Magnocellular Vasopressin Neurons Research Articles

Stationary organotypic cultures of oxytocin and vasopressin magnocellular neurones from rat and mouse hypothalamus.

Rat and mouse hypothalami from postnatal animals containing highly differentiated neurones survive very well in long-term (>15 days in vitro, DIV) stationary organotypic cultures. Magnocellular oxytocin (OT) and vasopressin (VP) neurones are present in identifiable paraventricular (PVN), supraoptic (SON) and accessory (ACC) nuclei in these cultures. After 15 DIV in standard medium immunocytochemistry revealed 427 +/- 63 OT cells and 217 +/- 27 VP cells per cultured rat hypothalamus, and 380 +/- 72 OT cells and 622 +/- 91 VP cells per cultured mouse hypothalamus. Following a 7-day adaptation period in standard culture medium containing serum, the rat slice-explants survived very well after subsequent transfer to defined, serum- free media (SFM) for an additional 8 days. The number of OT cells surviving in SFM was 612 +/- 147 OT cells per cultured rat hypothalamus. Only 0.5% of the magnocellular OT and VP neurones in the cultures appeared to express both peptides. Experiments on c-fos gene expression in these cultures showed that while only 12% of the magnocellular OT and VP neurones contained barely detectable Fos protein in their nuclei under control conditions, potassium depolarization of these cultures for 3 h produced intense c-fos expression in 87-91% of these cells. Thus, magnocellular neurones in these cultures are sufficiently stable and responsive to permit long-term physiological and gene expression studies to be done under defined media conditions.

Selective Inhibition of Magnocellular Vasopressin Neurons by Hypoosmolality: Effect on Histamine- and Stress-Induced Secretion of Adrenocorticotropin and Prolactin

We investigated the effect of selective inhibition of magnocellular arginine vasopressin (AVP) and oxytocin neurons on histamine (HA)- and restraint-stress-induced adrenocorticotropin (ACTH) and prolactin (PRL) secretion in conscious male rats. The inhibition of magnocellular neurons was obtained by inducing chronic hypoosmolality via continuous exposure of the rats to the AVP V₂ receptor agonist 1-deamino(8-D-arginine)vasopressin (DDAVP) which was released from osmotic pumps implanted subcutaneously. In DDAVP-treated rats, plasma osmolality and sodium concentration were 273 mosm/l and 130 mmol/l, respectively. In control rats, the corresponding values were 291 mosm/l and 139 mmol/l. HA (270 nmol) administered intracerebroventricularly or 5 min of restraint stress stimulated ACTH and PRL secretion 4- to 11-fold in normoosmolar rats. In hypoosmolar rats, the HA-induced ACTH response was inhibited more than 40% whereas the restraint-stress-induced ACTH response was unaffected. Conversely, the PRL response to HA in hypoosmolar rats was unaffected whereas the PRL response to restraint stress was inhibited by 40%. In summary, chronic hypoosmolality inhibits HA-induced ACTH and restraint-stress-induced PRL secretion indicating involvement of magnocellular AVP in these responses.

Analysis of angiotensin type 2 receptors in vasopressinergic neurons and pituitary in the rat

Previous functional studies indicated that an angiotensin type 2 (AT 2) receptor subtype may participate in the regulation of vasopressin release by angiotensin II (AngII). In the present study, AT 2 receptor-directed antiserum immunohistochemically detected AT 2 receptors within the hypothalamic paraventricular (PVN) and the supraoptic nuclei (SON) of the rat brain, more specifically, in identified vasopressinergic neurons. Considering the lack of AT 2 binding in the PVN and the SON using receptor autoradiography, we tested the hypothesis that these AT 2 receptors are transported to the posterior pituitary. Western blot analysis detected AT 2 immunoreactivity in the posterior pituitary. However, no AT 2 binding was detected in posterior pituitary membranes, and no AT 2 binding was detected with quantitative receptor autoradiography in the neurohypophysis. Thus, if AT 2 receptors are transported from the magnocellular vasopressin neurons to the posterior pituitary, their role in AngII regulation of vasopressin release at the neurohypophyseal terminals remains to be clarified.

Specific expression of secretogranin II in magnocellular vasopressin neurons of the rat supraoptic and paraventricular nucleus in response to osmotic stimulation

As secretogranin II is considered to be a marker for the regulated secretory pathway, its distribution in the hypothalamo-neurohypophyseal system of salt-loaded Wistar rats was studied in detail by immunocytochemistry. Although after an osmotic challenge both vasopressin and oxytocin neurons are stimulated, secretogranin II was exclusively expressed in a subpopulation of vasopressinergic magnocellular neurons in the supraoptic and paraventricular nucleus of Wistar rats. Secretogranin II was only surely visualized after a combination of osmotic challenge and blockade of axonal transport by colchicine treatment. When these pre-treatments were not performed, only punctate fibers situated around the magnocellular neurons within the paraventricular and supraoptic nucleus were observed. Oxytocinergic magnocellular neurons never displayed any secretogranin II immunoreactivity, not even during lactation and after colchicine treatment. These findings suggest that secretogranin II is of functional importance during enhanced secretory activity within vasopressinergic neurons.

The kidneys stimulate vasopressin release during hemorrhage in rats with chronic NTS lesions.

Elimination of baroreceptor afferent input to the brain produced by chronic lesion of nucleus of the solitary tract (NTS) does not alter vasopressin (VP) release during hypotensive hemorrhage in conscious rats. To investigate whether the kidneys play a critical role in stimulating VP release during hemorrhage in chronic NTS-lesioned rats, we examined the effects of removing potential signals arising from the kidneys. In NTS-lesioned rats, nephrectomy or renal denervation, but not captopril injection, markedly attenuated (but did not abolish) hemorrhage-induced VP release. In contrast, none of these manipulations attenuated the VP response in NTS-intact rats. Hemorrhage increased plasma renin activity in control and NTS-lesioned rats, and this response was not altered by renal denervation. In rats with NTS lesions and renal denervation, hemorrhage induced the expression of Fos in hypothalamic magnocellular VP neurons in a pattern similar to that of hemorrhage in intact rats. Collectively, these results indicate that in chronic NTS-lesioned rats an afferent signal arising from the kidneys stimulates VP release during hemorrhage, possibly through renal nerves. However, with the NTS intact or after the selective removal of arterial baroreceptor inputs, such a role for the kidneys is not apparent. Furthermore, in the absence of the NTS and renal nerves, another signal generated by hypotensive hemorrhage continues to stimulate VP neurons.

Chronic hyponatremia reduces survival of magnocellular vasopressin and oxytocin neurons after axonal injury

Axonal injury to hypothalamic magnocellular vasopressin (AVP) and oxytocin (OT) neurons causes degeneration of a substantial subpopulation of these neurons. In this study, we investigated the influence of osmolality on this injury-induced cell death. Normonatremic, chronically hypernatremic, and chronically hyponatremic rats received pituitary stalk compression (SC), which causes degeneration of AVP and OT terminals in the neurohypophysis. Twenty-one days after SC, rats were perfused and hypothalami were serially sectioned and alternately stained for AVP-neurophysin and OT-neurophysin immunoreactivities. Normonatremic and hypernatremic rats exhibited a triphasic pattern of water intake after SC, with peak intakes 3 times higher than those exhibited by sham-operated normonatremic rats. In contrast, hyponatremic SC rats exhibited peak water intakes of 600 ml/24 hr, approximately 9-10 times the water intakes of sham-operated normonatremic rats. In normonatremic rats, SC caused degeneration of 65% of the AVP neuron population in the SON and 73% in the PVN, but only 31% of the OT neuron population in the SON and 35% in the PVN. Similar results were found in hypernatremic rats after SC. However, in hyponatremic rats SC caused degeneration of 97% of the AVP neuron population in the SON and 93% in the PVN, and 90% of the OT neuron population in the SON and 84% in the PVN. Our results, therefore, demonstrate that injury-induced degeneration of magnocellular AVP and OT neurons is markedly exacerbated by chronic hypo-osmolar conditions, but neuronal survival is not enhanced by chronic hyperosmolar conditions.

Induction of glucocorticoid receptor expression in hypothalamic magnocellular vasopressin neurons during chronic hypoosmolality

The site of action for the modulation of hypothalamo-neurohypophyseal function by adrenal steroids is not known. Glucocorticoid receptors (GR) are an obvious potential site of action, but there have been conflicting reports of GR localization in magnocellular neurons. We confirm studies finding undetectable levels of GR in vasopressin or oxytocin neurons, but now report that chronic hypoosmolality induces the expression of GR in magnocellular vasopressin neurons, but not oxytocin neurons. These data support the hypothesis that the vasopressin gene can be directly inhibited by glucocorticoids, and that the induction of GR expression in magnocellular neurons may be part of a redundant set of mechanisms to suppress the expression of AVP during periods of prolonged hypoosmolality. This mechanism represents a novel form of steroid feedback control in brain.

Induction of glucocorticoid receptor expression in hypothalamic magnocellular vasopressin neurons during chronic hypoosmolality.

The site of action for the modulation of hypothalamo-neurohypophyseal function by adrenal steroids is not known. Glucocorticoid receptors (GR) are an obvious potential site of action, but there have been conflicting reports of GR localization in magnocellular neurons. We confirm studies finding undetectable levels of GR in vasopressin or oxytocin neurons, but now report that chronic hypoosmolality induces the expression of GR in magnocellular vasopressin neurons, but not oxytocin neurons. These data support the hypothesis that the vasopressin gene can be directly inhibited by glucocorticoids, and that the induction of GR expression in magnocellular neurons may be part of a redundant set of mechanisms to suppress the expression of AVP during periods of prolonged hypoosmolality. This mechanism represents a novel form of steroid feedback control in brain.

Grafted fetal suprachiasmatic nucleus cells survive much better in tissue pieces than in suspension

A comparison was made between the survival of fetal suprachiasmatic nucleus (SCN) grafted either in tissue pieces or as tissue suspension. Donor tissue was obtained from day 15, 16 or 17 Wistar fetuses, and stereotaxically placed in the dorsal thalamus of the brain of vasopressin(VP)-deficient Brattleboro adult rats. One month post-grafting, the suspension grafts largely failed to show the immunocytochemical presence of VP- and vasoactive intestinal polypeptide(VIP)-producing SCN cells, but solid piece grafts did. In the suspension grafts only a few tiny clusters of surviving VP SCN neurons were seen and hardly any VIP cells. This suggests that intrinsic contacts are necessary for SCN cells to survive and/or to express their genotype. Efferent VP fiber growth was observed to run from the tissue piece derived SCN grafts towards the periventricular area of the thalamus, a nearby SCN projection area. However, the number of VP efferents was very limited and not consistently present. Surprisingly, magnocellular VP neurons, also present in the fetal SCN grafts, showed an opposite survival pattern. Several of such cells were seen in day 15 suspension grafts, whereas hardly any were found in day 15 tissue piece grafts. These results indicate that the type of graft preparation might be important when survival of particular cells is desired.

Distribution of oxytocin and vasopressin neurons in the diencephalon of the Japanese horseshoe bat, Rhinolophus ferrumequinum. An immunohistochemical study.

The distribution of oxytocin (OXT) and vasopressin (VP) neurons in the diencephalon of the hibernating Japanese horseshoe bat, Rhinolophus ferrumequinum, was immunohistochemically investigated by the avidin-biotin complex method. Magnocellular OXT and VP neurons were localized mainly in the paraventricular nucleus and the supraoptic nucleus. In addition to these main nuclei, both kinds of magnocellular neurons were also found in the periventricular nucleus, perifornical area and lateral hypothalamic area. Extensively distributed parvocellular neurons containing only VP were observed in the rostral and middle portions of the suprachiasmatic nucleus. The size of OXT and VP magnocellular neurons was almost equal in the paraventricular and ventromedial supraoptic nuclei, whereas VP neurons were significantly larger than OXT neurons in the dorsolateral supraoptic nucleus. The OXT and VP cells in the ventral supraoptic nucleus showed a distinctive elliptical shape. Both OXT and VP fibers were distributed in the lateral habenular nucleus, stria medullaris thalami, lateral preoptic area, stria terminalis, and medial and supracapsular part of the bed nucleus of the stria terminalis. Moreover, OXT fibers were found in the substantia nigra, and VP fibers were noted in the nucleus reunions and the paraventricular nucleus of the thalamus.

Electrical activation and c‐fos mRNA expression in rat neurosecretory neurones after systemic administration of cholecystokinin.

1. The expression of c-fos mRNA in the rat hypothalamus was examined by in situ hybridization following systemic administration of cholecystokinin (CCK), a procedure known to activate magnocellular oxytocin neurons but not magnocellular vasopressin neurones. 2. Conscious male rats were given a single I.P. injection of 50 micrograms/kg CCK, c-fos mRNA signal was apparent in the supraoptic and paraventricular nuclei in rats killed 10 min after injection but not in uninjected or saline-(vehicle) injected rats. The density of c-fos mRNA at both sites was further elevated in rats killed 30 min or 60 min following injection, and was absent in rats killed 4 h after injection. 3. In the paraventricular nucleus the most dense expression of c-fos mRNA following CCK administration was in the medial, mainly parvocellular portion of the nucleus, in an area corresponding to the distribution of corticotrophin-releasing factor mRNA determined by in situ hybridization in adjacent sections. 4. The I.P. injection of CCK increased plasma oxytocin concentrations, measured by specific radioimmunoassay from 13 +/- 5 pg/ml in control rats to 107 +/- 9 pg/ml in the rats killed 10 min after injection, a similar response to that observed previously in urethane-anaesthetized rats. 5. In each of six urethane-anaesthetized rats, recordings were made from single neurones in the supraoptic nucleus, identified antidronomically as projecting to the posterior pituitary and identified electrophysiologically as putative oxytocin neurones. Following I.P. injection of 50 micrograms/kg CCK, the neurones increased their firing rate by a mean of 1.3 +/- 0.2 spikes/s averaged over the 10 min following injection. 6. From the appearance of c-fos mRNA in supraoptic neurones following CCK administration we conclude that this message is expressed in magnocellular oxytocin neurones, since vasopressin neuronal activity and vasopressin release is known to be unaffected by this stimulus, and since the supraoptic nucleus contains essentially only oxytocin neurones and vasopressin neurones. 7. We conclude that c-fos mRNA expression can be induced in supraoptic oxytocin neurones following brief and modest episodes of electrical activation, suggesting that c-fos may be involved in the gene regulation of these neurones under physiological conditions.

Central effects of oxytocin

Article de synthese sur les effets centraux de l'ocytocine chez les mammiferes: effets physiologiques et comportementaux; site cerebral et mecanisme d'action de l'ocytocine; sites de liaisons de l'ocytocine dans le cerveau et relation avec son activite biologique

Hereditary diabetes insipidus: an immunohistochemical study of the hypothalamus and pituitary gland

We report the histological findings in a case of hereditary diabetes insipidus (HDI) using vasopressin (VP) immunohistochemistry. The hypothalamus displayed a marked loss of magnocellular VP neurons, with preservation of the smaller cells. The neurohypophysis was severely atrophic with scanty immunoreactivity. Our results support the hypothesis that HDI results from a selective degeneration of VP neurons affecting chiefly the magnocellular elements projecting to the neurohypophysis. The sparing of the parvocellular component may reflect the projection of these neurons to non-pituitary targets.

Maintenance of LHRH and Oxytocin Neurons in Slice Explants Cultured in Serum-Free Media: Effects of Tetrodotoxin on Gene Expression

A variety of neuroendocrine cells survive and express specific neuropeptide genes for long periods of time in slice explant cultures in the presence of serum. However, before use of these slice explant cultures as experimental models for physiological and pharmacological studies on the regulation of neuropeptide gene expression, it is first necessary to evaluate their characteristics in defined (e.g. serum free) media and to control for the spontaneous electrical and synaptic activity of neurons in these cultures. In this study, brain slices from postnatal day 4 rats were cultured in serum-containing media (SCM) for 12 days to allow thinning, and then maintained in a serum-free, defined media (SFM) for 6 days. Culture slices transferred to SFM appeared healthy and numerous neuroendocrine neurons containing messenger RNA (mRNA) encoding for LHRH and magnocellular neurons containing mRNA encoding for oxytocin (OT) were detected using in situ hybridization histochemistry (ISHH). Each of these neuronal subtypes robustly produced their appropriate gene products as determined by immunocytochemical analysis. Abundant magnocellular OT neurons were found in cultures grown in either SCM or SFM. In contrast, magnocellular vasopressin (VP) neurons were rarely detected under these conditions. Inhibition of spontaneous electrical and synaptic activity in these slice explant cultures was effectively achieved by incubation for the last 2.5 days of culture in the presence of tetrodotoxin (TTX; 10(-6) M). Densitometric single cell analyses after ISHH was performed on both LHRH and OT cells. Comparisons of the density values (corresponding to mRNA levels), from these slice explants, found that: (1) cellular LHRH mRNA levels decreased in the absence of serum, whereas cellular OT mRNA levels did not significantly change under these conditions; (2) the presence of TTX in the media resulted in an overall decrease in cellular LHRH mRNA values in both SCM and SFM, and (3) the OT neurons in these slice cultures appear to be composed of a heterogeneous population, with one cell subtype responding to TTX with an increase in cellular OT mRNA levels. These data show that factors in serum and spontaneous electrical activity can differentially influence mRNA levels of LHRH cells and magnocellular OT neurons in culture.

Vasopressin mRNA Expression in Individual Magnocellular Neuroendocrine Cells of the Supraoptic and Paraventricular Nucleus in Response to Water Deprivation

Vasopressin neuroendocrine function involves the regulation of both secretion and synthesis from magnocellular neuroendocrine cells but the coordination of these two processes is poorly understood. To explore the temporal relationship between physiological stimulation and vasopressin mRNA levels we measured vasopressin mRNA content within individual magnocellular neurons of the supraoptic and paraventricular nucleus during the course of water deprivation. Analysis of autoradiographic silver grain densities from in situ hybridization of an [125I]dCTP-labeled oligonucleotide specific for vasopressin mRNA revealed a wide variety of resting vasopressin mRNA levels and differential responses to water deprivation in the magnocellular neuroendocrine cells. During water deprivation, the vasopressin mRNA content of the paraventricular nucleus increases rapidly and with shorter latency and greater incremental response than the supraoptic nucleus. Double-labeling experiments with combined in situ hybridization and immunocytochemistry identified a population of vasopressin immunoreactive cells which maintain very low basal levels of vasopressin mRNA. The location of these cells correlates with the location of increased silver grain densities during water deprivation. One subset of vasopressin magnocellular neurons failed to show high levels of vasopressin mRNA, indicating that all cells are not equally responsive to water deprivation. These patterns of vasopressin mRNA expression suggest the presence of functional subpopulations of vasopressin neuroendocrine cells which may reflect stimulus-specific patterns of afferent input to the supraoptic and paraventricular nucleus.

Metabolic mapping of functional activity in rat brain and pituitary after water deprivation.

Glucose utilization in rat brain and pituitary was measured in control and water-deprived rats by autoradiographic assessment of the metabolic trapping of radioactivity from [1-14C]glucose. Two days of water deprivation resulted in significant increases in hematocrit, plasma osmolality and vasopressin levels, indicating a functional activation of magnocellular vasopressin neurons. The uptake and retention of radioactivity from [1-14C]glucose in the dehydrated rats, compared to controls, was 103% greater in the magnocellular portion of the paraventricular nucleus and 74% greater in the supraoptic nucleus. Water deprivation also resulted in significant increases in glucose utilization (30-40%) in the lateral and anterior hypothalamic areas, somatosensory cortex and cingulate cortex. No change in glucose utilization after 2 days of water deprivation was apparent in the parvocellular paraventricular nucleus, periventricular nucleus of the hypothalamus, corpus callosum, organum vasculosum of the lamina terminalis (OVLT) or the subfornical organ (SFO). In the pituitary, glucose utilization was increased in the neural lobe but was unchanged in the anterior and intermediate lobes after water deprivation. Under the conditions of the present study, no increase in metabolic activity was apparent in 2 brain regions thought to be possible sources of osmoreception, the OVLT and SFO. These results do not support, but do not exclude, functional involvement of the OVLT and SFO in regulating the activity of magnocellular neurons of the paraventricular nucleus and supraoptic nucleus during chronic water deprivation.

Structural and functional relationships of grafted vasopressin neurons

Fetal vasopressin neurons were grafted into adult Long-Evans rats with vasopressin deficiencies created by neural lobe ablation one week prior to implantation. Control animals and recipients with ectopic grafts still possessed significant deficits in fluid regulation 6 weeks following implantation. However, recipients with grafts fused to the host median eminence and containing magnocellular vasopressin neurons juxtaposed to the host portal vasculature showed restored peripheral vasopressin activity as measured by normal urine volumes and osmolalities. These findings suggest that structural integration of grafted neuroendocrine tissue with the appropriate target in the host brain is a necessary prerequisite for physiological activity.

Vasopressin- and neurophysin-immunoreactive neurons in the septal region, medial amygdala and locus coeruleus in colchicine-treated rats

The distribution and morphology of neurons containing vasopressin, oxytocin and their associated neurophysins were examined immunohistochemically in rats given intracerebroventricular injections of colchicine. Under these conditions, numerous neurons containing vasopressin and neurophysin were found in several brain areas in addition to those previously described in the hypothalamus. Individual parvocellular vasopressin neurons were scattered in the medial and lateral septum and vertical limb of the nucleus of the diagonal band, while a large number of such neurons were found throughout both the bed nucleus of the stria terminals and the dorsal portion of the medial amygdala. In addition a small cluster of parvocellular vasopressin neurons was present adjacent to the top of the third ventricle in the posterior dorsal hypothalamic area and a number of such neurons were found in the ventral locus coeruleus and sub coeruleus. The mean diameters of these parvocellular vasopressin neurons ranged from 16.6 to 19.8 μm in the different regions, in contrast to the 25.4 μm mean diameter of hypothalamic magnocellular vasopressin neurons, or the 13.7 μm mean diameter of parvocellular vasopressin neurons in the suprachiasmatic nucleus. No vasopressin neurons were found in other brain and spinal cord regions under the conditions used in this study, although all regions were examined. No oxytocin neurons other than those previously described in the hypothalamus and immediately contiguous regions were found. Measurement of the mean diameter of oxytocin neurons showed that neurons in the caudal paraventricular nucleus were clearly smaller (18.9 μm) than magnocellular oxytocin neurons (24.8 μm) in other parts of the hypothalamus. These parvocellular oxytocin neurons with experimentally documented central connections were similar in both size and appearance to the parvocellular vasopressin neurons seen after colchicine treatment, which are potential sources of certain central vasopressin pathways. These findings indicate that there are at least two types of oxytocin neurons in the hypothalamus and several types of vasopressin neurons in a variety of different areas in the brain, many of which are outside of the hypothalamus.

Corticotropin-releasing factor-immunoreactive neurons of the paraventricular nucleus become vasopressin positive after adrenalectomy.

The immunoperoxidase technique was used to study the effect of adrenalectomy on vasopressin (VP) immunoreactivity in the hypothalamic paraventricular nucleus of rat. In control animals, relatively few VP-immunostained parvocellular neurons were found in addition to a large population of magnocellular VP neurons. Seven to 14 days after bilateral adrenalectomy, VP immunostaining increased markedly in specific subdivisions of the paraventricular nucleus. In contrast to normal animals, VP immunoreactivity was localized in a large number of parvocellular neurons. Colchicine treatment, on the other hand, did not significantly increase the number of VP-immunostained parvocellular neurons found in control rats. These observations suggest that adrenalectomy increases the number of VP-positive neurons and appears to increase the intensity of VP immunoreactivity specifically in parvocellular neurons. VP parvocellular neurons are confined to those paraventricular nucleus subdivisions that are known to project to the external zone of the median eminence. Moreover, their distribution pattern is very similar, if not identical, to that of the corticotropin-releasing factor (CRF) immunoreactive cells. Parvocellular neurons on adjacent thin sections could be stained for both CRF and VP. Thus, adrenalectomy seems to increase VP staining in CRF immunoreactive parvocellular neurons, which innervate the external zone of the median eminence.

Phasically firing neurones in the lateral hypothalamus of anaesthetized rats

Sixteen of 146 neurones from the rat lateral hypothalamus were observed to discharge in a ‘phasic’ pattern. These neurones were activated orthodromically but not antidromically by neural stalk stimulation, and were unaffected by systemic osmotic stimulation. They are therefore functionally and anatomically distinct from the phasically discharging magnocellular vasopressin neurones. Thus phasic discharge, which facilitates hormone release from vasopressin nerve terminals, also appears in other hypothalamic systems.