Hypothalamo-neurohypophysial System Research Articles

APeg3, a novel paternally expressed gene 3 antisense RNA transcript specifically expressed in vasopressinergic magnocellular neurons in the rat supraoptic nucleus

Vasopressin (VP) and oxytocin (OT) play critical roles in the regulation of salt and water balance, lactation, and various behaviors and are expressed at very high levels in specific magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS). In addition to the cell-specific expression of the VP and OT genes in these cells, there are other transcripts that are preferentially expressed in the VP or OT MCNs. One such gene, paternally expressed gene 3 (Peg3), is an imprinted gene expressed exclusively from the paternal allele that encodes a Kruppel-type zinc finger-containing protein involved in maternal behavior and is abundantly expressed in the VP-MCNs. We report here the robust expression in the VP-MCNs of an RNA, which we designate APeg3 that is transcribed in the antisense direction to the 3′ untranslated region of the Peg3 gene. The APeg3 mRNA is about 1 kb in size, and the full-length sequence of APeg3, as determined by 5′ and 3′ RACE, contains an open reading frame that predicts a protein of 93 amino acids and is predominantly expressed in VP-MCNs. Both Peg3 and APeg3 gene expression in the VP-MCNs increase during systemic hyperosmolality in vivo, demonstrating that both of these genes are osmoregulated.

Co-expression of vasopressin and androgen-binding protein in the rat hypothalamus

In previous studies we have observed the expression of androgen binding protein (ABP) in the rat hypothalamo-neurohypophysial system. With immunocytochemical double staining we found partial co-localization with oxytocin. In the present study we used antibodies to the anti-diuretic hormone arginine vasopressin (AVP) for co-localization with ABP in the rat hypothalamus. Both antigens were seen in the magnocellular paraventricular and supraoptic nuclei. Dense fiber networks with varicosities containing both AVP and ABP immunoreactivity were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed also co-existence in the parvocellular portion of the paraventricular nucleus and in the suprachiasmatic nucleus. ABP immunoreactive neurons in the preoptic region were devoid of AVP staining, AVP neurons in the bed nucleus of the stria terminalis stained only occasionally for ABP. We conclude that both the magnocellular and the parvocellular hypothalamic vasopressin systems are capable of expressing the steroid binding globulin, which is probably subject to axonal transport, along with the peptide hormone. Intrahypothalamic expression of ABP may be among the mechanisms necessary for rapid actions of steroids on hypothalamic neuroendocrine systems.

Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system

Glycine receptors are ionotropic receptors formed by either the homomeric assembly of ligand-binding α subunits or the heteromeric combination of an α subunit and the auxiliary β subunit. Glycine receptors in the brain are found at either pre- or post-synaptic sites. Rat supraoptic nucleus neurons express glycine receptors on the membrane of both their soma and dendrites within the supraoptic nucleus, and their axon terminals in the neurohypophysis. Taking advantage of the well-separated cellular compartments of this system, we correlated the structural properties of the receptors to their subcellular localization. Immunohistochemical study using the generic mAb4a antibody revealed that somatodendritic receptors were clustered, whereas axonal glycine receptors showed a more diffuse distribution. This was paralleled by the presence of clusters of the glycine receptor aggregating protein gephyrin in the supraoptic nucleus and its complete absence in the neurohypophysis. Moreover, another antibody recognizing the α1/α2 subunits similarly labeled the axonal glycine receptors, but did not recognize the somatodendritic receptor clusters of supraoptic nucleus neurons, indicative of structural differences between somatic and axonal glycine receptors. Furthermore, the subunits composing the somatic and axonal receptors have different molecular weight. Functional study further differentiated the two types of glycine receptors on the basis of their sensitivity to picrotoxin, identifying somatic receptors as α/β heteromers, and axonal receptors as α homomers. These results indicate that targeting of glycine receptors to axonal or somatodendritic compartment is directly related to their subunit composition, and set the hypothalamo-neurohypophysial system as an excellent model to study the mechanisms of targeting of proteins to various neuronal cellular compartments.

Regulation of Gene Expression in Magnocellular Neurons in Rat Supraoptic Nucleus during Sustained Hypoosmolality

Hypoosmolality produces a dramatic inhibition of vasopressin (VP) and oxytocin gene expression in the supraoptic nucleus (SON). This study examines the effect of sustained hypoosmolality on global gene expression in the oxytocin and VP magnocellular neurons of the hypothalamo-neurohypophysial system, to identify genes associated with the magnocellular neuron's adaptation to this physiological condition. Using laser microdissection of the SON, T7-based linear amplification of its RNA, and a 35,319-element cDNA microarray, we compare gene expression profiles between SONs in normoosmolar (control), 1-desamino-[8-D-arginine]-VP-treated normoosmolar, and hypoosmolar rats. We found 4959 genes with statistically significant differences in expression between normosmolar control and the hypoosmolar SONs, with 1564 of these differing in expression by more than 2-fold. These genes serve a wide variety of functions, and most were up-regulated in gene expression in hypoosmolar compared with control SONs. Of these, 90 were preferentially expressed in the SON, and 44 coded for transcription-related factors, of which 15 genes were down-regulated and 29 genes were up-regulated in the hypoosmolar rat SONs. None of these transcription-related factor genes significantly changed in expression after sustained 1-desamino-[8-D-arginine]-VP-treatment alone, indicating that these changes were associated with the hypoosmolar state and not due solely to a decreased activity in the SON. Quantitative in situ hybridization histochemistry was selectively used to confirm and extend these microarray observations. These results indicate that the hypoosmolar state is accompanied by a global, but selective, increase in expression of a wide variety of regulatory genes in the SON.

Role of Neurokinin 3 Receptors in Supraoptic Vasopressin and Oxytocin Neurons

Neurokinin 3 receptors (NK3-Rs) are expressed in the supraoptic nucleus (SON), and SON is innervated by substance P (SP)-expressing A1 neurons in the medulla. Because SP stimulates vasopressin (VP) and oxytocin release from explants of the hypothalamo-neurohypophyseal system (HNS), two hypotheses were tested: (1) SP-stimulated VP release is mediated by NK3-Rs, and (2) stimulation of the A1 pathway by hypotension activates SON NK3-Rs. Senktide, an NK3-R agonist, stimulated VP release from HNS explants, but neither a neurokinin 1 receptor antagonist [L732,138 (N-acetyl-L-tryptophan 3,5-bis(tri-fluoromethyl)benzyl ester)] nor two NK3-R antagonists (SB222200 and SB235375) prevented SP-stimulated VP release. Because the affinity of these antagonists for rat NK-Rs may limit their efficacy, NK3-R internalization was used to assess the ability of SP to activate SON NK3-Rs. Senktide, SP, or vehicle was microinjected above SON. The brain was perfused 5 min after injection and stained for NK3-R immunoreactivity. Using confocal microscopy, the number of NK3-R-immunoreactive (-IR) endosomes was counted in a 5.6(2) mu region of cytoplasm in SON neurons. Senktide, but not SP or vehicle, significantly increased the number of NK3-R-IR endosomes in the cytoplasm. When hypotension was induced with hydralazine, NK3-R internalization was observed within 5 min (p < 0.005). A decrease in cytoplasmic NK3-R immunoreactivity was observed within 15 min of hypotension. Unexpectedly, both senktide and hypotension resulted in translocation of NK3-R-IR immunoreactivity to the nucleus. Thus, although these studies do not identify SP as the NK3-R ligand, they do provide evidence for hypotension-induced release of an endogenous tachykinin in SON and evidence suggesting a role for NK3-Rs in transcription regulation.

Selective gene expression in magnocellular neurons in rat supraoptic nucleus.

Oxytocin- and vasopressin-producing magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system are the only neuronal phenotypes present in the rat supraoptic nucleus (SON). Laser microdissection of the SON, extraction and T7-based amplification of its RNAs, and analysis of the resulting cDNAs by hybridization on a 35, 319 element DNA microarray have provided a detailed composite view of the gene expression profile of the MCNs. The genes expressed in the SON were compared with those expressed in a reference tissue consisting of total hypothalamus, and this "expression ratio" indicated which genes were preferentially expressed in the SON. Of the 26,000 unique genes on the array, 1385 were found to be expressed in the SON at levels more than two times greater than in the hypothalamus as a whole. Of these, 123 were expressed > or =3.4-fold higher in the SON versus hypothalamus. Most of these preferentially expressed genes were not previously known to be expressed in the MCNs. Quantitative and double-label in situ hybridization histochemistry was used selectively to confirm a number of these microarray observations and to evaluate the osmotic regulation and cell-specific expression of these genes, respectively.

Neural activity protects hypothalamic magnocellular neurons against axotomy-induced programmed cell death.

Axotomy typically leads to retrograde neuronal degeneration in the CNS. Studies in the hypothalamo-neurohypophysial system (HNS) have suggested that neural activity is supportive of magnocellular neuronal (MCN) survival after axotomy. In this study, we directly test this hypothesis by inhibiting neural activity in the HNS, both in vivo and in vitro, by the use of tetrodotoxin (TTX). After median eminence compression to produce axonal injury, unilateral superfusion of 3 microM TTX into the rat supraoptic nucleus (SON), delivered with the use of a miniature osmotic pump for 2 weeks in vivo, produced a decrease in the number of surviving MCNs in the TTX-treated SON, compared with the contralateral untreated side of the SON. In vitro application of 2.5 microM TTX for 2 weeks to the SON in organotypic culture produced a 73% decrease in the surviving MCNs, compared with untreated control cultures. Raising the extracellular KCl in the culture medium to 25 mM rescued the MCNs from the axotomy- and TTX-induced cell death. These data support the proposal that after axotomy, neural activity is neuroprotective in the HNS.

The Activity of the Hypothalamo-Neurohypophysial System in Response to Acute Stressor Exposure: Neuroendocrine and Electrophysiological Observations

The present mini review focuses on stress-induced alterations of the electrical and secretory activity of vasopressin (AVP) and oxytocin (OXT) neurones originating within the supraoptic nucleus (SON) and constituting the hypothalamo-neurohypophysial system (HNS) in the male rat. Previously, it was thought that SON neurones are predominantly activated by osmotic and reproductive stimuli. However, recent findings also suggest a selective activation of AVP and/or OXT neurones in response to specific stressors. Inhibitory amino acids seem to participate at the level of the SON in the control of HNS activity during stress. Taurine, probably of glial origin, selectively inhibits the secretory activity of AVP neurones. In contrast, GABA, probably of neuronal origin, interferes with the release of OXT both from axon terminals into blood and from somata/dendrites into the extracellular fluid of the SON. Depending upon whether a defined stressor triggers taurine and/or GABA release within the SON the secretion of AVP and/or OXT from HNS neurones will be inhibited. These observations shed new light on the neurone–neurone and glial–neurone interactions that ensure an appropriate neuroendocrine stress response.

Nitric oxide modulation of the hypothalamo-neurohypophyseal system.

Nitric oxide (NO), a free radical gas produced endogenously from the amino acid L-arginine by NO synthase (NOS), has important functions in modulating vasopressin and oxytocin secretion from the hypothalamo-neurohypophyseal system. NO production is stimulated during increased functional activity of magnocellular neurons, in parallel with plastic changes of the supraoptic nucleus (SON) and paraventricular nucleus. Electrophysiological data recorded from the SON of hypothalamic slices indicate that NO inhibits firing of phasic and non-phasic neurons, while L-NAME, an NOS inhibitor, increases their activity. Results from measurement of neurohypophyseal hormones are more variable. Overall, however, it appears that NO, tonically produced in the forebrain, inhibits vasopressin and oxytocin secretion during normovolemic, isosmotic conditions. During osmotic stimulation, dehydration, hypovolemia and hemorrhage, as well as high plasma levels of angiotensin II, NO inhibition of vasopressin neurons is removed, while that of oxytocin neurons is enhanced. This produces a preferential release of vasopressin over oxytocin important for correction of fluid imbalance. During late pregnancy and throughout lactation, fluid homeostasis is altered and expression of NOS in the SON is down- and up-regulated, respectively, in parallel with plastic changes of the magnocellular system. NO inhibition of magnocellular neurons involves GABA and prostaglandin synthesis and the signal-transduction mechanism is independent of the cGMP-pathway. Plasma hormone levels are unaffected by i.c.v. 1H-[1, 2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor) or 8-Br-cGMP administered to conscious rats. Moreover, cGMP does not increase in homogenates of the neural lobe and in microdialysates of the SON when NO synthesis is enhanced during osmotic stimulation. Among alternative signal-transduction pathways, nitrosylation of target proteins affecting activity of ion channels is considered.

Neuronal, glial and synaptic remodeling in the adult hypothalamus: functional consequences and role of cell surface and extracellular matrix adhesion molecules

The adult hypothalamo-neurohypophysial system (HNS) undergoes activity-dependent morphological plasticity which modifies astrocytic coverage of its oxytocinergic neurons and their synaptic inputs. Thus, during physiological conditions that enhance central and peripheral release of oxytocin (OT), adjacent somata and dendrites of OT neurons become extensively juxtaposed, without intervening astrocytic processes and receive an increased number of synapses. The morphological changes occur within a few hours and are reversible with termination of stimulation. The reduced astrocytic coverage has direct functional consequences since it modifies extracellular ionic homeostasis, synaptic transmission, and the size and geometry of the extracellular space. It also contributes indirectly to neuronal function by permitting formation of synapses on neuronal surfaces freed of astrocytic processes. Overall, such remodeling is expected to potentiate activated neuronal firing, especially in clusters of tightly packed neurons, an anatomical arrangement characterizing OT neurons. This plasticity connotes dynamic cell interactions that must bring into play cell surface and extracellular matrix adhesive proteins like those intervening in developing neuronal systems undergoing neuronal–glial and synaptogenic transformations. It is worth noting, therefore, that adult HNS neurons and glia continue to express such molecules, including polysialic acid (PSA)-enriched neural cell adhesion molecule (PSA-NCAM) and the glycoprotein, tenascin-C. PSA is a large, complex sugar on the extracellular domain of NCAM considered a negative regulator of adhesion; it occurs in large amounts on the surfaces of HNS neurons and astrocytes. Tenascin-C, on the other hand, possesses adhesive and repulsive properties; it is secreted by HNS astrocytes and occurs in extracellular spaces and on cell surfaces after interaction with appropriate ligands. These molecules have been considered permissive factors for morphological plasticity. However, because of their localization and inherent properties, they may also serve to modulate the extracellular environment and in consequence, synaptic and volume transmission in a system in which the extracellular compartment is constantly being modified.

Neuroendocrine control of body fluid metabolism.

Mammals control the volume and osmolality of their body fluids from stimuli that arise from both the intracellular and extracellular fluid compartments. These stimuli are sensed by two kinds of receptors: osmoreceptor-Na+ receptors and volume or pressure receptors. This information is conveyed to specific areas of the central nervous system responsible for an integrated response, which depends on the integrity of the anteroventral region of the third ventricle, e.g., organum vasculosum of the lamina terminalis, median preoptic nucleus, and subfornical organ. The hypothalamo-neurohypophysial system plays a fundamental role in the maintenance of body fluid homeostasis by secreting vasopressin and oxytocin in response to osmotic and nonosmotic stimuli. Since the discovery of the atrial natriuretic peptide (ANP), a large number of publications have demonstrated that this peptide provides a potent defense mechanism against volume overload in mammals, including humans. ANP is mostly localized in the heart, but ANP and its receptor are also found in hypothalamic and brain stem areas involved in body fluid volume and blood pressure regulation. Blood volume expansion acts not only directly on the heart, by stretch of atrial myocytes to increase the release of ANP, but also on the brain ANPergic neurons through afferent inputs from baroreceptors. Angiotensin II also plays an important role in the regulation of body fluids, being a potent inducer of thirst and, in general, antagonizes the actions of ANP. This review emphasizes the role played by brain ANP and its interaction with neurohypophysial hormones in the control of body fluid homeostasis.

Regulation of vasopressin secretion by ETA and ETB receptors in compartmentalized rat hypothalamo-neurohypophysial explants.

The endothelins (ET) have been implicated in vasopressin (AVP) release in vivo and in vitro. The effects of ET in this system are complex, and the net AVP secretory response likely depends on a unique combination of ET isoform, ET receptor subtype, and neural locus. The purpose of these studies was to examine the role of ET receptor subtypes at hypothalamic vs. neurohypophysial sites on somatodendritic and neurohypophysial AVP secretion. Experiments were done in cultured explants of the hypothalamo-neurohypophysial system of Long Evans rats. Either the whole explant (standard) or only the hypothalamus or posterior pituitary (compartmentalized) was exposed to log dose increases (0.01-10 nM) of the agonists ET-1 (ET(A) selective), ET-3 (nonselective), or IRL-1620 (ET(B) selective) with or without selective ET(A) (BQ-123, 2-200 nM) or ET(B) (IRL-1038, 6-600 nM) receptor antagonism. In standard explants, ET-1 and ET-3 dose-dependently increased, whereas IRL-1620 decreased net AVP release. Hypothalamic ET(B) receptor activation increased both somatodendritic and neurohypophysial AVP release. At least one intervening synapse was involved, as tetrodotoxin blocked the response. Activation of ET(A) receptors at the hypothalamic level inhibited, whereas ET(A) receptor activation at the posterior pituitary stimulated, neurohypophysial AVP secretion. Antagonism of hypothalamic ET(A) receptors potentiated the stimulatory effect of ET-1 and ET-3 on neurohypophysial secretion, an effect not observed with ET(B) receptor-induced somatodendritic release of AVP. Thus the response of whole explants reflects the net result of both stimulatory and inhibitory inputs. The integration of these excitatory and inhibitory inputs endows the vasopressinergic system with greater plasticity in its response to physiological and pathophysiological states.

NO inhibition of the magnocellular neuroendocrine system in rats is independent of cGMP signaling pathway

Our objective was to test the hypothesis that the cGMP signal-transduction mechanism mediates nitric oxide's (NO) modulation of oxytocin (OT) and vasopressin (VP) secretion from the hypothalamo-neurohypophysial system. Three studies were conducted in adult male Sprague–Dawley rats: (1a) Euhydrated rats received an intracerebroventricular (icv) infusion (1 μl/min for 30 min) of artificial cerebrospinal fluid (aCSF), vehicle (2.6% dimethyl sulfoxide [DMSO]) or 1 H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) (0.05 μg/μl), an inhibitor of soluble guanylyl cyclase (sGC). ODQ did not affect basal levels of plasma VP or OT; (1b) Rats dehydrated for 24 h received aCSF or 8-Br-cGMP (icv), a membrane-permeable analog of cGMP, and plasma hormones were measured 2 min later. 8-Br-cGMP did not significantly change VP or OT levels; (2) Rats ingested water or 2% NaCl for 4 days, and NO synthase (NOS) and sGC activities were measured in posterior pituitaries, the anatomical site of hormone secretion. Salt loading enhanced ( P < 0.001) production of [ 14C]citrulline, the coproduct of NO synthesis, without altering cGMP; (3) One SON was microdialyzed with [ 14C]arginine and NOS and sGC activities were quantified in microdialysates during intravenous (iv) infusion of isotonic or hypertonic saline in awake and anesthetized rats. In awake rats, [ 14C]citrulline recovery, but not cGMP, increased ( P < 0.05) during intravenous infusion of both isotonic and hypertonic solutions, and after insertion of microdialysis probe itself. In anesthetized rats, however, where basal NOS activity is low, intravenous infusion of hypertonic, but not isotonic solution, increased [ 14C]citrulline recovery without affecting cGMP. Thus, in the forebrain, neither NO produced basally nor during osmotic stimulation depends on cGMP to modulate plasma vasopressin and oxytocin secretion.

Central neurokinin 3 receptors increase systemic oxytocin release: interaction with norepinephrine

Stimulation of central tachykinin receptors contributes to neuroendocrine functions of the hypothalamo-neurohypophyseal system. However, the specific role of each tachykinin receptor subtype has not been completely characterized. Specifically, while neurokinin 3 (NK3) receptor stimulation increases systemic vasopressin, the effects on oxytocin (OT) are not known. Therefore, the present studies investigated the effect of central NK3 receptor stimulation with senktide on release of systemic and central OT. Furthermore, since central NK3 receptors activate noradrenergic systems, which contribute to OT release, the effects of α-adrenergic receptor blockade on senktide-induced changes in OT release were evaluated. Female rats were implanted with a cannula in the third cerebral ventricle, and changes in plasma OT concentration determined before and following central administration of senktide in vehicle-treated rats, and animals following central administration of the α-adrenergic antagonist phentolamine. Other rats were implanted with microdialysis probes adjacent to the paraventricular nucleus (PVN), and dialysate and plasma OT concentrations were determined before and during administration of senktide through the dialysis probe. Central senktide increased systemic OT release, which was prevented by pretreatment with phentolamine. Furthermore, there was no detectable change in extracellular OT concentration in the PVN during dialysis administration of senktide. These data demonstrate that activation of central NK3 receptors stimulates systemic release of OT by activation of central noradrenergic systems, apparently without increasing intranuclear OT release in the PVN.

Transgenesis and the Study of Expression, Cellular Targeting and Function of Oxytocin, Vasopressin and Their Receptors

The neuropeptides oxytocin and vasopressin and the neurons in the hypothalamus that synthesize them have been a rich source for the exploration and understanding of both the brain and the endocrine system. Because of their large size and compact nuclear organization the magnocellular neurons of the hypothalamoneurohypophysial system have traditionally attracted scientists using state-of-the-art techniques, including the subject of this review, transgenesis. We discuss the role of transgenics in deciphering gene elements necessary for the appropriate expression of oxytocin and vasopressin and to deliver exogenous genes, such as green fluorescent protein, selectively to secretory granules in the neurons in the hypothalamoneurohypophysial system. Finally, we review the studies of mice whose genes for oxytocin and, most recently, for the oxytocin and vasopressin receptors have been knocked out through homologous recombination.

Distribution of androgen-binding protein in the rat hypothalamo-neurohypophyseal system, co-localization with oxytocin

Androgen-binding protein (ABP) is known to be expressed in the male and female rat hypothalamus. In the present study, we observed immunocytochemically ABP in neurons of the magnocellular hypothalamic nuclei, in the preoptic region and in the lateral hypothalamus. Dense fiber networks with varicosities, containing ABP immunofluorescence, were visible throughout the hypothalamus, the median eminence and in the posterior pituitary lobe. Double immunostaining revealed a partial coexistence of ABP—and oxytocin immunoreactivity in a portion of the magnocellular perikarya. ABP was isolated by affinity chromatography from hypothalamus homogenates. Western blots resulted in immunoreactive (IR) bands with an approximate molecular weight of 35 and 50 kDa. Mass spectrometry of these preparations confirmed the presence of ABP, which was almost identical to ABP isolated from rat testis. It is likely that ABP, expressed in magnocellular oxytocinergic neurons, is subject to axonal transport and release in the hypothalamo-neurohypophyseal system.

Nitric oxide is not involved in the control of vasopressin release during acute forced swimming in rats.

Neurons of the hypothalamo-neurohypophyseal system (HNS) are known to contain high amounts of neuronal nitric oxide (NO) synthase (nNOS). NO produced by those neurons is commonly supposed to be involved as modulator in the release of the two nonapeptides vasopressin (AVP) and oxytocin into the blood stream. Previous studies showed that forced swimming fails to increase the release of AVP into the blood stream while its secretion into the hypothalamus is triggered. We investigated here whether hypothalamically acting NO contributes to the control of the AVP release into blood under forced swimming conditions. Intracerebral microdialysis and in situ hybridization were employed to analyze the activity of the nitrergic system within the supraoptic nucleus (SON), the hypothalamic origin of the HNS. A 10-min forced swimming session failed to significantly alter the local NO release as indicated both by nitrite and, the main by-product of NO synthesis, citrulline levels in microdialysis samples collected from the SON. Microdialysis administration of NO directly into the SON increased the concentration of AVP in plasma samples collected during simultaneous forced swimming. In an additional experiment the effect of the defined stressor exposure on the concentration of mRNA coding for nNOS within the SON was investigated by in situ hybridization. Forced swimming increased the expression of nNOS mRNA at two and four hours after onset of the stressor compared to untreated controls. Taken together, our results imply that NO within the SON does not contribute to the regulation of the secretory activity of HNS neurons during acute forced swimming. Increased nNOS mRNA in the SON after forced swimming and the increase in AVP release in the presence of exogenous NO under forced swimming points to a possible role of NO in the regulation of the HNS under repeated stressor exposure.

Transgenic models for molecular and physiological studies in the central nervous system

Transgenic models for molecular and physiological studies in the central nervous system

Loose-patch clamp currents from the hypothalamo-neurohypophysial system of the rat.

The loose-patch clamp technique was used to study voltage-activated currents from the surface of rat neurohypophysial and hypothalamic regions in situ. In the neurohypophysis, depolarizing pulses of 4-8 ms duration yielded tetrodotoxin (TTX)-sensitive sodium currents, a 4-AP-sensitive "A"-type potassium current, and a long-lasting outward TEA- and tetrandrine-sensitive Ca(2+)-activated potassium current. All of these currents were elicited during the application of the pulse. With high external calcium there were long-lasting inward currents blocked by Ni(2+) and Cd(2+), identifying them as voltage-gated calcium currents. Depolarizing pulses of 0.3-0.7 ms duration yielded fast biphasic responses, of 1-3 ms duration, composed of mostly sodium and "A"-type potassium currents. With high external calcium there were fast inward currents blocked by Ni(2+) and Cd(2+), indicating that these were voltage-gated calcium currents. These responses have the characteristics of action potential currents: they were elicited after the cessation of the applied pulse and the "A" component is eliminated together with the sodium component upon application of TTX. Similar responses to long and short pulses were obtained from the surface of the associated magnocellular somata in the supraoptic nucleus, and their projections. The explant currents are similar to those previously characterized using conventional methods from somata and terminals.

Removal of PSA from NCAM affects the survival of magnocellular vasopressin- and oxytocin-producing neurons in organotypic cultures of the paraventricular nucleus.

The expression of the polysialic acid neural cell adhesion molecule (PSA-NCAM) in the hypothalamo-neurohypophyseal system has been correlated with morphofunctional plasticity. In this study, we investigated the role of PSA-NCAM in the survival of oxytocin (OT)- and vasopressin (VP)-producing magnocellular cells of this system. We used a recently developed organotypic slice culture model of the rat hypothalamic paraventricular nucleus (PVN) in which ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are potent survival factors for magnocellular neurons. We demonstrate by means of confocal microscopy that cultured magnocellular VP and OT neurons express strong immunoreactivity for PSA-NCAM. Removal of PSA from NCAM by the enzyme Endo N leads to a significant loss of both VP and OT neurons in the presence of low concentrations of CNTF. Endo N treatment did not change cell survival in the presence of LIF. These results suggest that, in addition to its role in neuro-glial plasticity, PSA-NCAM might also influence the trophic factor responsiveness of hypothalamic VP and OT neurosecretory cells.