Estrogen receptor-beta in oxytocin and vasopressin neurons of the rat and human hypothalamus: Immunocytochemical and in situ hybridization studies

Topographical distribution of estrogen receptor-beta (ER-beta)-synthesizing oxytocin (OT) and vasopressin (VP) neurons was studied in the hypothalamic paraventricular and supraoptic nuclei (PVH; SO) of ovariectomized rats. In distinct subregions, 45-98% of OT neurons and 88-99% of VP neurons exhibited ER-beta immunoreactivity that was confined to cell nuclei. Neuronal populations differed markedly with respect to the intensity of the ER-beta signal. Magnocellular OT neurons in the PVH, SO, and accessory cell groups typically contained low levels of the ER-beta signal; in contrast, robust receptor labeling was displayed by OT cells in the ventral subdivision of medial parvicellular subnucleus and in the caudal PVH (dorsal subdivision of medial parvicellular subnucleus and lateral parvicellular subnucleus). Estrogen receptor-beta signal was generally more intense and present in higher proportions of magnocellular and parvicellular VP vs. OT neurons of similar topography. Immunocytochemical observations were confirmed via triple-label in situ hybridization, an approach combining use of digoxigenin-, fluorescein-, and 35S-labeled cRNA hybridization probes. Further, ER-beta mRNA was also detectable in corticotropin-releasing hormone neurons in the parvicellular PVH. Finally, double-label immunocytochemical analysis of human autopsy samples showed that subsets of OT and VP neurons also express ER-beta in the human. These neuroanatomical studies provide detailed information about the topographical distribution and cellular abundance of ER-beta within subsets of hypothalamic OT and VP neurons in the rat. The variable receptor content may indicate the differential responsiveness to estrogen in distinct OT and VP neuronal populations. In addition, a relevance of these findings to the human hypothalamus is suggested.     

Oxytocin reduces anxiety via ERK1/2 activation: local effect within the rat hypothalamic paraventricular nucleus

The neuropeptide oxytocin (OT) modulates social behaviours and is an important anxiolytic substance of the brain. However, sites of action and the intracellular signalling pathways downstream of OT receptors (OTR) within the brain remain largely unknown. In the present studies, we localized the anxiolytic effect of OT by bilateral microinfusion of OT (0.01 nmol/0.5 microL) into the hypothalamic paraventricular nucleus (PVN) in male rats using both the elevated plus-maze and the light-dark box. Moreover, intracerebroventricular administration of OT, but not of the related neuropeptide vasopressin (VP), dose-dependently activated the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. Specifically, OT induced the phosphorylation of Raf-1, MEK1/2 and ERK1/2 in the hypothalamus in vivo and in hypothalamic H32 neurons via EGF receptors. OT-induced ERK1/2 phosphorylation was immunohistochemically localized within VP neurons of the PVN and the supraoptic nucleus. Importantly, the anxiolytic effect of OT within the PVN was prevented by local inhibition of the MAP kinase cascade with a MEK1/2 inhibitor (U0126, 0.5 nmol/0.5 microL) locally infused prior to OT, indicating the causal involvement of this intracellular signalling cascade in the behavioural effect of OT. OT effects within the hypothalamus may have far-reaching implications for the regulation of emotionality and social behaviours and, consequently, for the development of possible therapeutic strategies to treat affective disorders. Thus, OTR agonism or activation of the ERK1/2 cascade, specifically within the hypothalamus, may provide therapeutically relevant mechanisms.     

Evidence That IGF-I Acts as an Autocrine/Paracrine Growth Factor in the Magnocellular Neurosecretory System: Neuronal Synthesis and Induction of Axonal Sprouting

The ability of mature oxytocinergic (OT) and vasopressinergic (VP) neurons of the magnocellular neurosecretory system (MNS) to undergo axonal growth implies that one or more growth factors may be active in the adult MNS, yet little is known regarding their possible identity. One such potential factor is insulin-like growth factor I (IGF-I). We have examined the expression of IGF-I mRNA and IGF-I-immunoreactivity (IGF-I-ir) in the mature MNS and have also determined the in vivo response of OT and VP neurons to hypothalamic implants of IGF-I. In situ hybridization revealed moderate labeling of IGF-I mRNA in both the supraoptic (SON) and the paraventricular (PVN) nuclei of adult male rats. RT-PCR analysis confirmed the presence of authentic IGF-I mRNA in extracts of the basal hypothalamus. Faint IGF-I-ir was detected in scattered magnocellular neurons within both the PVN and the SON of normal rats, but IGF-I-ir was much more intense and the majority of MNS neurons including those in the accessory nuclei were immunoreactive in sections from rats given colchicine, as were some parvocellular neurons in the PVN. Confocal microscopy revealed that IGF-I-ir was present in both OT and VP neurons, but VP neurons contained the most intense IGF-I-ir. Finally, a dramatic growth response of OT but not of VP fibers was observed following implantation of polymer rods containing IGF-I into the hypothalamus. A dense OT fiber plexus grew along the cannula track and OT fibers invaded the leptomeninges ventral to the SON and encircled the rostral cerebral artery. To our knowledge this is the first demonstration of axonal sprouting by mature OT neurons in response to an identified growth factor and the first direct demonstration of sprouting in response to exogenous IGF-I in the adult CNS. These findings suggest that IGF-I is synthesized and transported by adult MNS neurons where it may act as an autocrine and/or paracrine growth factor.     

Localization of agmatine in vasopressin and oxytocin neurons of the rat hypothalamic paraventricular and supraoptic nuclei

Agmatine (decarboxylated l-arginine), an endogenous ligand of imidazoline and alpha(2) adrenoreceptors, is particularly enriched in the rat hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. The present study utilized light and electron microscopic immunocytochemical methods to determine the distribution and extent of colocalization of agmatine relative to subpopulations of vasopressin- (VP) and oxytocin- (OT) producing neurons in PVN and SON nuclei. By light microscopy, agmatine-immunoreactive perikarya were found in both the magnocellular and the parvocellular neuronal subdivisions of PVN and SON. Confocal and electron microscopy revealed that agmatine-immunoreactivity (I) within neuronal perikarya was associated with the nuclear membrane as well as mitochondria, Golgi complexes, endoplasmic reticula, and plasmalemma. Additionally, agmatine-I was identified in both axons and axonal terminals, which were enriched in large dense-core vesicles. Dual and triple immunocytochemical labeling experiments also demonstrated that agmatine coexists with VP or OT in most PVN and SON magnocellular neurons. Combinations of iontophoretic injections of Fluorogold into the dorsomedullary complex with immunocytochemical labeling revealed that many retrogradely labeled neurons in the parvocellular region of the PVN contained agmatine-I and either VP or OT. These findings provide evidence that agmatine may function as a modulator of both hypothalamically mediated neuroendocrine and autonomic responses.     

Vasopressin and oxytocin gene expression in intact rats and under catecholamine deficiency during ontogenesis

The development of the hypothalamic vasopressin (VP) and oxytocin (OT) systems has been studied in rats from the 16th embryonic day (E16) until the 11th postnatal day (P11). The VP and OT mRNA-producing neurons were identified on cryostat sections by in situ hybridization using oligonucleotide probes Iabeled by [³⁵S], [³H] or digoxigenin. Moreover, VP and OT gene expressions were evaluated either at E21 or at P11 following chronic depletion of catecholamines (CA). For this purpose, pregnant rats were daily injected with α-methyl-m(p)-tyrosine from gestational day 13 to 20, while neonates were daily injected with a-methyl-m(p)-tyrosine and neurotoxin 6-hydroxydopamine from postnatal day 2 to 10. No VP mRNA- or OT mRNA-expressing cells were observed in the hypothalamus of intact fetuses at E16, while 2 days later rather numerous VP and OT neurons occupied the anterior hypothalamus. One major bilateral group of VP and OT neurons was located in the supreoptic nucleus (SON). Less numerous labeled cab were found in the developing paraventricular nucleus (PVN). Some VP and OT neurons were also spread along the ventrolateral surface of the hypothalamus from the level of the median eminence, caudally, to the level of the optic nerves, rostrally. From E18 until birth, the OT neurons were localized in the dorsal portion of the SON, while its ventral portion was occupied by the VP neurons. The VP mRNA- and OT mRNA-expressing cells seemed to increase both in size and in number over the perinatal period. Frequent relatively long neuronal processes contained VP and OT mRNAs in fetuses and in newborns. When performed during the second half of the fetal life, the chronic depletion of CA did not cause any change in the VP and OT mRNA concentrations in the SON and PVN of fetuses. By contrast, similar treatment of neonates resulted in a significant increase of both mRNA levels in the SON. These data suggest that at least in the SON VP and OT gene expressions might be under the inhibitory control of CA during the neonatal period.     

Estrogen suppresses mu-opioid- and GABAB-mediated hyperpolarization of hypothalamic arcuate neurons.

The effects of estrogen on the response of hypothalamic arcuate neurons to mu-opioid and GABAB agonists were investigated. Intracellular recordings were made from arcuate neurons in slices prepared from ovariectomized guinea pigs that were pretreated with estrogen or vehicle. Estrogen shifted the dose-response curve to the mu-opioid agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) by 3.4-fold; the EC50 for DAMGO was 240 +/- 25 nM in estrogen-treated females versus 70 +/- 12 nM in the controls. The maximal hyperpolarization induced by DAMGO was equivalent in neurons from both groups. The Ke for the naloxone antagonism of the DAMGO response was similar in both groups, which would indicate that the affinity of the mu-receptor was unchanged. To explore where in the receptor/G-protein/K+ channel cascade estrogen may be acting to attenuate the mu-opioid-mediated hyperpolarization, the response to the GABAB agonist baclofen was also tested. Estrogen treatment also shifted the dose-response curve for the baclofen-induced hyperpolarization by 3.3-fold without altering the maximum hyperpolarization; the EC50 shifted from 11.0 +/- 4.0 microM to 36.0 +/- 5.0 microM. All of the neurons were identified after linking the intracellular biocytin with streptavidin-FITC, and a subpopulation of cells in both groups were immunoreactive for beta-endorphin. We conclude that estrogen decreases the functional coupling of the mu-opioid and GABAB receptors to the inwardly rectifying K+ channel possibly through an action on the G-protein.     

Differences in the properties of ionotropic glutamate synaptic currents in oxytocin and vasopressin neuroendocrine neurons.

Oxytocin (OT) and vasopressin (VP) hormone release from neurohypophysial terminals is controlled by the firing pattern of neurosecretory cells located in the hypothalamic supraoptic (SON) and paraventricular nuclei. Although glutamate is a key modulator of the electrical activity of both OT and VP neurons, a differential contribution of AMPA receptors (AMPARs) and NMDA receptors (NMDARs) has been proposed to mediate glutamatergic influences on these neurons. In the present study we examined the distribution and functional properties of synaptic currents mediated by AMPARs and NMDARs in immunoidentified SON neurons. Our results suggest that the properties of AMPA-mediated currents in SON neurons are controlled in a cell type-specific manner. OT neurons displayed AMPA-mediated miniature EPSCs (mEPSCs) with larger amplitude and faster decay kinetics than VP neurons. Furthermore, a peak-scaled nonstationary noise analysis of mEPSCs revealed a larger estimated single-channel conductance of AMPARs expressed in OT neurons. High-frequency summation of AMPA-mediated excitatory postsynaptic potentials was smaller in OT neurons. In both cell types, AMPA-mediated synaptic currents showed inward rectification, which was more pronounced in OT neurons, and displayed Ca2+ permeability. On the other hand, NMDA-mediated mEPSCs of both cell types had similar amplitude and kinetic properties. The cell type-specific expression of functionally different AMPARs can contribute to the adoption of different firing patterns by these neuroendocrine neurons in response to physiological stimuli.     

Smooth muscle-associated protein 8: distribution and biological activity in the rat brain

With the use of an antiserum directed against the human smooth muscle-associated protein 8 (SMAP8) fragment SMAP8(98-138), Western blot and immunohistochemical studies revealed SMAP8 expression in the rat brain. A band with a molecular size of about 45 kDa was detected in tissues from the rat hypothalamus and a weaker band from the cortex. SMAP8 immunoreactivity (irSMAP8) was detected in neurons of the hypothalamic paraventricular, supraoptic, and supraoptic retrochiasmatic nuclei; a few irSMAP8 cells were scattered in the zona incerta as well as the cerebral cortex. Immunoreactive cell processes were detected mostly in the internal layer of the median eminence. Double labeling the hypothalamic sections with SMAP8 and vasopressin (VP) or oxytocin (OT) antiserum revealed that a population of VP- and OT-immunoreactive neurons expressed irSMAP8. The biological activity of SMAP8 in rat central neurons was assessed by the calcium microfluorimetric Fura-2 method. SMAP8 (100 nM) elevated cytosolic calcium concentrations [Ca2+]i in a population of dissociated and cultured rat hypothalamic neurons; the response was eliminated in Ca2+-free saline. This is the first evidence of irSMAP8 in a population of VP/OT-containing hypothalamic neurons in the rat, and the peptide is biologically active in hypothalamic neurons, as evidenced by mobilization of extracellular Ca2+.     

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.     

Survival and regeneration of neurons of the supraoptic nucleus following surgical transection of neurohypophysial axons depend on the existence of collateral projections of these neurons to the dorsolateral hypothalamus

The aim of the present study was to compare the postlesional responses of vasopressin-producing (VP) and oxytocin-producing (OT) neurons of the supraoptic nucleus (SON) to transection of neurohypophysial axons. At different times after sectioning the median eminence of adult rats, immunocytochemical staining of both types of neuronal cell bodies and axons indicated that: (1) the number of OT neurons detected within the SON was only slightly decreased as compared with controls (−20%), whereas the number of VP neurons was severely decreased (−60%); and (2) the large majority of axonal sprouts that regenerated into the external layer of the median eminence were OT neurohypophysial axons. The injection of a retrograde tracer into various areas surrounding the SON further showed that numerous SON neurons could be retrogradely labeled when the injection was centered in the lateral hypothalamus dorsal to the SON. The immunocytochemical identification of these retrogradely labeled neurons demonstrated that most of them were OT neurons. When animals were subjected to median eminence transection and to a unilateral surgical cut placed in the lateral hypothalamus above the SON, the survival of both OT and VP neurons was dramatically reduced in the SON ipsilateral to the hypothalamic lesion, as compared to the contralateral SON. Taken together, these data indicate that OT (and to a lesser extent VP) neurons of the SON display collateral projections towards the lateral hypothalamus that protect them from retrograde degeneration following the lesion of their neurohypophysial projections.     

Combination of immunohistochemical and in situ hybridization methods to reveal tyrosine hydroxylase and oxytocin and vasopressin mRNAs in magnocellular neurons of obese Zucker rats

Co-localization of chemical messengers in the same neuron is linked to neurochemical plasticity and has been studied extensively [B. Meister, M.J. Villar, S. Ceccatelli, T. H?kfelt, Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: an immunohistochemical study using experimental manipulation, Neuroscience 37 (1990) 603-633; B. Meister, R. Cortés, M.J. Villar, M. Schalling, T. H?kfelt, Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels, Cell Tissue Res. 260 (1990) 279-297]. Obese Zucker rats display an example of such a phenomenon expressing an enzyme of catecholamine synthesis-tyrosine hydroxylase (TH)-in magnocellular neurons (MCN) of supraoptic and paraventricular nuclei of hypothalamus [S. Fetissov, F. Marsais, S. Nicola?dis, A. Calas, Expression of tyrosine hydroxylase in magnocellular hypothalamic neurons of obese (fa/fa) and lean heterozygous (Fa/fa) Zucker rats, Mol. Brain Res. 50 (1997) 314-318]. To understand the biological role of TH in MCN of obese Zucker rat, we studied TH expression in relation to the vasopressinergic and oxytocinergic neurons. We present a protocol of double labelling including immunohistochemical for TH and in situ hybridization for OT and VP mRNA. The described protocol can be applied for detection of co-localized expressions of a broad range of chemical brain messengers and proteins.     

Effects of osmotic pressure and brain-derived neurotrophic factor on the survival of postnatal hypothalamic oxytocinergic and vasopressinergic neurons in dissociated cell culture

Neurons from hypothalamic paraventricular nuclei (PVN) and supraoptic nuclei (SON) from postnatal day 6-8 rats were enzymatically dissociated and separately maintained in monolayer cultures for 14 days. The osmotic pressure of the culture medium, based on Neurobasal medium (Life Technologies), was varied (255, 300 and 330 mOsm/l) by adjustment using mannitol. The survival of oxytocin (OT), vasopressin (VP) and oxytocin-vasopressin (OT/VP) coexpressing neurons were studied under these varied conditions, and the identification of the cell phenotypes in the cultures was carried out by using double-label immunofluorescence. Under control osmolar conditions (300 mOsm/l) equivalent numbers of OT and VP neurons were found in the SON (P = 0.8398) and PVN (P = 0.4721) cultures. The OT neurons' survival did not change in 255 or 330 mOsm media in the SON cultures, but the VP neurons in the SON cultures were significantly increased in 255 mOsm/l medium as compared to control (300 mOsm/l) medium (P = 0.0088). No significant changes were found in VP neuron survival in SON cultures between the 300-330 mOsm/l media (P = 0.2372). Similar data were obtained for the VP neurons in PVN-derived cultures, but the OT neurons in these cultures survived significantly better at 300 mOs/l than at 255 mOsm/l (P<0.0001), but were not significantly different at 330 mOsm/l (P = 0.1208). In general, the VP neurons were more vulnerable than OT neurons to increases of culture medium osmolarity with respect to their survival. The number of OT/VP coexpressing neurons was greater in SON-derived cell cultures as compared to PVN-derived cell cultures, and their numbers were higher in the lower osmolarity media. The effects of adding brain-derived neurotrophic factor (BDNF) to the culture medium on survival were determined. BDNF significantly increased the numbers of all three types of neurons in both PVN and SON cell cultures (P = 0.0001-0.0060). The phenotypically identified cells, cultured in the 300 mOsm/l medium, responded by depolarization or hyperpolarization when transferred to hypertonic or hypotonic perfusion salines, respectively.     

Localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamus

Previous pharmacological studies have indicated that ATP receptors may be involved in the regulation of physiological functions in hypothalamus. In the present study, the distribution of P2X₂ receptor in the rat hypothalamus was studied with immunohistochemistry. It was shown that P2X₂ immunoreactivity-positive neurons and nerve fibres were localized in many hypothalamic nuclei. Intense labelling of both neuronal cell bodies and nerve fibres was observed in the paraventricular nucleus, arcuate nucleus, retrochiasmatic area, periventricular nucleus, and the ventral part of tuber cinereum area. In supraoptic, circular, and ventral tuberomammillary nuclei the neuronal cell bodies were strongly positive, but few nerve fibres were positive. Axons with strong P2X₂ immunoreactivity were found in the organum vasculosum of the lamina terminalis and median eminence. Some scattered positive neurons and nerve fibres were found in many hypothalamic nuclei including preoptic nucleus. The results of the present study demonstrated the existence of P2X receptors in hypothalamus, as a basis for detailed studies of the roles of P2X receptors in the regulation of hypothalamic functions.     

Hypothalamic regulation of food intake

The central regulation of the food intake is organized by a long-loop mechanism involving humoral signals and afferent neuronal pathways to the hypothalamus, obligatory processing in hypothalamic neuronal circuits, and descending commands through vagal and spinal neurons to the body. Receptors sensitive to glucose metabolism, body fat reserves, distension of the stomach, as well as neuropeptide and cannabinoid receptors have been identified and localized in the hypothalamus. Five groups of cells in the hypothalamus--arcuate, paraventricular, ventromedial and dorsomedial nuclei, and the dorsolateral hypothalamic area--contain neurons with either anorexic actions (alpha-MSH, CART peptide, corticotropin-releasing hormone, urocortin III, cholecystokinin, glucagon-like peptides) or that stimulate food intake (neuropeptide Y, agouti-related peptide, orexins, melanin concentrating hormone, galanin). Intrahypothalamic neuronal circuits exist between these peptidergic neurons including the arcuate-paraventricular and arcuate-dorsolateral hypothalamic projections. Circulating substances carrying signals connected to changes in body food homeostasis and energy balance (leptin, ghrelin, insulin, glucose) enter the hypothalamus mainly through the arcuate nucleus. Neurons in the medulla oblongata that express leptin and insulin receptors, as well as neuropeptide mediators project to the hypothalamus. Vica versa, hypothalamic neurons give rise to projections to autonomic centers in the brainstem and the spinal cord with potential for stimulation or inhibition of food intake, energy balance and ingestion behavior.     

Nocistatin inhibits food intake in rats

Nocistatin, a product of the same precursor as nociceptin/orphanin FQ (N/OFQ), has been shown to antagonize effects of N/OFQ. N/OFQ stimulates feeding, most probably by inhibiting activation of neurons containing oxytocin (OT) and vasopressin (VP), peptides considered as satiety factors, and implicated in the development of conditioned taste aversion (CTA). The present study was designed to investigate whether intracerebroventricularly (ICV) injected nocistatin (a) affects deprivation- and N/OFQ-induced feeding, (b) causes CTA, and (c) induces activation of hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, as well as OT and VP neurons present in these regions. C-Fos immunohistochemistry was used as a marker of cellular activation. Nocistatin (1-3 nmol) significantly reduced food intake in deprived rats during the first and second hour post-injection. Doses of 1-3 nmol suppressed N/OFQ-induced feeding. Nocistatin at the highest (3 nmol) dose did not cause CTA. It also did not affect activation of the PVN or SON. In nocistatin-treated animals, the percentage of Fos-positive OT and VP neurons was similar to controls. We conclude that nocistatin antagonizes the influence of N/OFQ on feeding and suppresses deprivation-induced food consumption through mechanisms other than aversion. Nocistatin does not, however, activate the PVN or SON. It does not exert its effects via VP or OT neurons.