Neurog2 Acts as a Classical Proneural Gene in the Ventromedial Hypothalamus and Is Required for the Early Phase of Neurogenesis.

The tuberal hypothalamus is comprised of the dorsomedial, ventromedial, and arcuate nuclei, as well as parts of the lateral hypothalamic area, and it governs a wide range of physiologies. During neurogenesis, tuberal hypothalamic neurons are thought to be born in a dorsal-to-ventral and outside-in pattern, although the accuracy of this description has been questioned over the years. Moreover, the intrinsic factors that control the timing of neurogenesis in this region are poorly characterized. Proneural genes, including Achate-scute-like 1 (Ascl1) and Neurogenin 3 (Neurog3) are widely expressed in hypothalamic progenitors and contribute to lineage commitment and subtype-specific neuronal identifies, but the potential role of Neurogenin 2 (Neurog2) remains unexplored. Birthdating in male and female mice showed that tuberal hypothalamic neurogenesis begins as early as E9.5 in the lateral hypothalamic and arcuate and rapidly expands to dorsomedial and ventromedial neurons by E10.5, peaking throughout the region by E11.5. We confirmed an outside-in trend, except for neurons born at E9.5, and uncovered a rostrocaudal progression but did not confirm a dorsal-ventral patterning to tuberal hypothalamic neuronal birth. In the absence of Neurog2, neurogenesis stalls, with a significant reduction in early-born BrdU+ cells but no change at later time points. Further, the loss of Ascl1 yielded a similar delay in neuronal birth, suggesting that Ascl1 cannot rescue the loss of Neurog2 and that these proneural genes act independently in the tuberal hypothalamus. Together, our findings show that Neurog2 functions as a classical proneural gene to regulate the temporal progression of tuberal hypothalamic neurogenesis.SIGNIFICANCE STATEMENT Here, we investigated the general timing and pattern of neurogenesis within the tuberal hypothalamus. Our results confirmed an outside-in trend of neurogenesis and uncovered a rostrocaudal progression. We also showed that Neurog2 acts as a classical proneural gene and is responsible for regulating the birth of early-born neurons within the ventromedial hypothalamus, acting independently of Ascl1 In addition, we revealed a role for Neurog2 in cell fate specification and differentiation of ventromedial -specific neurons. Last, Neurog2 does not have cross-inhibitory effects on Neurog1, Neurog3, and Ascl1 These findings are the first to reveal a role for Neurog2 in hypothalamic development.

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.

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.

Glucose regulates hypothalamic long-chain fatty acid metabolism via AMP-activated kinase (AMPK) in neurons and astrocytes.

Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance.

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.

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.

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.

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.

Use of calcium imaging for analysis of neuronal gap junction coupling.

We recently used Western blots for connexin 36 and neuronal dye coupling with neurobiotin to measure developmental decrease in neuronal gap junction coupling in cell cultures. To ask whether Ca2+ imaging also can be used to measure changes in the amount of neuronal gap junction coupling, we defined a Ca2+ coupling coefficient as the percentage of neurons with bicuculline-induced increases in intracellular Ca2+ that are suppressed by blocking gap junctions. We demonstrate in rat and mouse hypothalamic neuronal cultures that the Ca2+ coupling coefficient decreases during culture development, this decrease is prevented by manipulations that also prevent developmental decrease in neuronal gap junction coupling, and the coefficient is low in cultures lacking connexin 36. The results indicate that Ca2+ imaging is a useful tool to quantify the amount of neuronal gap junction coupling in cultures.

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.

Direct versus indirect actions of ghrelin on hypothalamic NPY neurons.

OBJECTIVES: Assess direct versus indirect action(s) of ghrelin on hypothalamic NPY neurons. MATERIALS AND METHODS: Electrophysiology was used to measure ion channel activity in NPY-GFP neurons in slice preparations. Ca2+ imaging was used to monitor ghrelin activation of isolated NPY GFP-labeled neurons. Immunohistochemistry was used to localize Trpm4, SUR1 and Kir6.2 in the hypothalamus. RESULTS: Acylated ghrelin depolarized the membrane potential (MP) of NPY-GFP neurons in brain slices. Depolarization resulted from a decreased input resistance (IR) in ~70% of neurons (15/22) or an increased IR in the remainder (7/22), consistent with the opening or closing of ion channels, respectively. Although tetrodotoxin (TTX) blockade of presynaptic action potentials reduced ghrelin-induced changes in MP and IR, ghrelin still significantly depolarized the MP and decreased IR in TTX-treated neurons, suggesting that ghrelin directly opens cation channel(s) in NPY neurons. In isolated NPY-GFP neurons, ghrelin produced a sustained rise of [Ca2+]c, with an EC50 ~110 pM. Pharmacologic studies confirmed that the direct action of ghrelin was through occupation of the growth hormone secretagogue receptor, GHS-R, and demonstrated the importance of the adenylate cyclase/cAMP/protein kinase A (PKA) and phospholipase C/inositol triphosphate (PLC/IP3) pathways as activators of 5' AMP-activated protein kinase (AMPK). Activation of isolated neurons was not affected by CNQX or TTX, but reducing [Na+]o suppressed activation, suggesting a role for Na+-permeable cation channels. SUR1 and two channel partners, Kir6.2 and Trpm4, were identified immunologically in NPY-GFP neurons in situ. The actions of SUR1 and Trpm4 modulators were informative: like ghrelin, diazoxide, a SUR1 agonist, elevated [Ca2+]c and glibenclamide, a SUR1 antagonist, partially suppressed ghrelin action, while 9-phenanthrol and flufenamic acid, selective Trpm4 antagonists, blocked ghrelin actions on isolated neurons. Ghrelin activation was unaffected by nifedipine and ω-conotoxin, inhibitors of L- and N-type Ca2+ channels, respectively, while Ni2+, mibefradil, and TTA-P2 completely or partially inhibited ghrelin action, implicating T-type Ca2+ channels. Activation was also sensitive to a spider toxin, SNX-482, at concentrations selective for R-type Ca2+ channels. Nanomolar concentrations of GABA markedly inhibited ghrelin-activation of isolated NPY-GFP neurons, consistent with chronic suppression of ghrelin action in vivo. CONCLUSIONS: NPY neurons express all the molecular machinery needed to respond directly to ghrelin. Consistent with recent studies, ghrelin stimulates presynaptic inputs that activate NPY-GFP neurons in situ. Ghrelin can also directly activate a depolarizing conductance. Results with isolated NPY-GFP neurons suggest the ghrelin-activated, depolarizing current is a Na+ conductance with the pharmacologic properties of SUR1/Trpm4 non-selective cation channels. In the isolated neuron model, the opening of SUR1/Trpm4 channels activates T- and SNX482-sensitive R-type voltage dependent Ca2+ channels, which could contribute to NPY neuronal activity in situ.

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.

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.

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.

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.

Hypothalamic glucose sensor: similarities to and differences from pancreatic beta-cell mechanisms.

Glucose-responsive neurons in the ventromedial hypothalamus (VMH) are stimulated when glucose increases from 5 to 20 mmol/l and are thought to play an essential role in regulating metabolism. The present studies examined the role of glucose metabolism in the mechanism by which glucose-responsive neurons sense glucose. The pancreatic, but not hepatic, form of glucokinase was expressed in the VMH, but not cerebral cortex, of adult rats. In brain slice preparations, the transition from 5 to 20 mmol/l glucose stimulated approximately 17% of the neurons (as determined by single-cell extracellular recording) from VMH but none in cortex. In contrast, most cells in both VMH and cortex were silent below 1 mmol/l and active at 5 mmol/l glucose. Glucosamine, 2-deoxyglucose, phloridzin, and iodoacetic acid blocked the activation of glucose-responsive neurons by the transition from 5 to 20 mmol/l glucose. Adding 15 mmol/l mannose, galactose, glyceraldehyde, glycerol, and lactate to 5 mmol/l glucose stimulated glucose-responsive neurons. In contrast, adding 15 mmol/l pyruvate to 5 mmol/l glucose failed to activate glucose-responsive neurons, although pyruvate added to 0 mmol/l glucose permitted neurons to maintain activity. Tolbutamide activated glucose-responsive neurons; however, diazoxide only blocked the effect of glucose in a minority of neurons. These data suggest that glucose-responsive neurons sense glucose through glycolysis using a mechanism similar to the mechanism of pancreatic beta-cells, except that glucose-responsive neurons are stimulated by glycerol and lactate, and diazoxide does not generally block the effect of glucose.

Neuropeptide-Y innervation of beta-endorphin-containing cells in the rat mediobasal hypothalamus: a light and electron microscopic double immunostaining analysis.

Central administration of neuropeptide-Y (NPY) inhibits pituitary LH release in ovariectomized rats and stimulates LH release in intact and ovariectomized rats pretreated with ovarian steroids. Although the precise neural mechanism of this dual effect of NPY is not known, experimental evidence suggests an underlying interaction between hypothalamic NPY and the inhibitory beta-endorphin (beta END) systems in the neuroendocrine regulation of pituitary LH release in the rat. The present study was undertaken to examine the morphological basis of the interaction between these two peptidergic systems in the hypothalamus. Sections of the mediobasal hypothalamus of colchicine-pretreated female rats were double immunostained for NPY and beta END and examined by light and electron microscopy. The light brown diaminobenzidine reaction was used to visualize beta END cells, while NPY neurons were labeled with a dark blue nickel ammonium sulfate-intensified diaminobenzidine reaction. Under the light microscope, a dense network of NPY-immunoreactive axons and axon terminals was observed in close apposition with beta END-immunoreactive neurons throughout the medial basal hypothalamus. Electron microscopic examination revealed that NPY-immunoreactive boutons formed axosomatic and axo-dendritic synaptic connections with beta END cells. A majority of these synaptic membrane specializations appeared asymmetrical [corrected]. In light of the previous evidence of excitatory and inhibitory effects on LH release and the existence of direct synaptic connections between NPY and LHRH neurons in the hypothalamus, the current results imply that the dual effects of NPY on LH secretion may involve modulation of LHRH secretion, both by the direct route and indirectly through the hypothalamic beta END system.

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.

Stimulatory effect of human, but not bovine, growth hormone expression on numbers of tuberoinfundibular dopaminergic neurons in transgenic mice.

Mice transgenic for heterologous and ectopic GH expression serve as models for studying the feedback effects of elevated nonregulated GH on hypothalamic hypophysiotropic neurons as well as on peripheral function. For example, hypothalamic somatostatin expression has been shown to be increased markedly in mice bearing either bovine (b) or human (h) GH transgenes. Human, but not bovine, GH has lactogenic properties in mice, and appears to stimulate PRL-inhibiting tuberoinfundibular dopaminergic (TIDA) neurons. The present study was designed to determine the effect of a lifelong excess of hGH on dopamine (DA) expression in and numbers of TIDA neurons. Male mice of four transgenic lines were examined. The transgenic animals bore constructs of either bGH or hGH fused to either metallothionein (MT) or phosphoenolpyruvate carboxykinase (PEPCK) promoters; brains of transgenic mice were compared morphologically with those of nontransgenic littermates. Formaldehyde-induced catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry were examined in alternate brain sections; cell number was quantified for TIDA neurons (area A12) and a nonhypophysiotropic diencephalic DA area, the medial zona incerta (A13). Body weights were higher (P < 0.01) in PEPCK-GH than in MT-GH transgenic mice, as were serum levels of heterologous GH in those lines. In MT-hGH, but not MT-bGH or PEPCK-bGH, transgenic mice, A12 perikaryal fluorescence was enhanced, and ME fluorescence was reduced compared with those in control animals. The reduced ME DA is likely to reflect stimulation of TIDA neurons, because A12 TH-immunoreactive neuron number was increased by 34% in MT-hGH mice compared with that in controls (P < 0.05). In mice bearing the PEPCK-hGH construct, A12 TH neuron number was increased 47% (P < 0.001) compared with that in littermate controls. There were no differences in A13 cell number among animals, and A12 cell numbers in mice expressing bGH did not differ from control values. These results suggest that although extremely high levels of circulating bGH do not stimulate TIDA neurons, lifelong high levels of hGH have a stimulatory and graded effect on developmental differentiation of these cells for TH and DA production, supporting the concept of PRL as a trophic factor for TIDA neurons.