulse generator, the reduction in the endogenous GABA A receptor activity does not have a significant effect in ovariectomized rats, suggesting a minor role of inhibitory GABA neurons in the control of pulsatile release of LHRH. In this study, we further analyzed the role of the GABA A receptor system in the regulation of LHRH pulse generator activity observed by recording the multiunit activity (MUA) in the hypothalamus of ovariectomized rats. An abrupt increase (volley) in the MUA in the arcuate nucleus (ARC)-median eminence region (ME) was accompanied by the initiation of a luteinizing hormone (LH) pulse, determined by measuring LH concentrations in blood sampled simultaneously. The injection of 1–3 mg/kg muscimol (MUS), a GABA A receptor agonist, decreased the basal MUA and delayed the occurrence of the next MUA volley, but it did not change the LH pulse amplitude. I.v. injection of 5 mg/kg bicuculline (BIC), a GABA A receptor antagonist, during continuous infusion of MUA (5 mg/kg/h) which decreased the basal MUA and prevented the MUA volley from occurring, evoked MUA volleys and LH pulses whose amplitudes were similar to those found before the MUS infusion. Injection of the same dose of BIC during the infusion of saline increased the MUA transiently, but this increase was not accompanied by an LH pulse and afterwards there occurred MUA volleys at ordinary intervals accompanied by LH pulses with unchanged pulse amplitudes. These results suggest that the GABA A receptor activity nhibits the LHRH pulse generator activity and that when the GABAergic activity is extremely high, a reduction in the GABA A receptor activity is able to cause activation of the LHRH pulse generator in the ovariectomized condition.

Mechanisms regulating neurohypophysial blood flow and function during isotonic volume expansion.

Regional cerebral and neurohypophysial blood flow responses to isotonic extracellular fluid volume expansion were studied in pentobarbital-anesthetized dogs using radiolabeled microspheres. Measurements were made at baseline and after increasing pulmonary capillary wedge pressure by 8 +/- 2 mmHg. Plasma arginine vasopressin (AVP) decreased by 50%, and neural lobe blood flow (NLBF) decreased by 47%. Blood flow through median eminence and other brain regions was unchanged. We investigated potential mechanisms responsible for the NLBF changes. Following control vagotomy, AVP concentration increased during the first hour and then returned to control values for hours 2 and 3, whereas NLBF was unchanged for the first hour and fell after 2 and 3 h. Vagus section abolished the decrease in NLBF but not the AVP response to volume expansion. The contribution of left atrial and pulmonary baroreceptors to this response was tested by inflation of a balloon in the left atria to produce a 13 +/- 1 mmHg rise in atrial pressure. This led to a 20% reduction in NLBF, a 35% reduction in AVP concentration, and a 270% increase in plasma atrial natriuretic factor (ANF). However, ANF release does not account for the NLBF changes, since intravenous ANF infusion had no effect on NLBF or AVP concentration. These data suggest that the NLBF response to volume expansion is mediated by the vagus with input from multiple cardiopulmonary baroreceptors.