Age-related changes of vasopressin content of microdissected areas of the rat brain

Vasopressin contents of posterior pituitaries and intra- and extrahypothalamic nuclei of brains from 3-month and 24-month-old Long-Evans rats and from Brattleboro rats heterozygous (HEDI) and homozygous (HODI) for diabetes insipidus were measured by radioimmunoassay.The pituitary content of vasopressin was highest in young rats and was significantly lower in HEDI tissue. The peptide was not detected in HODI pituitaries.In the brain regions studied both aged and HEDI rats showed reduced vasopressin contents as compared to young animals. In light of evidence of memory deficiencies in both HEDI and HODI rats, the possibility arises that memory decrements associated with senescence may be related to altered vasopressin synthesis and/or secretion occurring with aging of these neuronal systems.     

A non-equilibrium 24-hour vasopressin radioimmunoassay: development and basal levels in the rat brain

The monoamine levels in the locus coeruleus (LC) were determined by HPLC following specific lesions of the ventral mesencephalic tegmental-A10 regions (VMT-A10) and raphé dorsalis (RD). Only lesions in the VMT-A10 area decreased the dopamine (DA) content, which strongly suggests that the projection from this region to the LC is of dopaminergic nature. Lesions of the RD increased DA metabolism in the LC and provoked significant decreases in the serotonin (5-HT) levels.     

Comparison of the distribution of oxytocin and vasopressin in the rat brain

While immunohistochemistry has been used extensively to map both oxytocin (OT) and vasopressin (VP) pathways in the brain, little information is available concerning the quantitative distribution of these hormones--particularly oxytocin. We have isolated oxytocin from extrahypothalamic regions of the rat brain and shown it to behave identically with standard oxytocin in radioimmunoassay (RIA) and on high-performance liquid chromatography. Using sensitive RIA we have measured and compared levels of both oxytocin and vasopressin in the rat brain. Both hormones are widely distributed, with the largest amounts outside the hypothalamus being found in the locus coeruleus. Considerable quantities of both peptides (but particularly oxytocin) are found in mesencephalic, pontine and medullary nuclei. This distribution is similar to that of the catecholamines, and the possible interaction of oxytocin and vasopressin with catecholaminergic pathways in the central control of various functions is discussed.     

Regional distribution of luteinizing hormone-releasing hormone (LH-RH) in rat brain determined by microdissection and radioimmunoassay

We have re-examined the distribution of LH-RH in rat brain and in particular those rostral hypothalamic areas reported to contain perikarya by immunocytological techniques. Adult male rats were decapitated and 20 discrete brain structures microdissected. Tissue from single rats was homogenized in 50 μ10.1 N HCl and 40 μ1 of this acid extract was radioimmunoassayed for LH-RH using the R-42 antibody. Low but detectable concentrations which ranged from 0.2 to 1.0 pg/μg protein were found in the interstitial nucleus of the stria terminalis (ventral aspect), medial preoptic nucleus (dorsal and ventral aspects), lateral preoptic nucleus, anterior hypothalamic nucleus, paraventricular nucleus, ventromedial nucleus (medial and lateral aspects) and the dorsomedial nucleus. Slightly higher concentrations (1.0–4.0 pg/μg protein) were measured in a rostral, medial basal corridor made up of the suprachiasmatic preoptic nucleus, the suprachiasmatic nucleus and the retrochiasmatic area. LH-RH was not detected (i.e. < 0.8 pg/area) in the bed nucleus of the diagonal band of Broca, caudate nucleus, lateral septal nucleus or the cingulate cortex.     

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+.     

Cytodifferentiation of the supraoptic nucleus correlated with vasopressin synthesis in the rat

This study demonstrates that neurons in the supraoptic nucleus attain many of the prerequisites for functional activity prior to birth. Immunoassayable vasopressin (VP) was detected in the hypothalamo-neurohypophyseal system (HNS) of the rat as early as 17 days post-coitus (dpc). Vasopressin concentrations increased 3–6-fold daily from an average of 21 pg/animal on 17 dpc to 5984 pg/animal at 21 dpc. The daily increases were highly significant (P <0.001). Between 21 dpc and the morning of the day of birth on 22 dpc, a further significant increase (P <0.05) occurred to a mean level of 9672 pg VP/animal. Birth usually occurred on the afternoon of the 22nd day. Parturition did not seem to deplete VP stores in the HNS.Differentiation of the magnocellular neurons in the supraoptic nucleus closely paralleled the appearance and increases in VP. It was first possible to distinguish a supraoptic nucleus in the 17 dpc rat and to identify dense core granules in the developing neurons of the nucleus. Cytodifferentiation of the magnocellular neurons was essentially complete by 21 dpc. Synaptic contacts could not be found on the soma and dendrites of the supraoptic neurons until 21 dpc and were extremely rare throughout the period examined.     

Inhibition by morphine and beta-endorphin of vasopressin release evoked by electrical stimulation of the rat medial basal hypothalamus in vitro

The effect of opiates on vasopressin release from rat medial basal hypothalami was investigated in vitro. Electrical stimulation caused an increase in vasopressin release which was calcium-dependent and tetrodotoxin-sensitive. Morphine or β-endorphin decreased stimulation-evoked vasopressin release. The effect of morphine was reversed by naloxone.     

Long-term effects of ciliary neurotrophic factor on the survival of vasopressin magnocellular neurones in the rat supraoptic nucleus in vitro

The use of hypothalamic organotypic cultures for the long-term study of mechanisms in magnocellular neurones (MCNs) of the hypothalamic-neurohypophysial system has been limited by the relatively poor maintenance of the vasopressin MCNs in vitro. Recent studies have shown that addition of ciliary neurotrophic factor (CNTF) to the media significantly reduced the apoptosis of both oxytocin and vasopressin MCNs. Here, we studied various temporal factors in the CNTF treatment that can influence the efficacy of MCN survival. Immunohistochemistry was used to identify and count surviving vasopressin and oxytocin MCNs in the supraoptic nucleus (SON) in hypothalamic slices cultured in the presence of CNTF (10 ng/ml media) for various time intervals, and in situ hybridization for vasopressin mRNA was used to evaluate the vasopressin mRNA gene expression in the SON under the same conditions. The presence of CNTF in the medium for 10 days produced a maximal increase in the survival of vasopressin MCNs (by 11-fold) and in the survival of oxytocin-MCNs (by approximately four-fold) over controls. These effects persisted for an additional 7-10 days even in the absence of CNTF. The ability of CNTF to increase survival of the MCNs or increase vasopressin mRNA levels in the SON required that the CNTF be present during the initial 7-10 days of culture. CNTF failed to rescue vasopressin or oxytocin MCNs when added to the media only for the last 7 days of a total of 14 days in vitro. Similar results were observed when SON vasopressin mRNA levels were measured. These results indicate that the presence of CNTF is required at the outset to rescue the vasopressin and oxytocin MCN from axotomy induced apoptosis, and that, after 10 days in CNTF, the MCNs no longer require the CNTF for survival.     

Development of histamine-immunoreactive neurons in the rat brain

This study was undertaken to reveal the cellular stores of histamine in developing rat brain and to determine the stage of development during which the histamine-immunoreactive neurons can first be detected. Rats from embryonal day 12 to postnatal day 14 were studied. The brains were fixed in 4% 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and standard immunofluorescence technique was used. The first histamine-immunoreactive neurons were seen on embryonic day 13 in the border of mesencephalon and metencephalon. On embryonic day 15 immunoreactive neurons were detected in ventral mesencephalon and rhombencephalon. In caudal, tuberal, and postmammillary caudal magnocellular nuclei histamine-immunoreactive neurons were first detected on embryonic day 20 while those in the hindbrain had disappeared. Histamine-immunoreactive nerve fibers were first detected on embryonic day 15 in rhombencephalon and mesencephalon and in some areas of diencephalon including the mammillary bodies and frontal cortex. On embryonic day 18 the number of immunoreactive nerve fibers in the hindbrain had decreased considerably, but the olfactory bulb, septal and hypothalamic area, and the cerebral cortex showed immunoreaction in fibers. The density of histamine-immunoreactive fiber networks increased until postnatal day 14 when an adultlike pattern of neurons and fibers had developed. Histamine-immunoreactive neurons are present in embryonal CNS and they develop extensive projections to various brain areas.     

Extrahypothalamic vasopressin in human brain

The distribution of arginine vasopressin immunoreactivity in post-mortem human brain was examined using a radioimmunoassay. The highest concentration was found in the hypothalamus but substantial amounts of vasopressin-like immunoreactivity were also found in the locus coeruleus, periaqueductal grey, substantia nigra compacta and reticulata and in lower concentrations in the globus pallidus. The extrahypothalamic vasopressin was immunologically and chromatographically similar to hypothalamic vasopressin. The possibility arises that the high levels of vasopressin in the locus coeruleus may relate to an effect on noradrenergic transmission.     

Localization of vasopressin (V1a) receptor binding and mRNA in the rhesus monkey brain

Centrally released arginine vasopressin (AVP) has been associated with various behavioural and cognitive effects, such as scent marking, aggression, and memory, which are believed to be mediated by the V1a subtype of the vasopressin receptor. Although the distribution of V1a receptors is conserved in a few brain regions, the pattern of expression of this receptor is, in general, highly species-specific. We have used receptor autoradiography with the linear V1a receptor ligand (125I-Phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2) to characterize the pattern of receptor binding in the rhesus monkey brain. Brain sites of V1a receptor synthesis were defined using in-situ hybridization. The regions of highest V1a receptor density included the prefrontal, cingulate, pyriform, and entorhinal cortex, as well as the presubiculum and mamillary bodies. In addition, V1a receptor binding and mRNA were detected in several regions reported to have V1a receptor in most rodents, including the amygdala, bed nucleus of the stria terminalis, lateral septum, hypothalamus and the brainstem. The distribution is consistent with a role for vasopressin in higher cognitive functions, especially memory, in primates.     

Abdominal surgery induces Fos immunoreactivity in the rat brain

Previous neuropharmacological studies indicate that brain peptides are involved in mediating gastric stasis induced by abdominal surgery. Central pathways activated by abdominal surgery were investigated in the rat by using Fos protein as a marker of neuronal activation. Abdominal surgery (laparotomy alone or combined with cecal manipulation) was performed under brief enflurance anesthesia (7–8 minutes), and 1 hour later rats were killed and brains processed for Fos immunoreactivity. Double labeling with Fos and arginine vasopressin, oxytocin, or tyrosine hydroxylase antibodies was also performed. Abdominal surgery induced Fos staining in the nucleus tractus solitarii, paraventricular and supraoptic nuclei of the hypothalamus, locus coeruleus, and ventrolateral medulla. After abdominal surgery, 18–25% of vasopressin and 18–33% of oxytocin-labeled cells were found to be Fos positive in the paraventricular nucleus and 15% of activated cells in the nucleus tractus solitarii were positive for tyrosine hydroxylase immunoreactivity. Enflurane alone induced c-fos expression in the same brain area; however, the number of Fos-positive cells and double-labeled cells were decreased two-to fivefold and three- to eightfold, respectively, compared with the abdominal surgery groups. These data show that abdominal surgery induced activation of specific hypothalamic, pontine, and medullary neurons. These findings may have implications for the understanding of central mechanisms involved in mediating gastric ileus following abdominal surgery. © 1994 Wiley-Liss, Inc.     

The effects of ionotropic agonists of excitatory amino acids on the release of arginine vasopressin in rat hypothalamic slices

The effects of ionotropic excitatory amino acids agonists on the release of vasopressin from rat hypothalamic slices were studied. Incubation with increasing doses of NMDA, kainate or AMPA decreased the release of vasopressin in a dose-dependent manner. The values of the IC50 were 1.0, 9.6, or 3.7 x 10-8 M, respectively. The inhibitory effect of the various excitatory amino acids tested was blocked by coincubation with their respective antagonists. Vasopressin secretion was stimulated to 140.3 +/- 7.6% of controls when the slices were obtained from chronically (7 days) salt-loaded rats. Addition of 1 x 10-7 M NMDA or 1 x 10-6 M kainate to the incubation medium antagonized the salt loading-induced increase in vasopressin release. Incubation with 1 x 10-4 M tetrodotoxin did not change basal vasopressin release, but it blocked the decrease in vasopressin secretion induced by 1 x 10-7 M NMDA or 1 x 10-6 M kainate or 1 x 10-6 M AMPA. Incubation with 1 x 10-5 M phaclophen (a GABAB antagonist) and 1 x 10-5 M bicuculline (a GABAA antagonist) was without effect on basal vasopressin secretion while it reversed the inhibition of vasopressin release induced by 1 x 10-7 M NMDA. Incubation with 1 x 10-6 M GABA alone decreased vasopressin secretion to 64.6 +/- 6.9% of control values. The inhibitory effect of GABA did not change when 1 x 10-7 M NMDA was added to the incubation medium. These findings demonstrate that ionotropic excitatory amino acids agonists inhibit vasopressin secretion from hypothalamic slices. They strongly suggest that this inhibitory effect is mediated through local GABAergic interneurones.     

Development of neurophysin-containing neurons in primary cultures of rat hypothalami is related to the age of the embryo: morphological study and comparison of in vivo and in vitro neurophysins, oxytocin, and vasopressin content

The morphological development of immunocytochemically identified neurophysin neurons and the evolution of neuropeptide content (neurophysins, vasopressin, and oxytocin) were studied in primary cultures of hypothalami obtained from 15- to 19-day-old embryos. According to their perikaryal surface, two populations of neurons were distinguished: large and small cells. Full development (defined by the perikaryal surface) of these neurons was reached at day 21 only in cultures from 15- or 16-day-old embryos. These two types of neurons may correspond to the magnocellular and parvocellular neurons described in vivo. Total neurophysins, vasopressin, and oxytocin content were measured by specific radioimmunoassays. Ontogeny of neurophysins and vasopressin showed a good correlation between cells cultured from 15- to 16-day-old embryos and hypothalami from age-matched rats. However, oxytocin was never detected in any of the cultures whatever the age of the embryos. Under our experimental conditions, hypothalamic primary cultures from 15- to 16-day-old embryos therefore appeared to be suitable for studying the differentiation and regulation of neurophysin- and vasopressin-containing neurons.     

Subtle sex differences in vasopressin mRNA expression in the embryonic mouse brain

Arginine vasopressin (AVP) is a neuropeptide which acts centrally to modulate numerous social behaviors. One receptor subtype through which these effects occur is the AVP 1a receptor (AVPR1A). The modulatory effects of Avp via the AVPR1A varies by species as well as sex, since both AVP and the AVPR1A tend to be expressed more prominently in males. Beyond these neuromodulatory effects there are also indications that the AVP system may play a role in early development to, in part, organize sex‐specific neural circuitry that is important to sexually dimorphic social behaviors in adulthood. However, to date, AVP's role in early development is poorly understood, particularly with respect to its differential effect on males and females. In order to determine the timing and distribution of the AVP system in early brain development, we examined the brains of male and female C57BL/6J mice between embryonic day (E) 12.5 and postnatal day (P) 2 and quantified Avp and Avpr1a mRNA using qPCR and AVPR1A protein using receptor autoradiography. The mRNA for Avp was measurable in males and females starting at E14.5, with males producing more than females, while Avpr1a mRNA was found as early as E12.5, with no difference in expression between sexes. AVPR1A binding was observed in both sexes starting at E16.5, and while there were no observed sex differences, binding density and the number of neuroanatomical areas did increase over time. These data are significant as they provide the first whole‐brain characterization of the vasopressin system in the embryonic mouse. Further, these findings are consistent with data from other species, that have documented a sex difference in the vasopressin system during early brain formation.     

Localization of immunoreactive angiotensin II in the hippocampus and striatum of rat brain

Angiotensin II (ANG II) was estimated by radioimmunoassay in extracts of rat brain. In extracts of whole brain the mean content was 108 ± 16fmol/g and estimates of ANG II in kidney and plasma were similar to previous reports. A regional distribution of ANG II was determined. Hippocampus had the highest concentration and cortex the lowest. The concentrations relative to cortex were: hippocampus, 8; striatum, 5; cerebellum, 4; hypothalamus:thalamus:septum:midbrain (HTSM), 3; and medulla, 3.     

Demonstration of glucocorticoid receptor-like immunoreactivity in glucocorticoid-sensitive vasopressin and corticotropin-releasing factor neurons in the hypothalamic paraventricular nucleus

Many parvocellular neurons in the paraventricular nucleus of the hypothalamus express high levels of corticotropin releasing factor (CRF) or vasopressin following adrenalectomy. To determine whether glucocorticoids feed back directly on these neurons, a mouse monoclonal antibody directed against the rat liver glucocorticoid receptor was used in combination with polyclonal antisera directed against either vasopressin or CRF to permit simultaneous visualization of either peptide with glucocorticoid receptor-like immunoreactivity (IR). Rats were adrenalectomized (ADX) for 2 weeks to optimize numbers of vasopressin - and CRF-IR neurons. Six hours prior to sacrifice, a separate group of adrenalectomized rats was treated with corticosterone (40 mg/kg). This short-term replacement resulted in nuclear localization of glucocorticoid receptor-like-IR but did not attenuate the increased numbers of CRF- and vasopressin-IR neurons observed after adrenalectomy. It was therefore possible to visualize vasopressin- or CFR-IR and nuclear glucocorticoid receptor-like-IR simultaneously. Cell counts of double-labeled neurons in the paraventricular nucleus of the hypothalamus (PVH) demonstrated that glucocorticoid receptor-like-IR is colocalized in virtually all the CRF and vasopressin immunoreactive parvocellular neurons studied, which respond to adrenalectomy by increased peptide expression. These data suggest that a major feedback effect of glucocorticoids on the hypothalamic-pituitary-adrenal axis is exerted directly within nuclei of CRF and vasopressin neurons.     

Perfusion of vasopressin within the rat brain suppresses prostaglandin E-hyperthermia

These experiments were undertaken to determine whether arginine vasopressin (AVP) could suppress a prostaglandin hyperthermia and to localize sites of these actions in the rat. Prostaglandin E2 (PGE2) sensitive sites were localized in the ventral-septal area by microinjecting 200 ng/0.5 microliter of prostaglandin E2. During perfusion with an artificial CSF, PGE2 injected into the lateral cerebral ventricle evoked a hyperthermia of more than 1 degree C. Perfusion of 6.5 micrograms/ml of AVP markedly attenuated the PGE2-induced hyperthermia. These results suggest that AVP suppresses PGE2-induced hyperthermia in sites in which PGE2 evokes an increase in core temperature.