Recurrent glucose deprivation leads to the preferential use of lactate by neurons in the ventromedial hypothalamus.

Increased GABAergic output in the ventromedial hypothalamus (VMH) contributes to counterregulatory failure in recurrently hypoglycemic (RH) rats, and lactate, an alternate fuel source in the brain, contributes to this phenomenon. The current study assessed whether recurring bouts of glucose deprivation enhanced neuronal lactate uptake and, if so, whether this influenced γ-aminobutyric acid (GABA) output and the counterregulatory responses. Glucose deprivation was induced using 5-thioglucose (5TG). Control rats received an infusion of artificial extracellular fluid. These groups were compared with RH animals. Subsequently, the rats underwent a hypoglycemic clamp with microdialysis. To test whether 5TG affected neuronal lactate utilization, a subgroup of 5TG-treated rats was microinjected with a lactate transporter inhibitor [cyano-4-hydroxycinnamate (4CIN)] just before the start of the clamp. Both RH and 5TG raised VMH GABA levels, and this was associated with impaired counterregulatory responses. 4CIN reduced VMH GABA levels and restored the hormone responses in the 5TG group. We then evaluated [14C]lactate uptake in hypothalamic neuronal cultures. Recurring exposure to low glucose increased monocarboxylate transporter-2 mRNA expression and augmented lactate uptake. Taken together, our data suggest that glucose deprivation, per se, enhances lactate utilization in hypothalamic neurons, and this may contribute to suppression of the counterregulatory responses to hypoglycemia.

Daily, circadian and seasonal changes of rhodopsin-like encephalic photoreceptor and its involvement in mediating photoperiodic responses of Gambel's white-crowned Sparrow, Zonotrichia leucophrys gambelii.

Extra-retinal, non-pineal, encephalic photoreceptors (EP) play important roles in mediating development of the reproductive system by the annual change in day length (photoperiodic gonadal response - PGR) in birds. However, the distribution of rhodopsin-like EPs and their functional daily, circadian and seasonal changes are still unclear in the avian brain. This study identifies two novel groups of rhodopsin-immunoreactive cells in the nucleus paraventricularis magnocellularis (PVN) of the hypothalamus and in the medial basal hypothalamus (MBH) in a seasonally breeding species, Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii). In the PVN, rhodopsin-ir cell number showed both daily and circadian changes with more labeled cells apparent in the night phase in photosensitive birds, while only circadian changes were observed involving fewer labeled cells in the night phase in photorefractory birds. Single long day photo-stimulation significantly decreased the rhodopsin-ir cell number only in photosensitive birds, coincident with a rise in plasma levels of luteinizing hormone (LH). In the MBH, rhodopsin-ir cell number did not show daily, circadian or single long day induced changes in either photoperiodic states. But, overall these rhodopsin expressing neurons significantly increased from photosensitive to photorefractory states. In the median eminence (ME), more intense rhodopsin-ir was detected in photorefractory birds compared to photosensitive birds. For expression of GnRH and vasoactive intestinal polypeptide (VIP), seasonal differences were found with opposite relationships, consistent with previous studies. Our results suggest different roles of the two groups of rhodopsin-like EPs in the regulation of PGR in white-crowned sparrows.

Activation of orexin/hypocretin neurons is associated with individual differences in cued fear extinction.

Identifying the neurobiological mechanisms that underlie differential sensitivity to stress is critical for understanding the development and expression of stress-induced disorders, such as post-traumatic stress disorder (PTSD). Preclinical studies have suggested that rodents display different phenotypes associated with extinction of Pavlovian conditioned fear responses, with some rodent populations being resistant to extinction. An emerging literature also suggests a role for orexins in the consolidation processes associated with fear learning and extinction. To examine the possibility that the orexin system might be involved in individual differences in fear extinction, we used a Pavlovian conditioning paradigm in outbred Long-Evans rats. Rats showed significant variability in the extinction of cue-conditioned freezing and extinction recall, and animals were divided into groups based on their extinction profiles based on a median split of percent freezing behavior during repeated exposure to the conditioned cue. Animals resistant to extinction (high freezers) showed more freezing during repeated cue presentations during the within trial and between trial extinction sessions compared with the group showing significant extinction (low freezers), although there were no differences between these groups in freezing upon return to the conditioned context or during the conditioning session. Following the extinction recall session, activation of orexin neurons was determined using dual label immunohistochemistry for cFos in orexin positive neurons in the hypothalamus. Individual differences in the extinction of cue conditioned fear were associated with differential activation of hypothalamic orexin neurons. Animals showing poor extinction of cue-induced freezing (high freezers) had significantly greater percentage of orexin neurons with Fos in the medial hypothalamus than animals displaying significant extinction and good extinction recall (low freezers). Further, the freezing during extinction learning was positively correlated with the percentage of activated orexin neurons in both the lateral and medial hypothalamic regions. No differences in the overall density of orexin neurons or Fos activation were seen between extinction phenotypes. Although correlative, our results support other studies implicating a role of the orexinergic system in regulating extinction of conditioned responses to threat.

The Distribution of Substance P and Kisspeptin in the Mediobasal Hypothalamus of the Male Rhesus Monkey and a Comparison of Intravenous Administration of These Peptides to Release GnRH as Reflected by LH Secretion.

Substance P (SP) was recently reported to be expressed in human kisspeptin/neurokinin B/dynorphin (KNDy) neurons and to enhance KNDy neuron excitability in the mouse hypothalamus. We therefore examined (1) interactions of SP and kisspeptin in the mediobasal hypothalamus of adult male rhesus monkeys using immunofluorescence, and (2) the ability of SP to induce LH release in GnRH-primed, agonadal juvenile male monkeys. SP cell bodies were observed only occasionally in the arcuate nucleus (Arc), but more frequently dorsal to the Arc in the region of the premammillary nucleus. Castration resulted in an increase in the number of SP cell bodies in the Arc but not in the other regions. SP fibers innervated the Arc, where they were found in close apposition with kisspeptin perikarya in the periphery of this nucleus. Beaded SP axons projected to the median eminence, where they terminated in the external layer and intermingled with beaded kisspeptin axons. Colocalization of the two peptides, however, was not observed. Although close apposition between SP fibers and kisspeptin neurons suggest a role for SP in modulating GnRH pulse generator activity, i.v. injections of SP failed to elicit release of GnRH (as reflected by LH) in the juvenile monkey. Although the finding of structural interactions between SP and kisspeptin neurons is consistent with the notion that this tachykinin may be involved in regulating pulsatile GnRH release, the apparent absence of expression of SP in KNDy neurons suggests that this peptide is unlikely to be a fundamental component of the primate GnRH pulse generator.

Alcohol alters insulin-like growth factor-1-induced transforming growth factor ß1 synthesis in the medial basal hypothalamus of the prepubertal female rat.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) and transforming growth factor ß1 (TGFß1) are produced in hypothalamic astrocytes and facilitate luteinizing hormone-releasing hormone (LHRH) secretion. IGF-1 stimulates release by acting directly on the LHRH nerve terminals and both peptides act indirectly through specific plastic changes on glial/tanycyte processes that further support LHRH secretion. Because the relationship between these growth factors in the hypothalamus is not known, we assessed the ability of IGF-1 to induce TGFß1 synthesis and release and the actions of alcohol (ALC) on this mechanism prior to the onset of puberty. METHODS: Hypothalamic astrocytes were exposed to medium only, medium plus IGF-1 (200 ng/ml), or medium plus IGF-1 with 50 mM ALC. After 18 hours, media were collected and assayed for TGFß1. For the in vivo experiment, prepubertal female rats were administered either ALC (3 g/kg) or water via gastric gavage at 07:30 hours and at 11:30 hours. At 09:00 hours, saline or IGF-1 was administered into the third ventricle. Rats were killed at 15:00 hours and the medial basal hypothalamus (MBH) was collected for assessment of TGFß1, IGF-1 receptor (IGF-1R), and Akt. RESULTS: IGF-1 induced TGFß1 release (p < 0.01) from hypothalamic astrocytes in culture, an action blocked by ALC. In vivo, IGF-1 administration caused an increase in TGFß1 protein compared with controls (p < 0.05), an action blocked by ALC as well as a phosphatidylinositol 3 kinase/Akt inhibitor. IGF-1 stimulation also increased both total (p< 0.01) and phosphorylated (p)-IGF-1R (p < 0.05) protein levels, and phosphorylated (p)-Akt levels (p < 0.01), which were also blocked by ALC. CONCLUSIONS: This study shows that ALC blocks IGF-1 actions to stimulate synthesis and release of hypothalamic TGFß1, total and p-IGF-1R, and p-Akt levels further demonstrating the inhibitory actions of ALC on puberty-related events associated with hypothalamic LHRH release.

The medio-basal hypothalamus as a dynamic and plastic reproduction-related kisspeptin-gnrh-pituitary center in fish.

Kisspeptin regulates reproductive events, including puberty and ovulation, primarily via GnRH neurons. Prolonged treatment of prepubertal striped bass females with kisspeptin (Kiss) 1 or Kiss2 peptides failed to enhance puberty but suggested a gnrh-independent pituitary control pathway. Kiss2 inhibited, but Kiss1 stimulated, FShß expression and gonadal development, although hypophysiotropic gnrh1 and gnrh receptor expression remained unchanged. In situ hybridization and immunohistochemistry on brains and pituitaries revealed a differential plasticity between the 2 kisspeptin neurons. The differences were most pronounced at the prespawning phase in 2 regions along the path of gnrh1 axons: the nucleus lateralis tuberis (NLT) and the neurohypophysis. Kiss1 neurons appeared in the NLT and innervated the neurohypophysis of prespawning males and females, reaching Lh gonadotropes in the proximal pars distalis. Males, at all reproductive stages, had Kiss2 innervations in the NLT and the neurohypophysis, forming large axonal bundles in the former and intermingling with gnrh1 axons. Unlike in males, only preovulatory females had massive NLT-neurohypophysis staining of kiss2. Kiss2 neurons showed a distinct appearance in the NLT pars ventralis-equivalent region only in spawning zebrafish, indicating that this phenomenon is widespread. These results underscore the NLT as important nuclei for kisspeptin action in 2 facets: 1) kisspeptin-gnrh interaction, both kisspeptins are involved in the regulation of gnrh release, in a stage- and sex-dependent manner, especially at the prespawning phase; and 2) gnrh-independent effect of Kiss peptides on the pituitary, which together with the plastic nature of their neuronal projections to the pituitary implies that a direct gonadotropic regulation is plausible.

Role of microglia in ethanol's apoptotic action on hypothalamic neuronal cells in primary cultures.

BACKGROUND: Microglia are the major inflammatory cells in the central nervous system and play a role in brain injuries as well as brain diseases. In this study, we determined the role of microglia in ethanol's apoptotic action on neuronal cells obtained from the mediobasal hypothalamus and maintained in primary cultures. We also tested the effect of cAMP, a signaling molecule critically involved in hypothalamic neuronal survival, on microglia-mediated ethanol's neurotoxic action. METHODS: Ethanol's neurotoxic action was determined on enriched fetal mediobasal hypothalamic neuronal cells with or without microglia cells or ethanol-activated microglia-conditioned media. Ethanol's apoptotic action was determined using nucleosome assay. Microglia activation was determined using OX6 histochemistry and by measuring inflammatory cytokines secretion from microglia in cultures using enzyme-linked immunosorbent assay (ELISA). An immunoneutralization study was conducted to identify the role of a cytokine involved in ethanol's apoptotic action. RESULTS: We show here that ethanol at a dose range of 50 and 100 mM induces neuronal death by an apoptotic process. Ethanol's ability to induce an apoptotic death of neurons is increased by the presence of ethanol-activated microglia-conditioned media. In the presence of ethanol, microglia showed elevated secretion of various inflammatory cytokines, of which TNF-α shows significant apoptotic action on mediobasal hypothalamic neuronal cells. Ethanol's neurotoxic action was completely prevented by cAMP. The cell-signaling molecule also prevented ethanol-activated microglial production of TNF-α. Immunoneutralization of TNF-α prevented the microglia-derived media's ability to induce neuronal death. CONCLUSIONS: These results suggest that ethanol's apoptotic action on hypothalamic neuronal cells might be mediated via microglia, possibly via increased production of TNF-α. Furthermore, cAMP reduces TNF-α production from microglia to prevent ethanol's neurotoxic action.

Cell organization of the rat pars tuberalis. Evidence for open communication between pars tuberalis cells, cerebrospinal fluid and tanycytes.

The pars tuberalis (PT) is the only pituitary region in close contact with the medial-basal hypothalamus and bathed by cerebrospinal fluid (CSF). Although PT has long been recognized as an endocrine gland, certain aspects of its structure remain obscure. The present investigation has been designed to gain information concerning (1) the cellular organization of PT, (2) the PT/median eminence spatial relationship and (3) the exposure of various cell compartments of PT to CSF. Non-endocrine cells (S100-reactive) appear as the organizer of the PT architecture. The apical poles of these cells line large cistern-like cavities and the processes of these cells establish a close spatial relationship with PT-specific secretory cells, portal capillaries and tanycytes. The cisterns are also endowed with clusters of ciliated cells and with a highly electron-dense and PAS-reactive content. The unique spatial organization of endocrine and non-endocrine cells of the PT supports a functional relationship between both cell populations. PT endocrine cells display a hallmark of PT-specific cells, namely, the paranuclear spot, which is a complex structure involving the Golgi apparatus, a large pool of immature secretory granules and a centriole from which originates a single 9+0 cilium projecting to the intercellular channels. Horseradish peroxidase (HRP) injected into the CSF readily reaches the intercellular channels of PT and the inner channel of the single cilium and is incorporated by the endocytic machinery of the secretory cells. The PT endocrine cells, through their single 9+0 cilium, may act as sensors of the CSF. HRP also reaches the lumen of the cisterns, indicating that this PT compartment is also exposed to CSF. PT endocrine cells establish direct cell-to-cell contacts with hypothalamic beta(1) tanycytes, suggesting a second means of brain-PT communication.

Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse.

In female mammals, increased ovarian estradiol (E(2)) secretion triggers GnRH release from neurons in the basal forebrain, which drives LH secretion from the pituitary and subsequently induces ovulation. However, the neural circuits that activate this preovulatory GnRH/LH surge remain unidentified. Neurotensin is expressed in neurons of the anteroventral periventricular nucleus (AVPV), a region thought to be critical for generating the preovulatory GnRH/LH surge. E(2) induces neurotensin (Nts) gene expression in this region, and blockade of neurotensin signaling reduces the LH surge in the rat. We postulated that neurotensin signaling plays a similar role in generating the E(2)-induced GnRH/LH surge in mice. We used in situ hybridization (ISH) to determine whether E(2) induces Nts expression in the mouse and found evidence to support this proposition. Next, we determined that the neurotensin receptor (Ntsr2) is present in many GnRH-expressing neurons. Since the kisspeptin gene (Kiss1) is expressed in the AVPV and is responsive to E(2), we predicted that some neurons in this region express both Kiss1 and Nts; however, by double-label ISH, we observed no coexpression of the two mRNAs. We also postulated that Nts mRNA expression would increase in parallel with the E(2)-induced LH surge and that the central (icv) administration of neurotensin would stimulate LH secretion and activation of GnRH neurons but found no evidence to support either of these hypotheses. Together, these findings suggest that, although neurotensin neurons in the AVPV are targets for regulation by E(2), neurotensin does not appear to play a direct role in generating the GnRH/LH surge in the mouse.

Presence of delta opioid receptors on a subset of hypothalamic gonadotropin releasing hormone (GnRH) neurons.

Opioid peptides exert an inhibitory effect on hypothalamic gonadotropin releasing hormone (GnRH) secretion mainly by interacting with mu-opioid receptors. Although a direct role for opioids via delta-opioid receptors (DORs) has been suggested, the presence of these receptors on GnRH neurons has never been demonstrated. In the present study, we determined the distribution of DORs in the basal hypothalamus of rat with special focus on their relation to GnRH neurons. Double-labelling immunofluorescence and confocal microscopy revealed that DORs are exclusively present in a subpopulation of GnRH nerve terminals, with the highest density in the external layer of the median eminence. We then studied the functional characteristics of DORs in an immortalized GnRH-secreting neuronal cell line (GT1-1) known to endogenously express this receptor. Here, pertussis toxin pretreatment abolished the delta-agonist (DPDPE) inhibitory effect on cAMP accumulation. We also analyzed the type of G proteins involved in the signal transduced by the DOR and showed that GT1-1 cells express the inhibitory Go and Gi2 alpha-subunits. However, only Go was down-regulated under chronic DPDPE exposure. Finally, since DOR is expressed postnatally in brain, we compared GnRH neuronal cells immortalized at different developmental stages (the more mature GT1-1 and GT1-7 cells, versus the more immature GN11 cells), evidencing that only mature neurons express DOR. In conclusion, our study indicates that a direct control of opioids via delta-receptors occurs on GnRH neurons and validates the use of GT1 cells to further investigate the nature of the DOR present on GnRH neurons.

Ontogeny of the projections from the anteroventral periventricular nucleus of the hypothalamus in the female rat.

Neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) mediate a variety of autonomic functions. In adults they primarily innervate neuroendocrine nuclei in the periventricular zone of the hypothalamus, including the paraventricular and arcuate nuclei (PVH, ARH). Ascending projections from the AVPV also provide inputs to the ventrolateral septum (LSv) and the principal division of the bed nuclei of the stria terminalis (BSTp). Consistent with a role in regulating preovulatory luteinizing hormone secretion, rostral projections from the AVPV contact gonadotropin-releasing hormone (GnRH) neurons surrounding the vascular organ of the lamina terminalis (OVLT). To study the development of these pathways, we placed implants of the lipophilic tracers DiI and CMDiI into the AVPV of female rats ranging in age from embryonic day 19 (E19) through adulthood. The earliest projections targeted a population of GnRH neurons, with apparent contacts from labeled fibers observed as early as E19. These connections appeared to be fully developed before birth, as similar numbers of appositions from AVPV projections onto the GnRH-immunoreactive cells were observed at all ages examined. Caudal projections were delayed relative to projections to the OVLT. Labeled AVPV fibers reached the PVH during the first postnatal week, and fibers targeting the BSTp and LSv were not observed until the second and third postnatal weeks, respectively. Labeled AVPV fibers were not seen in the ARH of animals at any age. Our results demonstrate that projections from the AVPV develop with both spatial and temporal specificity, innervating each target with a unique developmental profile.

Hypothalamic tanycytes: a key component of brain-endocrine interaction.

Tanycytes are bipolar cells bridging the cerebrospinal fluid (CSF) to the portal capillaries and may link the CSF to neuroendocrine events. During the perinatal period a subpopulation of radial glial cells differentiates into tanycytes, a cell lineage sharing some properties with astrocytes and the radial glia, but displaying unique and distinct morphological, molecular, and functional characteristics. Four populations of tanycytes, alpha(1,2) and beta(1,2), can be distinguished. These subtypes express differentially important functional molecules, such as glucose and glutamate transporters; a series of receptors for neuropeptide and peripheral hormones; secretory molecules such as transforming growth factors, prostaglandin E(2), and the specific protein P85; and proteins of the endocytic pathways. This results in functional differences between the four subtypes of tanycytes. Thus, alpha(1,2) tanycytes do not have barrier properties, whereas beta(1,2) tanycytes do. Different types of tanycytes use different mechanisms to internalize and transport cargo molecules; compounds internalized via a clathrin-dependent endocytosis would only enter tanycytes from the CSF. There are also differences in the neuron-tanycyte relationships; beta(1,2) tanycytes are innervated by peptidergic and aminergic neurons, but alpha(1,2) tanycytes are not. Important aspects of the neuron-beta(1) tanycyte relationships have been elucidated. Tanycytes can participate in the release of gonadotropin-releasing hormone (GnRH) to the portal blood by expressing estrogen receptors, absorbing molecules from the CSF, and providing signal(s) to the GnRH neurons. Removal of tanycytes prevents the pulse of GnRH release into the portal blood, the peak of luteinizing hormone, and ovulation. The discovery in tanycytes of new functional molecules is opening a new field of research. Thus, thyroxine deiodinase type II, an enzyme generating triiodothyronine (T(3)) from thyroxine, appears to be exclusively expressed by tanycytes, suggesting that these cells are the main source of brain T(3). Glucose transporter-2 (GLUT-2), a low-affinity transporter of glucose and fructose, and ATP-sensitive K(+) channels are expressed by tanycytes, suggesting that they may sense CSF glucose concentrations.

Sex and estrogenic effects on coexpression of mRNAs in single ventromedial hypothalamic neurons.

Regulated gene expression in single neurons can be linked to biophysical events and behavior in the case of estrogen-regulated gene expression in neurons in the ventrolateral portion of the ventromedial nucleus (VMN) of the hypothalamus. These cells are essential for lordosis behavior. What genes are coexpressed in neurons that have high levels of mRNAs for estrogen receptors (ERs)? We have been able to isolate and measure certain mRNAs from individual VMN neurons collected from rat hypothalamus. Large numbers of neurons express mRNA for ERalpha, but these neurons are not identical with the population of VMN neurons expressing the likely gene duplication product, ERbeta. An extremely high proportion of neurons expressing either ER also coexpress mRNA for the oxytocin receptor (OTR). This fact matches the known participation of oxytocin binding and signaling in sexual and affiliative behaviors. In view of data that ER and OTR can signal through PKCs, we looked at coexpression of selected PKCs in the same individual neurons. The most discriminating analysis was for triple coexpression of ERs, OTR, and each selected PKC isoform. These patterns of triple coexpression were significantly different for male vs. female VMN neurons. Further, individual neurons expressing ERalpha could distribute their signaling across the various PKC isoforms differently in different cells, whereas the reverse was not true. These findings and this methodology establish the basis for systematic linkage of the brain's hormone-sensitive signaling pathways to biophysical and behavioral mechanisms in a well studied mammalian system.

Prolactin-regulated tyrosine hydroxylase activity and messenger ribonucleic acid expression in mediobasal hypothalamic cultures: the differential role of specific protein kinases.

Prolactin secretion from the anterior pituitary is tightly regulated by feedback onto the hypothalamic neuroendocrine dopaminergic (NEDA) neurons. Prolactin stimulates these neurons to synthesize and secrete dopamine, which acts via the pituitary portal vasculature to inhibit prolactin secretion from the pituitary lactotrophs. Despite the physiological importance of this feedback, relatively little is known about the signaling mechanisms responsible for prolactin activation of NEDA neurons. This issue has been examined here using a cell culture preparation of the fetal rat mediobasal hypothalamus. Prolactin stimulated a time- and concentration-dependent increase in catecholamine synthesis, which was maximal after 60-120 min (1 microg/ml prolactin) and inhibited by the prolactin antagonist Delta1-9-G129R-hPRL. This prolactin response was accompanied by a rise in the site-specific (ser-19, -31, and -40) phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Consistent with this observation, the prolactin-induced increase in catecholamine synthesis was abolished by inhibitors of protein kinase A and protein kinase C (PKC). Prolactin incubation also resulted in a PKC-dependent activation of the MAPK pathway, although this was not required for the stimulation of catecholamine synthesis. In addition to increasing TH phosphorylation and catecholamine synthesis, prolactin also increased TH mRNA expression. In contrast to catecholamine synthesis, this latter response was not suppressed by inhibition of protein kinase A or PKC. These results indicate that although prolactin controls catecholamine synthesis in NEDA neurons by regulating both TH activity and TH mRNA expression, it employs distinct, nonoverlapping, signaling pathways to achieve these ends.

Excitatory effects of ATP on rat dorsomedial hypothalamic neurons.

We investigated P2X purinoceptors in rat dorsomedial hypothalamic (DMH) neurons using nystatin-perforated patch-clamp recordings and fura-2 microfluorometry. Adenosine triphosphate (ATP) concentration-dependently evoked an inward current and increased cytosolic Ca(2+) ([Ca](i)). The rise in [Ca](i) was dependent on external Ca(2+) and Na(+), was blocked by Ca(2+) channel antagonists and had pharmacological properties consistent with P2X2 receptors. These results suggest that P2X receptor-mediated depolarization activates voltage-gated Ca(2+) channels, resulting in an increase in [Ca](i).

Evidence that dynorphin plays a major role in mediating progesterone negative feedback on gonadotropin-releasing hormone neurons in sheep.

Endogenous opioid peptides (EOP) mediate progesterone-negative feedback in many species, but the specific EOP systems involved remain unresolved. We first addressed this question in sheep by determining the role of different EOP receptor subtypes in the medial basal hypothalamus (MBH) and preoptic area (POA). Local administration of EOP receptor antagonists to luteal phase ewes indicated that kappa-, but not micro- or delta-, receptors mediate the inhibition of LH secretion in the MBH. In contrast, both kappa- and micro-, but not delta-receptor, antagonists increased LH pulse frequency when placed in the POA. We next examined close appositions between dynorphin (kappa ligand) and beta-endorphin (micro ligand) containing varicosities and GnRH perikarya in luteal phase ewes using dual immunocytochemistry and light microscopy. Approximately 90% of MBH GnRH neurons had close associations by dynorphin-containing varicosities, but only 40-50% of GnRH perikarya elsewhere had such close associations. In contrast, the percentage of beta-endorphinergic varicosities close to GnRH neurons was similar among all regions. Electron microscopic analysis demonstrated both dynorphinergic synapses and beta-endorphinergic synapses onto GnRH perikarya. These and other data lead to the hypothesis that dynorphin neurons play a major role in progesterone-negative feedback in the ewe and that this inhibition may be exerted directly on GnRH perikarya within the MBH, whereas dynorphin and beta-endorphin input to GnRH neurons in the POA provide redundancy to this system or are involved in other actions of progesterone or estradiol in the control of the GnRH surge.

Mating induces gonadotropin-releasing hormone neuronal activation in anosmic female ferrets.

In females of both spontaneously and induced ovulating species, pheromones from male conspecifics can directly stimulate GnRH neuronal activity, thereby inducing pituitary LH secretion and stimulating the onset of estrus. However, whether pheromones contribute to the steroid- or mating-induced preovulatory activation of GnRH neurons is less clear. Previous studies in the ferret, an induced ovulator, raised the possibility that olfactory cues contribute to the ability of genital-somatosensory stimulation to activate GnRH neurons in the mediobasal hypothalamus (MBH). In the present study the percentage of GnRH neurons colabeled with Fos-immunoreactivity (IR), used as a marker for neuronal activation, was investigated in the MBH of mated gonadectomized, estradiol-treated female ferrets in which both nares were occluded. In addition, the percentage of GnRH neurons colabeled with Fos-IR was examined in the MBH of gonadectomized, estradiol-treated female ferrets exposed to male bedding. Bilateral nares occlusion successfully blocked mating or odor-induced increments in Fos-IR in central olfactory regions, including the cortical and medial amygdala. By contrast, the percentage of GnRH neurons expressing Fos-IR did not differ between mated nares- and sham-occluded females. Exposure to male bedding alone failed to induce Fos-IR in MBH GnRH neurons. Thus, the mating-induced preovulatory activation of GnRH neurons in the female ferret's MBH appears to rely solely on genital-somatosensory as opposed to olfactory inputs.

Progesterone increases mRNA levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and type I PACAP receptor (PAC(1)) in the rat hypothalamus.

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates pituitary hormone biosynthesis and secretion through its cognate receptors. PACAP also plays an important role in the regulation of ovarian steroid biosynthesis. If so, there might be a feedback regulation of hypothalamic PACAP synthesis by the pituitary and by ovarian steroids. In the present study, we used RNase protection assays to determine changes in mRNA levels of PACAP and type I PACAP receptor (PAC(1)) under the conditions of ovariectomy and replacement with ovarian steroids. Progesterone (P) alone or in combination with estradiol (E) induced significant increases in PACAP mRNA level in the medial basal hypothalamus (MBH) and PAC(1) mRNA levels in MBH and the preoptic area (POA). This finding suggests that feedback regulation takes place between the ovary and hypothalamic PACAP neurons. P is known to be a major regulatory feedback factor for hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons, but P receptor is not present in these neurons. Therefore, we examined a possible involvement of PACAP in the feedback regulatory pathway of P to LHRH neurons. After an antisense PAC(1) oligodeoxynucleotide (ODN) was i.c.v.-injected into the third ventricle of E and P-treated rats, LHRH mRNA levels were determined. The ODN markedly decreased the P-induced increase in the LHRH mRNA level. Taken together, the present data suggest that PACAP may play a role as a mediator in the regulation of LHRH synthetic machinery by stimulatory feedback of P.

A second look at the barriers of the medial basal hypothalamus.

The cell bodies of hypothalamic secretory neurons are localized in areas protected by the blood-brain barrier (BBB), whereas their axon terminals are localized in the median eminence, which lacks a BBB. This implies a complex barrier system, allowing neurons of the central nervous system to secrete into the blood stream without making the BBB leaky. In the present study, three experimental protocols were applied to clarify certain relevant aspects of the barriers operating in the medial basal hypothalamus of the rat. We established that the milieu of the arcuate nucleus is exposed to both the ventricular and the subarachnoidal cerebrospinal fluid (CSF). The median eminence milieu, the perivascular space of the portal vessels, and the subarachnoid space appear to be in open communication; also, beta2-tanycytes establish an efficient barrier between the median eminence milieu and the ventricular CSF. Similarly, beta1-tanycytes establish a lateral barrier, separating the intercellular space of the median eminence from that of the arcuate nucleus. We also found that the glucose transporter I (GLUT I), a BBB marker, is localized throughout the whole plasma membrane of beta1-tanycytes, but is missing from beta2-tanycytes. Expression of GLUT I by tanycytes progressively develops during the first postnatal weeks; while the degree of damage of the arcuate nucleus by administration of monosodium glutamate, at different postnatal intervals, parallels that of the GLUT I immunoreactivity of beta1-tanycytes. An explanation is offered for the selective destruction of the arcuate neurons by the parenteral administration of monosodium glutamate to infant rats.

The distribution of progesterone receptor immunoreactivity and mRNA in the preoptic area and hypothalamus of the ewe: upregulation of progesterone receptor mRNA in the mediobasal hypothalamus by oestrogen.

The distribution of progesterone receptors (PR) was mapped in the hypothalamus of the ewe using immunocytochemistry. These results were confirmed using in situ hybridization with a sheep-specific 35S-labelled riboprobe. In addition, the effect of oestrogen on the level of PR mRNA in the hypothalamus was examined in ovariectomized (OVX) ewes following treatment with an oestrogen implant or without treatment. PR immunoreactive (-ir) cells were readily detected in OVX animals. Labelled cells were observed in four main hypothalamic regions: the preoptic area (POA), including the organum vasculosum of the lamina terminalis, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and the arcuate nucleus (ARC) (including the region ventral to the mamillary recess). In addition, lightly stained PR-ir cells were observed in the supraoptic nucleus and a few PR-ir cells were also found in the diagonal band of Broca. No PR-ir cells were found in the brainstem. PR mRNA-containing cells were found in the same hypothalamic regions as the PR-ir cells. Image analysis of emulsion-dipped slides following in situ hybridization indicated that oestrogen treatment increased (P<0.01) the mean number of silver grains/cell and the density of labelled cells in the VMN and ARC but had no effect on the level of PR mRNA expression in the POA or PeN. The distribution of PR-containing cells in the hypothalamus is similar to that described in other species and all cells were located in nuclei that contain large populations of oestrogen receptor-containing cells. These include regions implicated in the regulation of reproductive neuroendocrine function, and reproductive behaviour. Oestrogen and progesterone synergize to inhibit GnRH secretion and the present results suggest that these functions may involve cells of the VMN and ARC, with oestrogen acting to upregulate PR.