Effects of Morris water maze testing on the neuroendocrine stress response and intrahypothalamic release of vasopressin and oxytocin in the rat

Adult male Wistar rats were trained in the Morris water maze (MWM) on 3 consecutive days to find a visible platform. Concomitantly, microdialysis samples from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei were collected in order to monitor local release of the neuropeptides vasopressin (AVP) and oxytocin (OXT), respectively, during controllable swim stress. Additionally, a separate set of animals was equipped with chronic jugular venous catheters to collect blood samples for analyzing plasma concentrations of corticotropin (ACTH) and corticosterone during training in the MWM. As measured by microdialysis, swimming in the MWM caused a significantly increased release of AVP within the PVN and of OXT within the SON on each of the 3 test sessions. In contrast to OXT in the SON, basal AVP concentrations in the PVN tended to rise from day to day. Plasma ACTH and corticosterone were found to be similarly elevated in response to MWM exposure on each of the test sessions. Taken together, these data demonstrate that testing in the MWM is not only associated with a significant activation of the hypothalamo-pituitary-adrenal axis but also with an intrahypothalamic release of AVP and OXT. If compared with findings using repeated forced swimming as an uncontrollable stressor (Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R., Engelmann, M., 1998. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 85, 1209-1222), the present results suggest that (1) similarities in the release profiles of AVP in the PVN and plasma hormone levels are fairly independent from the controllability of the stressor and seem, thus, to primarily relate to the physical demands of the task, whereas (2) the different intra-SON OXT release profiles might be linked to the controllability of the stressor.     

Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction.     

Centrally administered adrenomedullin 2 activates hypothalamic oxytocin-secreting neurons, causing elevated plasma oxytocin level in rats

We examined the effects of intracerebroventricular (i.c.v.) administration of adrenomedullin 2 (AM2) on plasma oxytocin (OXT) and arginine vasopressin (AVP) levels in conscious rats. Plasma OXT levels were markedly increased 5 min after i.c.v. administration of AM2 (1 nmol/rat) compared with vehicle and remained elevated in samples taken at 10, 15, 30, and 60 min. By contrast, plasma AVP levels were not significantly elevated in samples taken between 5 and 180 min after i.c.v. administration of AM2 except at the 30-min time point. Fos-like immunoreactivity (Fos-LI) was observed in various brain areas, including the paraventricular (PVN) and the supraoptic nuclei (SON) after i.c.v. administration of AM2 (2 nmol/rat) in conscious rats (measured at 90 min post-AM2 infusion). Dual immunostaining for OXT/Fos and AVP/Fos showed that OXT-LI neurons predominantly exhibited nuclear Fos-LI compared with AVP-LI neurons in the PVN and the SON. In situ hybridization histochemistry showed that i.c.v. administration of AM2 (0.2, 1, and 2 nmol/rat) caused marked induction of the expression of the c-fos gene in the PVN and the SON. This induction was significantly reduced by pretreatment with both the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) (3 nmol/rat) and the AM receptor antagonist AM-(22-52) (27 nmol/rat). These results suggest that centrally administered AM2 mainly activates OXT-secreting neurons in the PVN and the SON, at least in part through the CGRP and/or AM receptors with marked elevation of plasma OXT levels in conscious rats.     

Oxytocin actions within the supraoptic and paraventricular nuclei: differential effects on peripheral and intranuclear vasopressin release

In response to forced swimming (FS), AVP is released somato-dendritically within the supraoptic nucleus (SON) and paraventricular nucleus (PVN), but not from neurohypophyseal terminals into blood. Together with AVP, oxytocin (OXT) is released within the SON and PVN. Here, we studied the role of intra-SON and intra-PVN OXT in the regulation of local AVP release and into the blood in male rats. Within the SON, bilateral retrodialysis of an OXT receptor antagonist (OXT-A) increased local AVP release in response to FS [60 s, 21 degrees C, vehicle twofold, not significant (ns); OXT-A: 15-fold increase, P < 0.05] without significantly affecting basal AVP release. In addition, local OXT-A elevated plasma AVP secretion under basal conditions (twofold increase, P < 0.05) without further elevation after FS. Within the PVN, exposure to FS elevated local AVP release, reaching significance only in the OXT-A group (vehicle: 1.4-fold, ns; OXT-A: 1.6-fold increase, P = 0.050). Bilateral OXT-A into the PVN did not affect peripheral AVP secretion either under basal or stress conditions. Basal ACTH concentrations tended to be elevated by local OXT-A within the PVN (1.7-fold increase, P = 0.076). In contrast, the swim-induced ACTH secretion was attenuated after retrodialysis of OXT-A within both the SON (at 5 min) and PVN (at 15 min) (P < 0.05 both) compared with vehicle. The results demonstrate a receptor-mediated effect of OXT within the SON and PVN on local and neurohypophyseal AVP release, which depends upon the activity conditions. Further, while exerting an inhibitory effect on hypothalamo-pituitary-adrenal axis activity under basal conditions, hypothalamic OXT is essential for an adequate acute ACTH response.     

Changes of hypothalamic and plasma vasopressin in rats with deoxycorticosterone-acetate induced salt appetite.

Mineralocorticoids play a predominant role in development of salt appetite and hypertension. Since vasoactive peptides could mediate the central effects of mineralocorticoids, we evaluated changes of immunoreactive (IR) arginine vasopressin (AVP) in the paraventricular (PVN) and supraoptic (SON) hypothalamic nucleus during DOCA-induced salt appetite. In one model, rats having free access to water and 3% NaCl during 9 (prehypertensive stage) or 21 days (hypertensive stage) received DOCA (s.c., 10 mg/rat/in alternate days). A decrease in the IR cell area, number of IR cells and staining intensity was obtained in magnocellular PVN of rats treated during 9 days. After 21 days IR cell area and number of cells in the PVN also decreased, but staining intensity of remaining cells was normal. The same parameters were unchanged in the SON. In another model, animals treated with DOCA during 9 days had only access to 3% NaCl or water. The IR cell area in PVN and SON significantly increased in mineralocorticoid-treated and control animals, both drinking 3% NaCl. Staining intensity (PVN and SON) and number of IR cells (PVN) also augmented in DOCA-treated animals drinking salt respect of a group drinking water. Plasma AVP in rats treated with DOCA and offered salt and water, exhibited a 2-2.5 fold increase at the time of salt appetite induction. Plasma AVP was substantially higher in rats drinking salt only, while the highest levels were present in salt-drinking DOCA-treated rats. Thus, peptide depletion in the PVN may be due to increased release, because reduced levels of hypothalamic and posterior pituitary AVP were measured in this model. In rats drinking salt only the substantial increase of IR AVP in the PVN and SON, may be due to dehydration and hyperosmosis. Because DOCA-salt treated rats showed higher AVP levels in the PVN compared to untreated rats drinking salt only, it is possible that DOCA sensitized PVN cells to increase AVP production. The results suggest the vasopressinergic system could mediate some central functions of mineralocorticoids.     

Microdialysis with High NaCl Causes Central Release of Amino Acids and Dopamine

Abstract: Recent studies have shown that the neuropeptides arginine-8-vasopressin (AVP) and oxytocin (OXT) are released within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to microdialysis of these nuclei with high-NaCl perfusion media. These results suggest an inherent osmosensitivity of SON and PVN neurons. To investigate whether the observed release of AVP/OXT is a unique phenomenon to these neuropeptides, several brain regions were examined for the release of amino acids or dopamine in response to high- or low-NaCl stimulation. Urethane-anesthetized male Sprague-Dawley rats were perfused with five-ion solution using U-shaped microdialysis probes. Samples were collected at 30-min intervals and analyzed for amino acids and dopamine by HPLC. In the dialysates of all perfusion areas, including the SON, PVN, hippocampus, and striatum, concentrations of Asp, Glu, Ser, Gln, Gly, taurine (Tau), and γ-aminobutyric acid (GABA) were significantly increased during perfusion with high-NaCl medium. This release was found to be dose dependent when tested in the hippocampus and striatum with perfusion medium containing 0.5 or 1.0 M NaCl. However, only the release of Glu and Ser was found to be Ca²⁺ dependent. In contrast, the use of mannitol, a nonionic osmolyte, for perfusions in the striatum in concentrations of 0.5 and 1 M resulted in reduced levels of amino acids in the dialysates (Glu, Ser, Gln, and Tau). Low-NaCl perfusion medium (0.01 M) resulted in significantly increased Glu, Tau, Gly, and GABA levels in the striatum. In addition, dopamine levels in striatal dialysates were significantly increased during stimulation with 1 M NaCl. These results indicate that stimulation with high NaCl concentrations affects the release of several neurotransmitters and is not specific for AVP and OXT. The described phenomenon of the release of amino acids in response to this stimulation seems to be a response to the changed ionic concentration rather than to the osmolality. In light of these findings shown for amino acids and dopamine as well as those previously reported for AVP, OXT, and angiotensin, it would appear that sensitivity to tonicity changes brought about by microdialysis may be a feature of many transmitter systems.     

Oxytocin, but not arginine vasopressin is involving in the antinociceptive role of hypothalamic supraoptic nucleus

Our pervious study has demonstrated that the hypothalamic supraoptic nucleus (SON) plays a role in pain modulation. Oxytocin (OXT) and arginine vasopressin (AVP) are the important hormones synthesized and secreted by the SON. The experiment was designed to investigate which hormone was relating with the antinociceptive role of the SON in the rat. The results showed that (1) microinjection of l-glutamate sodium into the SON increased OXT and AVP concentrations in the SON perfusion liquid, (2) pain stimulation induces OXT, but not AVP release in the SON, and (3) intraventricular injection (pre-treatment) with OXT antiserum could inhibit the pain threshold increase induced by SON injection of l-glutamate sodium, but administration of AVP antiserum did not influence the antinociceptive role of SON stimulation. The data suggested that the antinociceptive role of the SON relates to OXT rather than AVP.     

Genomic Effects of Cold and Isolation Stress on Magnocellular Vasopressin mRNA-Containing Cells in the Hypothalamus of the Rat

We assessed the effects of cold and isolation stress on arginine vasopressin (AVP) mRNA in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Vasopressin mRNA levels were determined by in situ hybridization histochemistry at the cellular level. In posterior magnocellular neurons of the PVN isolation stress for 7 or 14 days increased vasopressin mRNA levels 28 and 29%, respectively, compared to group-housed controls. No significant alterations in vasopressin gene expression were observed in the SON after 7 or 14 days of isolation stress. Scattered magnocellular AVP mRNA-expressing cells of the medial parvocellular PVN showed increases of 19 and 34% after 7 and 14 days of isolation, respectively. We also studied the effect of cold or combined cold and isolation stress on vasopressin gene expression in the PVN and SON. Cold stress for 3 h daily for 4 consecutive days increased AVP mRNA levels in the posterior magnocellular PVN by 15%. Cold-isolated animals showed an increase of 21%. No significant effect on AVP mRNA levels in the SON was observed. In contrast to the posterior magnocellular PVN, cold or cold-isolation stress increased AVP mRNA in magnocellular neurons of the medial parvocellular region of the PVN by 25 and 43%, respectively, relative to control rats. These results suggest that psychological and metabolic stress may be added to the list of stressors that activate the hypothalamo-neurohypophysial system.     

Locus Coeruleus Lesions Decrease Oxytocin and Vasopressin Release Induced by Hemorrhage

The role of the noradrenergic nucleus Locus Coeruleus (LC) on hemorrhage-induced vasopressin (AVP) and oxytocin (OT) secretion was examined. Rats with LC lesion were submitted to three 1-min hemorrhage sessions at 5-min intervals; 15% of the total blood volume was withdrawn in each session. OT and AVP were measured in plasma, paraventricular (PVN) and supraoptic (SON) nuclei and in posterior pituitary (PP). LC Lesion did not affect basal plasma AVP or OT levels, but partly blocked the increase in plasma AVP and OT induced by hemorrhage. Hemorrhage produced decreases in content of AVP and OT in the PVN and SON and increased levels in the PP. These responses were attenuated in the lesioned group, but only in the PVN and PP. Data suggest a stimulatory role of the inputs from LC to PVN neurons on hemorrhage-induced OT and AVP secretion and that, this pathway is critical in the hypo-volemic neuroendocrine reflex.Special Issue Dedicated to Miklós Palkovits.     

BiP mRNA expression is upregulated by dehydration in vasopressin neurons in the hypothalamus in mice

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone that facilitates the proper folding of newly synthesized secretory and transmembrane proteins. Here we report that BiP mRNA was expressed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in wild-type mice under basal conditions. Dual in situ hybridization in the SON and PVN demonstrated that BiP mRNA was expressed in almost all the neurons of arginine vasopressin (AVP), an antidiuretic hormone. BiP mRNA expression levels were increased in proportion to AVP mRNA expression in the SON and PVN under dehydration. These data suggest that BiP is involved in the homeostasis of ER function in the AVP neurons in the SON and PVN.     

Variations in concentration of oxytocin and vasopressin in the paraventricular nucleus of the hypothalamus during the estrous cycle in rats

Oxytocin (OT) and arginine-8-vasopressin (AVP) were measured by radioimmunoassay in micropunched hypothalamic neurosecretory nuclei of estrous cycling female Sprague-Dawley rats. In the paraventricular nucleus (PVN): the concentration (pg/microgram protein) of OT was significantly higher in rats in diestrus than during proestrus, estrus, or metestrus, while the concentration during metestrus was significantly greater than in proestrus and estrus; the concentration of AVP was significantly lower in animals in estrus than during the other three stages; because the paraventricular OT levels dropped before proestrus, the AVP/OT ratio was significantly greater in animals in proestrus than in diestrus, metestrus, and estrus. In the supraoptic nucleus (SON) a similar trend was noted: the concentration of OT was highest during diestrus, and AVP was lowest during estrus, though neither was significantly different from other stages. Because the OT and AVP cycles in the SON were asynchronous, the ratio of AVP to OT was significantly higher in proestrus than in metestrus or diestrus and significantly greater in estrus than during diestrus. In contrast to these two areas, peptide concentrations did not vary significantly across the estrous cycle in other sites of nonapeptide synthesis, i.e. the anterior commissural nucleus (ACN) and the suprachiasmatic nuclei (SCN).     

Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100μg/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4, 6 or 24h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24h after surgery, and in both hypothalamic nuclei only at 6h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood.     

Estradiol potentiates hypothalamic vasopressin and oxytocin neuron activation and hormonal secretion induced by hypovolemic shock

Estrogen receptors are located in important brain areas that integrate cardiovascular and hydroelectrolytic responses, including the subfornical organ (SFO) and supraoptic (SON) and paraventricular (PVN) nuclei. The aim of this study was to evaluate the influence of estradiol on cardiovascular and neuroendocrine changes induced by hemorrhagic shock in ovariectomized rats. Female Wistar rats (220-280 g) were ovariectomized and treated for 7 days with vehicle or estradiol cypionate (EC, 10 or 40 μg/kg, sc). On the 8th day, animals were subjected to hemorrhage (1.5 ml/100 g for 1 min). Hemorrhage induced acute hypotension and bradycardia in the ovariectomized-oil group, but EC treatment inhibited these responses. We observed increases in plasma angiotensin II concentrations and decreases in plasma atrial natriuretic peptide levels after hemorrhage; EC treatment produced no effects on these responses. There were also increases in plasma vasopressin (AVP), oxytocin (OT), and prolactin levels after the induction of hemorrhage in all groups, and these responses were potentiated by EC administration. SFO neurons and parvocellular and magnocellular AVP and OT neurons in the PVN and SON were activated by hemorrhagic shock. EC treatment enhanced the activation of SFO neurons and AVP and OT magnocellular neurons in the PVN and SON and AVP neurons in the medial parvocellular region of the PVN. These results suggest that estradiol modulates the cardiovascular responses induced by hemorrhage, and this effect is likely mediated by an enhancement of AVP and OT neuron activity in the SON and PVN.     

烫伤对大鼠下丘脑视上核、室旁核AVP神经元的影响──免疫电镜观察及形态计量分析

为研究加压素与应激的关系,我们曾应用免疫细胞化学（ＰＡＰ）方法结合图像分析技术观察到烫伤应激后２小时视上核、室旁核加压素神经元免疫反应阳性物质的总体积减小最明显。本文采用免疫电镜技术,进一步观察了烫伤后２小时视上核、室旁核加压素神经元超微结构的变化,并运用体视学方法定量分析了对照组及烫伤后２小时组视上核、室分校加压素神经元各２４０张照片的粗面内质网、高尔基复合体及线粒体的体积密度（Ｖｖ）、面积密度（Ｓｖ）、面数密度（Ｎａ）等。结果显示：烫伤后２小时组视上核、室旁核加压素神经元的粗面内质网的Ｖｖ．Ｓｖ显著增大,说明合成功能已开始增强,但是,高尔基复合体的Ｖｖ、Ｓｖ及阳性分泌颗粒却显著减少,因而引起总体积的缩小。从超微结构水平进一步证实加压素参与应激过程。     

Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats

Central administration of either adrenomedullin 2 (AM2) or adrenomedullin (AM) activates hypothalamic oxytocin (OXT)-secreting neurons in rats. We compared AM2 with AM, given intracerebroventricularly (icv), across multiple measures: (1) plasma OXT levels in conscious rats; (2) blood pressure, heart rate and circulating catecholamine levels in urethane-anesthetized rats; and (3) the expression of the c-fos gene in the supraoptic (SON) and the paraventricular nuclei (PVN). We also tested the effects of the AM receptor antagonist, AM(22-52) and calcitonin gene-related peptide (CGRP) antagonist, CGRP(8-37) on these measures. Plasma OXT levels at 10 min after icv injection of AM (1 nmol/rat) were increased (compared with vehicle), but OXT levels after AM2 (1 nmol/rat) were nearly double the levels seen after AM injection. OXT levels remained elevated at 30 min. Pretreatment with AM(22-52) (27 nmol/rat) and CGRP(8-37) (3 nmol/rat), nearly abolished the increase in plasma OXT level after AM injection, but partially blocked OXT level changes due to AM2. Increases in blood pressure, heart rate and circulating catecholamines were all greater in response to central AM2 than to AM at the same dose. In situ hybridization histochemistry showed that both AM2 and AM induced expression of the c-fos gene in the SON and the PVN, but AM(22-52)+CGRP(8-37) could only nearly abolish the effects of centrally administered AM. These results suggest that the more potent central effects of AM2 and only partial blockade by AM/CGRP receptor antagonists may result from its action on an additional, as yet unidentified, specific receptor in the central nervous system.     

Effects of experimental hypothyroidism on the development of the hypothalamo-pituitary-adrenal axis in the rat

Hypothyroid pups were obtained by adding methimazole in the mother's drinking water from day 15 of gestation and sacrificed at 4, 8 or 15 days. Circulating corticosterone decreased at all ages, while CBG concentrations diminished at day 4, increased at day 8 and did not change at day 15 in hypothyroid rats. As opposed to controls, plasma ACTH concentrations decreased steadily with age while there was an accumulation of ACTH in the anterior pituitary of hypothyroid 15-day-old rats. Anterior pituitary POMC contents were unaffected by the treatment. In the hypothalamic PVN, CRF mRNA levels in the total population of CRF-synthesizing cells and in the CRF+/AVP+ subpopulation were below those of controls whatever the age considered while AVP mRNA in the CRF+/AVP+ subpopulation did not change at day 4 and decreased at day 8 and 15 in hypothyroid animals. Both the number of cell bodies expressing detectable levels of CRF mRNA and the percentage of CRF and AVP colocalization decreased at day 4 and were unchanged thereafter. CRF and AVP immunoreactivity in the zona externa of the median eminence increased with age but was not affected by methimazole treatment. The concentration of AVP mRNA in the magnocellular cell bodies of the PVN and the SON as well as AVP immunoreactivity in the zona interna of the median eminence were not changed by the treatment at days 4 and 8. In hypothyroid 15-day-old rats, SON AVP mRNA increased, AVP immunoreactivity decreased while plasma osmolality was enhanced. In conclusion, our data demonstrate that experimental hypothyroidism impairs specifically the maturation of hypothalamic parvocellular CRF and AVP gene expression during the stress hyporesponsive period. These observations suggest that the physiological peak in plasma thyroxine concentrations that occur between day 8-12 may participate in the maturation of hypothalamic CRF- and AVP-synthesizing cells.     

Colchicine-induced increases in immunoreactive neuropeptide levels in hypothalamus: use as an index of biosynthesis.

The colchicine-induced accumulation of vasopressin (AVP) and oxytocin (OXT) has recently been applied to estimate the synthesis and turnover rates for these neuropeptides in whole rat hypothalamus. In the present studies, this pharmacologic procedure has been examined as a potential method for estimating hypothalamic somatostatin (SRIF) synthesis rate, and evaluated further for its utility in estimating nonapeptide synthesis in individual hypothalamic nuclei. Adult male rats received a single injection of colchicine (8 micrograms) into the third ventricle under pentobarbital anesthesia. Twenty-four hr later, immunoreactive (IR) levels of AVP and OXT increased considerably, as previously noted. Hypothalamic IR-SRIF levels, however, were unaffected. The absolute increases in IR-AVP and IR-OXT were greatest in the supraoptic nucleus (SON), with smaller increments in the para/periventricular hypothalamus (PVH) and the median eminence (ME). IR-SRIF levels showed no changes in the PVH or the ME. As a test, the method was applied to the detection of changes in AVP synthesis in diabetic rats. The colchicine procedure reported increases in AVP synthesis in both the SON and PVH in diabetic animals, a result compatible with that obtained previously for whole hypothalamus using radiolabeled procedures. Together, the results indicate that the colchicine procedure is useful in detecting changes in the syntheses of some (AVP and OXT) but not all (SRIF) neuropeptides, and that when applicable, the method is sufficiently sensitive to detect changes in small hypothalamic regions. The method may prove useful in estimating changes in peptide synthesis analogous to that used for serotonin and dopamine; e.g., 5-hydroxytryptophan and dopa accumulation following inhibition of aromatic L-amino acid decarboxylase.     

Differential effects of water and saline intake on water deprivation-induced c-Fos staining in the rat

We studied c-Fos staining in adult male rats after 48 h of water deprivation and after 46 h of water deprivation with 2 h of access to water or physiological saline. Controls were allowed ad libitum access to water and physiological saline. For immunocytochemistry, anesthetized rats were perfused with a commercially available antibody for c-Fos. Dehydration significantly increased plasma vasopressin (AVP), osmolality, plasma renin activity (PRA), hematocrit, and sodium concentration and decreased urinary volume. Fos staining was significantly increased in the median preoptic nucleus, organum vasculosum of the lamina terminalis, supraoptic nucleus (SON), and magnocellular and parvocellular paraventricular nucleus (PVN), as well as the area postrema, nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVL). Rehydration with water significantly decreased AVP levels and Fos staining in the SON, PVN, and RVL and significantly increased Fos expression in the perinuclear zone of the SON, NTS, and parabrachial nucleus. Rehydration with water was associated with decreased urinary sodium concentration and hypotonicity, and hematocrit and PRA were comparable to levels seen after dehydration. After rehydration with saline, plasma osmolality, hematocrit, and PRA were not different from control, but plasma AVP and urinary sodium concentration were increased. In the SON, Fos staining was significantly increased, with a great percentage of the Fos cells also stained for oxytocin compared with water deprivation. Changes in Fos staining were also observed in the NTS, RVL, parabrachial nucleus, and PVN. Rehydration with water or saline produces differential effects on plasma AVP, Fos staining, and sodium concentration.