Arginine Vasopressin Cells Research Articles

A vasopressin circuit that modulates mouse social investigation and anxiety-like behavior in a sex-specific manner

One of the largest sex differences in brain neurochemistry is the expression of the neuropeptide arginine vasopressin (AVP) within the vertebrate brain, with males having more AVP cells in the bed nucleus of the stria terminalis (BNST) than females. Despite the long-standing implication of AVP in social and anxiety-like behaviors, the circuitry underlying AVP's control of these behaviors is still not well defined. Using optogenetic approaches, we show that inhibiting AVP BNST cells reduces social investigation in males, but not in females, whereas stimulating these cells increases social investigation in both sexes, but more so in males. These cells may facilitate male social investigation through their projections to the lateral septum (LS), an area with the highest density of sexually differentiated AVP innervation in the brain, as optogenetic stimulation of BNST AVP → LS increased social investigation and anxiety-like behavior in males but not in females; the same stimulation also caused a biphasic response of LS cells ex vivo. Blocking the vasopressin 1a receptor (V1aR) in the LS eliminated all these responses. Together, these findings establish a sexually differentiated role for BNST AVP cells in the control of social investigation and anxiety-like behavior, likely mediated by their projections to the LS.

Bile Duct Ligation Changes the Inhibitory Control of Vasopressin Neurons in Male Rats

Dilutional hyponatremia typically is caused by increased Arginine Vasopressin (AVP) secretion. This increases the risk of complications and mortality in patients with liver cirrhosis. Liver cirrhosis in male rats increases AVP release but the underlying mechanisms are still under investigation. In other models of chronic AVP release, changes in chloride homeostasis alters the inhibitory control of AVP neurons in the supraoptic nucleus (SON) causing GABA mediated excitation instead of inhibition. In the bile duct ligation (BDL) model of cirrhosis, changes in either intracellular chloride concentration or cell firing activity have not been directly tested. We hypothesize that liver cirrhosis increases intracellular chloride concentration leading to a decrease in GABA‐mediated inhibition of SON neurons from male rats.To test this hypothesis, we injected an adeno‐associated virus with a vasopressin promoter and a ratiometric chloride indicator, AAV2‐0VP1‐ClopHensorN into the SON of adult male Sprague‐Dawley rats. The rats either received BDL surgery, which was used to model liver cirrhosis, or a sham ligation surgery. All BDL rats had significantly higher liver weight to body weight ratio (n = 10‐20, p < 0.05), lower plasma osmolality (n = 10‐20, p < 0.001) and lower hematocrit (n = 10‐20, p < 0.05) as compared to sham rats. All rats were anesthetized with isoflurane (2‐3%), their brains were collected, and slices containing the SON were prepared. Loose‐cell patch recordings were made at 31±1oC using standard artificial cerebrospinal fluid. Labeled AVP cells and unlabeled SON cells were tested with focal application of muscimol (100µM), a GABAA receptor agonist.Cells from BDL rats showed impaired GABAA‐mediated inhibition in response to muscimol, as compared to those from sham rats. All putative AVP SON neurons from sham rats (4/4) showed decreased activity following muscimol while half of the putative AVP SON neurons (7/14) from BDL rats were excited (p < 0.001). Similar results were observed in experiments were the injections missed the SON. In these experiments, all SON neurons from sham rats were inhibited by muscimol (26/26) while thirty‐five percent of SON neurons from BDL rats (16/45) were excited by muscimol (p = 0.001). ClopHensorN based chloride imaging showed that muscimol application was associated with decreased intracellular chloride concentrations in putative AVP cells from BDL rats as compared to sham rats (n = 31‐32, p = 0.001). The results show that liver cirrhosis impairs the normal inhibitory response of SON neurons to GABAA receptor activation which could contribute to increased cell activity and dilutional hyponatremia in male rats.

Knockdown of sexually differentiated vasopressin expression in the bed nucleus of the stria terminalis reduces social and sexual behaviour in male, but not female, mice.

The neuropeptide arginine-vasopressin (AVP) has long been implicated in the regulation of social behaviour and communication, but the sources of AVP release relevant for behaviour have not been precisely determined. Ablations of the sexually dimorphic AVP cells within the bed nucleus of the stria terminalis (BNST), which are more numerous in males, affect social behaviour differently in males and females. However, it is unknown whether these behavioural effects are caused by a reduction of AVP or of other factors associated with these cells. To test the role of AVP specifically, we used an shRNA viral construct to knock down AVP gene expression within the BNST of wild-type male and female mice, using scrambled sequence virus as a control, and evaluated subsequent changes in social behaviours (social investigation, ultrasonic vocalization (USV), scent marking, copulation, and aggression), or anxiety-like behaviours (elevated plus maze). We observed that, in males, knockdown of AVP expression in the BNST strongly reduced investigation of novel males, aggressive signalling towards other males (tail rattling, USV), and copulatory behaviour, but did not alter attack initiation, other measures of social communication, or anxiety-like behaviours. In females, however, BNST AVP knockdown did not alter any of these behaviours. These results point to differential involvement of AVP derived from the BNST in social behaviour.

Transcription Factor TonEBP Stimulates Hyperosmolality-Dependent Arginine Vasopressin Gene Expression in the Mouse Hypothalamus.

The hypothalamic neuroendocrine system is strongly implicated in body energy homeostasis. In particular, the degree of production and release of arginine vasopressin (AVP) in the hypothalamus is affected by plasma osmolality, and that hypothalamic AVP is responsible for thirst and osmolality-dependent water and metabolic balance. However, the osmolality-responsive intracellular mechanism within AVP cells that regulates AVP synthesis is not clearly understood. Here, we report a role for tonicity-responsive enhancer binding protein (TonEBP), a transcription factor sensitive to cellular tonicity, in regulating osmosensitive hypothalamic AVP gene transcription. Our immunohistochemical work shows that hypothalamic AVP cellular activity, as recognized by c-fos, was enhanced in parallel with an elevation in TonEBP expression within AVP cells following water deprivation. Interestingly, our in vitro investigations found a synchronized pattern of TonEBP and AVP gene expression in response to osmotic stress. Those results indicate a positive correlation between hypothalamic TonEBP and AVP production during dehydration. Promoter and chromatin immunoprecipitation assays confirmed that TonEBP can bind directly to conserved binding motifs in the 5’-flanking promoter regions of the AVP gene. Furthermore, dehydration- and TonEBP-mediated hypothalamic AVP gene activation was reduced in TonEBP haploinsufficiency mice, compared with wild TonEBP homozygote animals. Therefore, our result support the idea that TonEBP is directly necessary, at least in part, for the elevation of AVP transcription in dehydration conditions. Additionally, dehydration-induced reductions in body weight were rescued in TonEBP haploinsufficiency mice. Altogether, our results demonstrate an intracellular machinery within hypothalamic AVP cells that is responsible for dehydration-induced AVP synthesis.

Viral rescue of magnocellular vasopressin cells in adolescent Brattleboro rats ameliorates diabetes insipidus, but not the hypoaroused phenotype

Dysregulated arousal often accompanies neurodevelopmental disorders such as attention deficit hyperactivity disorder and autism spectrum disorder. Recently, we have found that adolescent homozygous Brattleboro (Hom) rats, which contain a mutation in the arginine vasopressin (AVP) gene, exhibit lower behavioral arousal than their heterozygous (Het) littermates in the open field test. This hypoaroused phenotype could be due to loss of AVP in magnocellular cells that supply AVP to the peripheral circulation and project to limbic structures or parvocellular cells that regulate the stress axis and other central targets. Alternatively, hypoarousal could be a side effect of diabetes insipidus – polydipsia and polyuria seen in Hom rats due to loss of AVP facilitation of water reabsorption in the kidney. We developed a viral-rescue approach to “cure” magnocellular AVP cells of their Brattleboro mutation. Infusion of a recombinant adeno-associated virus (rAAV) containing a functional Avp gene and promoter (rAAV-AVP) rescued AVP within magnocellular cells and fiber projections of the paraventricular nucleus of the hypothalamus (PVN) of male and female adolescent Hom rats. Furthermore, water intake was markedly reduced, ameliorating the symptoms of diabetes insipidus. In contrast, open field activity was unaffected. These findings indicate that the hyporaoused phenotype of adolescent Hom rats is not due to the loss of AVP function in magnocellular cells or a side effect of diabetes insipidus, but favors the hypothesis that central, parvocellular AVP mechanisms underlie the regulation of arousal during adolescence.

Sexually Dimorphic Vasopressin Cells Modulate Social Investigation and Communication in Sex-Specific Ways.

The neuropeptide arginine vasopressin (AVP) has long been implicated in the regulation of social behavior and communication, but precisely which AVP cell groups are involved is largely unknown. To address whether the sexually dimorphic AVP cell group in the bed nucleus of the stria terminalis (BNST) is important for social communication, we deleted BNST AVP cells by viral delivery of a Cre-dependent caspase-3 cell-death construct in AVP-iCre-positive mice using AVP-iCre negative littermate as controls, and assessed social, sexual, aggressive and anxiety-related behaviors. In males, lesioning BNST AVP cells reduced social investigation of other males and increased urine marking (UM) in the presence of a live female, without altering ultrasonic vocalizations (USVs), resident-intruder aggression, copulatory behavior, anxiety, or investigation of females or their odor cues. In females, which have significantly fewer AVP cells in the BNST, these injections influenced copulatory behavior but otherwise had minimal effects on social behavior and communication, indicating that these cells contribute to sex differences in social behavioral function.

Vasopressin infusion into the lateral septum of adult male rats rescues progesterone-induced impairment in social recognition

It is well established that social recognition memory is mediated, in part, by arginine vasopressin (AVP). AVP cells within the bed nucleus of the stria terminalis (BST) and medial amygdala (MeA) send AVP-ergic projections to the lateral septum (LS). We have demonstrated that progesterone treatment decreases AVP immunoreactivity within the BST, the MeA and the LS, and that progesterone treatment impairs social recognition. These data suggested that progesterone may impair social recognition memory by decreasing AVP. In the present experiment, we hypothesized that infusions of AVP into the LS would rescue the progesterone-induced impairment in social recognition within adult male rats. One week after adult male rats underwent cannula surgery, they were given systemic injections of either a physiological dose of progesterone or oil control for 3days. Four hours after the last injection, we tested social recognition memory using the social discrimination paradigm, a two-trial test that is based on the natural propensity for rats to be highly motivated to investigate novel conspecifics. Immediately after the first exposure to a juvenile, each animal received bilateral infusions of either AVP or artificial cerebrospinal fluid into the LS. Our results show that, as expected, control animals exhibited normal social discrimination. In corroboration with our previous results, animals given progesterone have impaired social discrimination. Interestingly, animals treated with progesterone and AVP exhibited normal social discrimination, suggesting that AVP treatment rescued the impairment in social recognition caused by progesterone. These data also further support a role for progesterone in modulating vasopressin-dependent behavior within the male brain.

Activation of vasopressin neurons leads to phenotype progression in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI) is a rare disease that is inherited in an autosomal dominant manner. In a previous study, we made a mouse model for FNDI, which showed progressive polyuria accompanied by inclusion bodies in the arginine vasopressin (AVP) neurons formed by aggregates in the endoplasmic reticulum. The present study was conducted to determine whether the activities of AVP neurons are related to the phenotype progression in the FNDI model. In the first experiment, female heterozygous mice were administered either desmopressin (dDAVP) or a vehicle (control) subcutaneously with osmotic minipumps for 30 days. The dDAVP treatment significantly decreased the urine volume, AVP mRNA expression, and inclusion bodies in the AVP neurons. Urine volume in the dDAVP group remained significantly less than the control for 14 days even after the minipumps were removed. In the second experiment, the males were fed either a 0.2% Na or 2.0% Na diet for 6 mo. Urine AVP excretion was significantly increased in the 2.0% Na group compared with the 0.2% Na group for the first 2 mo but gradually decreased thereafter. Throughout the experiments, urine volume increased progressively in the 2.0% Na group but not in the 0.2% Na group. Immunohistochemical analyses revealed that inclusion bodies in the AVP cells had significantly increased in the 2.0% Na compared with the 0.2% Na group. These data demonstrated that activation of AVP neurons could accelerate the aggregate formation as well as the progression of the polyuria in the FNDI model mice.

Progressive polyuria without vasopressin neuron loss in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, is mostly caused by mutations in the gene of neurophysin II (NPII), the carrier protein of arginine vasopressin (AVP). Previous studies suggest that loss of AVP neurons might be the cause of polyuria in FNDI. Here we analyzed knockin mice expressing mutant NPII that causes FNDI in humans. The heterozygous mice manifested progressive polyuria as do patients with FNDI. Immunohistochemical analyses revealed that inclusion bodies that were not immunostained with antibodies for mutant NPII, normal NPII, or AVP were present in the AVP cells in the supraoptic nucleus (SON), and that the size of inclusion bodies gradually increased in parallel with the increases in urine volume. Electron microscopic analyses showed that aggregates existed in the endoplasmic reticulum (ER) as well as in the nucleus of AVP neurons in 1-mo-old heterozygous mice. At 12 mo, dilated ER filled with aggregates occupied the cytoplasm of AVP cells, while few aggregates were found in the nucleus. Analyses with in situ hybridization revealed that expression of AVP mRNA was significantly decreased in the SON in the heterozygous mice compared with that in wild-type mice. Counting cells expressing AVP mRNA in the SON indicated that polyuria had progressed substantially in the absence of neuronal loss. These data suggest that cell death is not the primary cause of polyuria in FNDI, and that the aggregates accumulated in the ER might be involved in the dysfunction of AVP neurons that lead to the progressive polyuria.

Regional differences in circadian period within the suprachiasmatic nucleus

In mammals, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nucleus (SCN), which is composed of multiple, single-cell oscillators. Isolated SCN tissue shows clear circadian oscillation in release of arginine vasopressin (AVP) in organotypic slice cultures. Previously, we reported that the oscillators in the dorsal SCN have shorter periods than those in the ventral part. Here, we examined whether a correlation between the period and the rostral–caudal co-ordination could exist. The rostral, central and caudal SCN were cultured separately and the periods of circadian rhythms of AVP release were measured. The rostral and caudal parts of the SCN showed shorter periods than the central SCN. Together with previous findings, it is suggested that the shorter period region originates from AVP containing areas, while the longer period region corresponds with vasoactive intestinal polypeptide (VIP) containing cells. In our VIP-immunoreactive slices, the application of VIP antagonists shortened the periods of the AVP-releasing rhythm. These data indicate that the oscillators in AVP cells have short periods and are entrained by VIP cells to form a single integrated rhythm.

Different signaling molecules responsible for IL-1β-induced oxytocinergic and vasopressinergic neuron activation in the hypothalamic paraventricular nucleus of the rat

It has been well known that oxytocin (OT)-ergic and arginine vasopressin (AVP)-ergic neurons located in the hypothalamic paraventricular nucleus (PVN) and superoptic nucleus (SON) are two kinds of neuroendocrine cells with diverse functions. It has also been demonstrated that immune stimuli can activate these neurons to secret OT and AVP. However, the intracellular signal transduction molecules responsible for the activation of these OT-ergic and AVP-ergic neurons in PVN by immune stimuli are still unclear. In this experiment, the roles of Fos, a protein product of immediate early gene c-fos, and extracellular signal-regulated protein kinase (ERK) 1/2, a signal transduction molecule of mitogen-activated protein kinase (MAPK) family, in these processes were studied in the PVN of the rat following IL-1β stimulation. The Sprague–Dawley rats were received either 750 ng/kg IL-1β or equal volume normal saline (NS) injection intravenously (i.v.), and perfused transcardially by 4% paraformaldehyde 3 h later. Fos and phosphorylated ERK1/2 (pERK1/2)-immunoreactivity (-ir) was observed in PVN by ABC immunohistochemical staining. Meanwhile, the double staining for OT/Fos, AVP/Fos, OT/pERK1/2 and AVP/pERK1/2 were also processed. The ABC immunohistochemical staining results showed that after an i.v. injection of IL-1β, the expressions of Fos and pERK1/2 increased evidently in the PVN. Double-staining results showed that a large number of OT-ir cells contained strong Fos-ir products in their nuclei, while only a few of OT cells were double labeled with pERK1/2. As to AVP neurons, great quantities of AVP cells were strongly double labeled with pERK1/2 while there were nearly no Fos-ir nuclei in AVP-ir cells. We conclude from these results that the intracellular IL-1β-induced events in OT and AVP neurons in PVN are quite different. The OT neurons are mainly activated via Fos without involvement of ERK1/2 pathway, while the latter, but not Fos, involves the intracellular event in AVP neurons activated by IL-1β.

Progesterone Treatment of Adult Male Rats Suppresses Arginine Vasopressin Expression in the Bed Nucleus of the Stria Terminalis and the Centromedial Amygdala

The steroid sensitive vasopressin cells of the bed nucleus of the stria terminalis (BST) and centromedial amygdala (CMA) are involved in numerous behavioural and physiological functions. These cells are known to be greatly influenced by gonadal steroids. Castration reduces and testosterone replacement restores arginine vasopressin (AVP)-immunoreactive (-ir) labelling and AVP mRNA expression in the BST and CMA. Gonadal steroids appear to act directly in AVP-expressing cells within the BST and CMA, because the majority of AVP-ir cells in these areas contain oestrogen and androgen receptor immunoreactivity. Recently, we have localised progestin receptor immunoreactivity in virtually all of the AVP-ir cells in the BST and CMA. To understand the role played by progestin receptors in AVP cells within the BST and CMA, we treated male rats with 1 mg of progesterone or oil for 5 days, and then examined AVP immunoreactivity within the brain. We found that progesterone decreased AVP-ir labelling within the BST and CMA, as well as in two of the projection sites of these cells, the lateral septum and lateral habenula. Progesterone treatment did not alter testosterone secretion from the testes, nor did it alter adult male sexual behaviour. These data illustrate an additional mechanism by which the AVP cells in the BST and CMA can be regulated. These data also suggest that progesterone may act in the male brain to influence behaviours that are AVP-dependent.

Co-localization and distribution of corticotrophin-releasing hormone, arginine vasopressin and enkephalin in the paraventricular nucleus of sheep: A sex comparison

The paraventricular nucleus (PVN) is integral to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and contains cells producing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin. We used immunohistochemistry to map these peptides and to resolve the extent of co-localization within PVN cells in intact and gonadectomized male and female sheep. Immunoreactive (ir) CRH, AVP and enkephalin cells were mapped in two rams and two ewes at 180 μm intervals throughout the rostro-caudal extent of the PVN. Similar distributions of AVP-ir cells occurred in both sexes whereas CRH-ir and enkephalin-ir cells extended more rostrally in rams. In groups ( n=4) of intact and gonadectomized sheep of both sexes, co-localization and distribution of neuropeptides was influenced by sex and gonadectomy. Males had more AVP and CRH cells than females. Intact animals had more AVP cells than gonadectomized animals. There were no differences between groups in the number or percentage of cells that stained for both CRH and AVP or in the number of cells that stained for both CRH and enkephalin. Differences were observed in the percentage of enkephalin cells that contained CRH with males having a greater percentage of co-localized cells than did females. Differences were also observed in the number and percentage of cells that stained for both enkephalin and AVP; the number of cells that stained for both neuropeptides was greater in males than in females and greater in intact animals than in gonadectomized animals. Differences were observed in the percentage of AVP cells that contained enkephalin, and in the percentage of enkephalin cells that contained AVP with males having a greater percentage of co-localized cells than did females. We conclude that sex and gonadal status affect peptide distribution in the PVN of the sheep which may provide an anatomical basis for sex differences in HPA axis responses to stress.

Regulation of Vasopressin Gene Expression by cAMP and Glucocorticoids in Parvocellular Neurons of the Paraventricular Nucleus in Rat Hypothalamic Organotypic Cultures

Arginine vasopressin (AVP) in the parvocellular neurons of the paraventricular nucleus (PVN) is known to play an important role in the hypothalamo-pituitary-adrenal axis. In the present study, we examined how cAMP and glucocorticoids regulate AVP gene expression in the parvocellular neurons of the PVN in rat hypothalamic organotypic cultures with in situ hybridization. AVP heteronuclear (hn) RNA, an indicator for gene transcription, was induced in the PVN with incubation of forskolin as reported previously, and AVP mRNA was increased by forskolin in the presence of the gene transcription inhibitor 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB). These data indicate that cAMP could increase not only gene transcription but also mRNA stability. Dexamethasone treatment, in contrast, significantly decreased AVP mRNA expression levels in the PVN, but this inhibitory action was abolished in the presence of DRB or the sodium channel blocker tetrodotoxin (TTX). However, when the hypothalamic slices were treated with forskolin, dexamethasone decreased AVP mRNA expression even in the presence of DRB and/or TTX. Furthermore, AVP hnRNA expression induced by forskolin was attenuated by dexamethasone treatment in the presence of TTX. These data indicate that dexamethasone could act on AVP cells independently of action potentials to decrease mRNA stability and to suppress AVP gene transcription during stimulation by cAMP. Thus, it was demonstrated that: (1) cAMP upregulates AVP gene transcriptionally and post-transcriptionally, (2) the mode of action of glucocorticoids was dependent on whether the cells were stimulated by cAMP, and (3) the interactions between cAMP and glucocorticoids encompass both gene transcription and mRNA stability.

Direct stimulation of arginine vasopressin gene transcription by cAMP in parvocellular neurons of the paraventricular nucleus in organotypic cultures.

The regulation of arginine vasopressin (AVP) gene transcription in the paraventricular nucleus (PVN) was studied in rat hypothalamic organotypic cultures using intronic in situ hybridization. While AVP heteronuclear (hn) RNA was not detected in the PVN under basal conditions, a marked induction of AVP hnRNA was observed after 2 and 3 h incubation of slices with forskolin. In contrast to the stimulatory effects of forskolin, phorbol 12-myristate 13-acetate (PMA) was completely ineffective in inducing AVP hnRNA in the PVN at any time examined (1-3 h). Forskolin-induced AVP hnRNA expression was unaffected by blockage of neurotransmission by the sodium channel inhibitor, tetrodotoxin, indicating that forskolin acts directly on AVP cells in the PVN. Dual staining in situ hybridization of forskolin-stimulated hypothalamic sections using both radio labeled AVP hnRNA and digoxigenin-labeled CRH mRNA probes revealed colocalization of both transcripts, indicating AVP hnRNA is expressed in the parvocellular neurons. The data demonstrate that cAMP directly activates AVP gene transcription in parvocellular neurons of the PVN.

Functional significance of colocalization of PACAP and catecholamine in nerve terminals.

Medullary neurons containing pituitary adenylate cyclase-activating polypeptide (PACAP) and noradrenalin (NA) project to the hypothalamus and they are involved in the regulation of arginine vasopressin (AVP) neurons. At the ultrastructural level, PACAP immunoreactivity was detected in the granular vesicles in catecholaminergic nerve terminals that made synaptic contact with AVP neurons. Both PACAP (at least 1 nM) and NA (at least 1 microM) induced large increases in the cytosolic Ca2+ concentration ([Ca2+]i) in isolated AVP cells. PACAP at 0.1 nM and NA at 0.1 microM had little effects, if any, on [Ca2+]i. However, when 0.1 nM PACAP and 0.1 microM NA were combined, they evoked large increase in [Ca2+]i in AVP neurons. An inhibitor of protein kinase A (PKA) completely inhibited the PACAP-induced increase in [Ca2+]i, but only partly inhibited the NA-induced increase in [Ca2+]i. In AVP cells that were prelabeled with quinacrine, PACAP and NA acted synergistically to induce a loss of quinacrine fluorescence, indicating secretion of neurosecretory granules in AVP neurons. The results suggest that PACAP and NA, coreleased from the same nerve terminals, act in synergy to evoke calcium signaling and secretion in AVP neurons, and that the synergism is mediated by the interaction between cAMP-PKA pathway an as yet unidentified factor "X" linked to L-type Ca2+ channels. The synergism between PACAP and NA may contribute to the regulation of AVP secretion under physiological conditions.

Stimulating Effect of HIV-1 Coat Protein gp120 on Corticotropin-Releasing Hormone and Arginine Vasopressin in the Rat Hypothalamus: Involvement of Nitric Oxide

Subjects with human immunodeficiency virus type 1 (HIV-1) infection display increased activity of the hypothalamo-pituitary-adrenal (HPA) axis, which may play a role in both HIV-related neurodegenerative processes and disease progression. It has been speculated that the HIV coat protein gp120 may be responsible for these changes, and previous experimental evidence in both transgenic and nontransgenic mice supports this view. We speculated that one of the effects of gp120 in the CNS is to act within the hypothalamus to affect both corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), the principal regulators of HPA axis. We therefore administered i.p. gp120 (100 ng/rat) or vehicle to male Wistar rats and then detected Fos protein (an index of neuronal activation), CRH, and AVP immunoreactivity in the cellular compartments of the hypothalamic paraventricular nucleus (PVN). In addition, we tested the direct effect of various concentrations of gp120 on the release of CRH and AVP from rat hypothalamic explants maintained in vitro. Any modulation of gp120 effects by nitric oxide (NO) pathways was also sought by coadministering i.p. to rats or adding to the hypothalamic preparations the NO synthase inhibitor NG-methyl-l-arginine (l-NMMA). Gp120 induced the expression of Fos protein in both the parvo- and the magnocellular PVN, which was significantly attenuated by l-NMMA 10−6 nM/L (P < 0.001 vs gp120 alone). Double immunochemistry showed costaining for Fos protein and CRH or AVP in the PVN following gp120; the number of double-labeled CRH and AVP cells for Fos protein was markedly reduced (P < 0.001) by coadministration of l-NMMA 10−6 nM/L. In the in vitro studies, addition of gp120 to the hypothalamic explants in the dose range of 10 pM–1 nM resulted in a clear stimulation of both CRH and AVP release (P < 0.05–0.001 compared to control); in the presence of l-NMMA at 10-fold higher concentrations the stimulatory effect of gp120 on the release of both peptides was completely lost. It would therefore appear that gp120 activates CRH and AVP-producing neurons in the hypothalamic PVN and stimulates the release of both peptides in vitro via NO-dependent mechanisms. These findings, in line with previous evidence, further suggest that the increased activity of the HPA axis associated with HIV infection may be of central origin, due to the effects of gp120 on hypothalamic CRH and AVP release.

Severe loss of vasopressin-immunoreactive cells in the suprachiasmatic nucleus of aging voles coincides with reduced circadian organization of running wheel activity

Aging leads to a decrease in circadian organization of behavior. Whether this general observation is related to the finding that in older subjects the arginine–vasopressin (AVP) system in the suprachiasmatic nucleus (SCN) has deteriorated is an unsolved question. Here we assessed circadian organization of running wheel behavior and numbers of AVP cells in the SCN of old voles ( n=12, 11.5 months of age) and compared the results with data from young voles ( n=16, 4.5 months of age). A third of the young voles, but three-quarter of the old voles lost circadian rhythmicity. Analysis of daily onset to onset periodicity of running wheel activity at the age of 5 and 10 months in individual voles revealed a significant loss of precision of circadian rhythmicity at the higher age. The number of AVP cells in the SCN of old voles decreased substantially, over 78% compared to young voles in general. AVP cell numbers, however, cannot be directly correlated with the state of rhythmicity in old voles; in one of the three circadian rhythmic old voles the SCN contained the least AVP cells. This study does not support the idea of a causal relationship between aging induced reduction in AVP cells in the SCN and the presence of circadian rhythmicity in behavior.

Fetal tissue containing the suprachiasmatic nucleus restores multiple circadian rhythms in old rats.

The suprachiasmatic nucleus (SCN) is the major circadian pacemaker in mammals. When fetal tissue containing the SCN is transplanted into young rats whose circadian rhythms have been abolished by SCN lesions, the rhythms gradually reappear. Circadian rhythms in many rats deteriorate or disappear with age. The rationale of the present study was that old rats with poor circadian rhythms resemble young rats with SCN lesions. If there is a similar mechanism underlying this resemblance, then fetal tissue containing the SCN should restore rhythms in old rats. Therefore, we implanted such tissue into the third ventricle of intact aged rats with poor circadian rhythms. Body temperature, locomotor activity, and/or drinking were measured simultaneously within subjects. Grafts and hosts were stained immunocytochemically for vasoactive intestinal polypeptide (VIP), arginine vasopressin (AVP), and neuropeptide Y (NPY). Of 23 SCN grafts, 14 were viable (cells observable with Nissl or peptide staining). In 7 of the 14 aged hosts, up to three circadian rhythms were improved or restored. VIP cells were always observable, which was not the case for AVP cells or NPY fibers. In the other seven hosts, no circadian rhythm was improved. Compared with the successful grafts, these unsuccessful grafts had similar amounts of AVP and NPY staining but significantly less VIP cell and/or fiber staining. Fetal cerebellar grafts, which do not contain any of the three peptides, did not improve or restore any rhythms. Thus the degeneration of circadian rhythms in aged rats may be due, at least in part, to deterioration of the aged SCN and in particular, to a loss of function of VIP-containing neurons.

Vasopressinergic Innervation of the Bovine Pineal Gland: Is There a Local Source for Arginine Vasopressin?

Although the presence of arginine vasopressin (AVP) in the mammalian pineal has been characterized biochemically, the source of this nonapeptide hormone remains enigmatic. Most earlier data pointed to an extrapineal origin, although some recent evidence suggests intrapineal synthesis of AVP. The present study examined this issue using a combination of immunohistochemistry with antibodies against both the AVP and neurophysin moieties of the AVP precursor polypeptide, together with polymerase chain reaction (PCR) assay for the specific mRNA. Furthermore, the effects of AVP on melatonin production by monolayer cultures of bovine pinealocytes were examined. Bovine pineal glands possessed numerous neurophysin- and AVP-immunopositive nerve fibers, mainly in the distal part of the gland. However, no positively stained perikarya were observed. As a positive control perikarya of AVP cells were easily identifiable in the magnocellular cells of the bovine hypothalamus. Nevertheless, a highly sensitive PCR assay specific for full-length AVP mRNA did indicate the presence of AVP gene transcripts in both bovine and ovine pineal glands, using two different primer combinations. This suggests either that there are AVP perikarya in the pineal whose peptide contents are below the level of detection by immunohistochemistry or that gene transcripts may be present in AVP nerve axons, as in the posterior pituitary. AVP had significant inhibitory effects on noradrenaline-provoked melatonin secretion in vitro. These results indicate that AVP released by nonsympathetic nerve fibers terminating in the bovine pineal gland may act to modulate melatonin production.