Vasopressin mRNA Levels Research Articles

Disorders in the gut and liver are involved in depression contagion between isosexual post-stroke depression mice and the healthy cohabitors

Despite the accumulated evidence that pair housing could attenuate post-stroke depression (PSD), but less attention has been paid to the healthy cohabitors, and the underlying mechanisms remain unclear. This study aimed to determine whether there is depressive contagion between PSD mice and their healthy cohabitors. PSD was induced by middle cerebral artery occlusion (MCAO) plus restraint stress for four weeks. Three days after MCAO, the mice were restrained two hours per day and isosexually pair-housed for four weeks. The results showed that, compared with the partners pair housed with normal control mice (Ctrl group), the partners pair housed with PSD mice (CH group) displayed depressive-like behaviors, including decreased sucrose preference rate, significantly shorter duration in the center arena and reduced total distance in the open-field test, and extended immobile time in forced swimming test and tail-suspension test without sex differences. Regarding the change in the body weight, only the males showed a significant reduction on days 17 and 24 after treatment. Furthermore, the CH group showed significantly increased corticosterone and decreased oxytocin (OXT) levels in serum, while the mRNA levels of OXT, vasopressin and oxytocin receptor were remarkably upregulated in the hypothalamus of the CH group. However, there was no significant change in the vasopressin receptor V1a. Interestingly, compared with the Ctrl group, there was a significant decrease in butyrate in serum of the CH group. Consistently, they had mild liver dysfunction with increased alanine transaminase, extended hepatic sinus surrounded by enhanced SLC22A9, and significantly increased Iba1-positive macrophages. Moreover, the expression of tight junction protein (Occludin and ZO-1) obviously decreased in the colon with increasing Iba1-positive cells. These results suggest that isosexual pair-housing with PSD mice causes the healthy partners to develop depressive-like behaviors with disturbances in the gut and liver.

Expression of vasopressin and its receptors in migraine-related regions in CNS and the trigeminal system: influence of sex

BackgroundHypothalamus is a key region in migraine attacks. In addition, women are disproportionately affected by migraine. The calcitonin gene-related peptide (CGRP) system is an important key player in migraine pathophysiology. CGRP signaling could be a target of hormones that influence migraine. Our aim is to identify the expression of vasopressin and its receptors in the brain and in the trigeminovascular system with focus on the migraine-related regions and, furthermore, to examine the role of sex on the expression of neurohormones in the trigeminal ganglion.MethodsRat brain and trigeminal ganglia were carefully harvested, and protein and mRNA levels were analyzed by immunohistochemistry and real-time PCR, respectively.ResultsVasopressin and its receptors immunoreactivity were found in migraine-related areas within the brain and, in the trigeminal ganglion, predominantly in neuronal cytoplasm. There were no differences in the number of positive immunoreactivity cells expression of CGRP and vasopressin in the trigeminal ganglion between male and female rats. In contrast, the number of RAMP1 (CGRP receptor), oxytocin (molecular relative to vasopressin), oxytocin receptor and vasopressin receptors (V1aR and V1bR) immunoreactive cells were higher in female compared to male rats. Vasopressin and its receptors mRNA were expressed in both hypothalamus and trigeminal ganglion; however, the vasopressin mRNA level was significantly higher in the hypothalamus.ConclusionsA better understanding of potential hormonal influences on migraine mechanisms is needed to improve treatment of female migraineurs. It is intriguing that vasopressin is an output of hypothalamic neurons that influences areas associated with migraine. Therefore, vasopressin and the closely related oxytocin might be important hypothalamic components that contribute to migraine pathophysiology.

Renal transcriptome profiles in mice reveal the need for sufficient water intake irrespective of the drinking water type

This study sought to characterize the impact of long-term dehydration in terms of physiological and biochemical parameters, as well as renal transcriptomes. Furthermore, we assessed whether consumption of specific types of water elicit more beneficial effects on these health parameters. To this end, C57BL/6 mice were either provided water for 15 min/day over 2 and 4 weeks (water restricted; RES), or ad libitum access to distilled (CON), tap, spring, or purified water. Results show that water restriction decreases urine output and hematocrit levels while increasing brain vasopressin mRNA levels in RES mice compared to control mice (CON). Meanwhile, blood urea nitrogen and creatinine levels were higher in the RES group compared to the CON group. Kidney transcriptome analysis further identified kidney damage as the most significant biological process modulated by dehydration. Mechanistically, prolonged dehydration induces kidney damage by suppressing the NRF2-signaling pathway, which targets the cytoprotective defense system. However, type of drinking water does not appear to impact physiological or blood biochemical parameters, nor the renal transcriptome profile, suggesting that sufficient water consumption is critical, irrespective of the water type. Importantly, these findings also inform practical action for environmental sustainability by providing a theoretical basis for reducing bottled water consumption.

Sex Differences in Escalated Methamphetamine Self-Administration and Altered Gene Expression Associated With Incubation of Methamphetamine Seeking.

Methamphetamine (METH) use disorder is prevalent worldwide. There are reports of sex differences in quantities of drug used and relapses to drug use among individuals with METH use disorder. However, the molecular neurobiology of these potential sex differences remains unknown. We trained rats to self-administer METH (0. 1 mg/kg/infusion, i.v.) on an fixed-ratio-1 schedule for 20 days using two 3-hour daily METH sessions separated by 30-minute breaks. At the end of self-administration training, rats underwent tests of cue-induced METH seeking on withdrawal days 3 and 30. Twenty-four hours later, nucleus accumbens was dissected and then used to measure neuropeptide mRNA levels. Behavioral results show that male rats increased the number of METH infusions earlier during self-administration training and took more METH than females. Both male and female rats could be further divided into 2 phenotypes labeled high and low takers based on the degree of escalation that they exhibited during the course of the METH self-administration experiment. Both males and females exhibited incubation of METH seeking after 30 days of forced withdrawal. Females had higher basal mRNA levels of dynorphin and hypocretin/orexin receptors than males, whereas males expressed higher vasopressin mRNA levels than females under saline and METH conditions. Unexpectedly, only males showed increased expression of nucleus accumbens dynorphin after METH self-administration. Moreover, there were significant correlations between nucleus accumbens Hcrtr1, Hcrtr2, Crhr2, and Avpr1b mRNA levels and cue-induced METH seeking only in female rats. Our results identify some behavioral and molecular differences between male and female rats that had self-administered METH. Sexual dimorphism in responses to METH exposure should be considered when developing potential therapeutic agents against METH use disorder.

Hypothalamic-pituitary-adrenal axis hypersensitivity in female rats on a post-weaning high-fat diet after chronic mild stress.

A high-fat diet (HFD) is highly correlated to obesity, metabolic diseases and certain behavioral changes. However, the effects of post-weaning HFD in rats during puberty and the role of the hypothalamic-pituitary-adrenal (HPA) axis in this process have remained elusive. The present study hypothesized that the HPA axis mediates the behavioral alterations induced by a post-weaning HFD. To investigate this, female rats were divided into two groups, one of which was fed a HFD from postnatal weeks (PWs) 4-12, while the other group received standard chow. Rats in each group were then subdivided into two subgroups each, and from PW 9-12, animals from one of the two subgroups were subjected to chronic mild stress (CMS), while the other subgroup received no stress. At PW 12, the body weight of rats receiving a HFD but no DMS was significantly higher than that in the control group. The frequency of crossing and rearing in the open field test and the time in the center of the Y-maze were decreased following CMS. Total time to escape was decreased in rats receiving HFD and after CMS. The serum levels of adrenocorticotropic hormone and corticosterone were increased in rats receiving an HFD and after CMS, and the mRNA levels of corticotropin-releasing hormone and arginine vasopressin in the hypothalamus were increased in the HFD + CMS group compared to that in the control group. The mRNA expression of glucocorticoid receptor (GR) in the hippocampi of rats in the HFD + CMS group was significantly decreased and the mineralocorticoid receptor/GR ratio was increased compared to that in the groups receiving either CMS or a HFD. In conclusion, these results indicated that female rats fed a post-weaning HFD showed HPA axis hypersensitivity under CMS, which may mediate behavioral alterations.

Upregulation of Orexin in the Paraventricular Nucleus Contributes to Salt Sensitive Hypertension

Orexin and orexin receptors are extensively expressed in the hypothalamic paraventricular nucleus (PVN) and the hyperactivity of PVN orexin contributes to increased sympathetic nerve activity (SNA) and elevated blood pressure in several animal models. However, the role of PVN orexin in the development of salt sensitive hypertension (SSHTN) is unknown. In this study, we tested the hypothesis that PVN orexin expression is increased in SSHTN and contributes to the development of SSHTN via regulating vasopressin secretion. Eight‐week‐old Dahl salt sensitive (DS) rats were divided into two groups and were fed either a high salt (HS, 8% NaCl) or normal salt (NS, 0.4% NaCl) diet. Six weeks following diet treatment, rats were anesthetized and their mean arterial pressure (MAP) and sympathetic nerve activity (SNA) response to orexin receptor blockade were recorded. In a separate group of rats PVN orexin mRNA levels were measured. The results showed that HS intake induced significant increases in MAP (HS: 146±2 vs. NS: 112±5; n=5; P<0.05) and PVN orexin mRNA levels (HS: 3.8±0.37 vs. NS: 1.0±0.68; n=3; P<0.05). Bilateral PVN microinjection of orexin receptor 1 (OX1R) antagonist SB408124 (30 pmol/50nl) resulted in a greater reduction in arterial blood pressure in DS rats with a HS intake compared to their cohorts with a NS diet (HS: −16±5 vs. NS: −4±4 mmHg; n=5–6; P<0.05). OX1R blockade also increased splanchnic SNA (HS: 48.2±12.6%) vs. NS: 35.82±9%; P=0.2) and renal SNA (HS: 26.7±7.5% vs. NS: 17.4±9.5%; P=0.2) in DS rats with either HS or NS diet. Incubation of orexin A resulted in a dose‐dependent increase in vasopressin mRNA levels (100nM: 2.9‐fold; 1μM; 5.6 fold; 10μM: 18‐fold) in primary cultured neurons from the hypothalamus containing the PVN of neonatal Sprague Dawley rats. These orexin A‐induced increases of vasopressin mRNA were abolished by OX1R blockade. These results coupled with the evidence that plasma vasopressin is upregulated in DS rats under the condition of HS‐induced hypertension suggest that increased PVN orexin expression may be involved in the development of SSHTN through regulating vasopressin secretion.Support or Funding InformationThis study is supported by AHA 11SDG7420029 and NIH R15‐HL122952.

Differential Changes in Expression of Stress- and Metabolic-Related Neuropeptides in the Rat Hypothalamus during Morphine Dependence and Withdrawal

Chronic morphine treatment and naloxone precipitated morphine withdrawal activates stress-related brain circuit and results in significant changes in food intake, body weight gain and energy metabolism. The present study aimed to reveal hypothalamic mechanisms underlying these effects. Adult male rats were made dependent on morphine by subcutaneous implantation of constant release drug pellets. Pair feeding revealed significantly smaller weight loss of morphine treated rats compared to placebo implanted animals whose food consumption was limited to that eaten by morphine implanted pairs. These results suggest reduced energy expenditure of morphine-treated animals. Chronic morphine exposure or pair feeding did not significantly affect hypothalamic expression of selected stress- and metabolic related neuropeptides - corticotropin-releasing hormone (CRH), urocortin 2 (UCN2) and proopiomelanocortin (POMC) compared to placebo implanted and pair fed animals. Naloxone precipitated morphine withdrawal resulted in a dramatic weight loss starting as early as 15–30 min after naloxone injection and increased adrenocorticotrophic hormone, prolactin and corticosterone plasma levels in morphine dependent rats. Using real-time quantitative PCR to monitor the time course of relative expression of neuropeptide mRNAs in the hypothalamus we found elevated CRH and UCN2 mRNA and dramatically reduced POMC expression. Neuropeptide Y (NPY) and arginine vasopressin (AVP) mRNA levels were transiently increased during opiate withdrawal. These data highlight that morphine withdrawal differentially affects expression of stress- and metabolic-related neuropeptides in the rat hypothalamus, while relative mRNA levels of these neuropeptides remain unchanged either in rats chronically treated with morphine or in their pair-fed controls.

The impact of maternal synthetic glucocorticoid administration in late pregnancy on fetal and early neonatal hypothalamic-pituitary-adrenal axes regulatory genes is dependent upon dose and gestational age at exposure.

In this study, we determined the gene and/or protein expression of hypothalamic-pituitary-adrenal (HPA) axis regulatory molecules following synthetic glucocorticoid exposures. Pregnant sheep received intramuscular saline or betamethasone (BET) injections at 104 (BET-1), 104 and 111(BET-2) or 104, 111 and 118 (BET-3) days of gestation (dG). Samples were collected at numerous time-points between 75 dG and 12 weeks postnatal age. In the BET-3 treatment group, fetal plasma cortisol levels were lower at 145 dG than controls and gestational length was lengthened significantly. The cortisol:adrenocorticotropic hormone (ACTH) ratio in fetal plasma of control and BET-3 fetuses rose significantly between132 and 145 dG, and remained elevated in lambs at 6 and 12 weeks of age; this rise was truncated at day 145 in fetuses of BET-3 treated mothers. After BET treatment, fetal and postnatal pituitary proopiomelanocortin mRNA levels were reduced from 109 dG to 12 weeks postnatal age; pituitary prohormone convertase 1 and 2 mRNA levels were reduced at 145 dG and postnatally; hypothalamic arginine vasopressin mRNA levels were lowered at all time-points, but corticotrophin-releasing hormone mRNA levels were reduced only in postnatal lambs. Maternal BET increased late fetal and/or postnatal adrenal mRNA levels of ACTH receptor and 3β hydroxysteroid dehydrogenase but decreased steroidogenic acute regulatory protein and P450 17-α hydroxylase. The altered mRNA levels of key HPA axis regulatory proteins after maternal BET injections suggests processes that may subserve long-term changes in HPA activity in later life after prenatal exposure to synthetic glucocorticoids.

Role of Bcl-2 in the regulation of CREB activity and vasopressin expression in hypothalamic neurons

For the last years, data have appears that indicate that functions of the antiapoptotic protein Bcl-2 are not restricted by its role in prevention of apoptosis. Earlier we showed that Bcl-2 participated in regulation of synthesis and secretion of vasopressin by neurons of magnocellular hypothalamic nuclei. Based on our previous works about effect of administration of the Bcl-2 blocker HA-14-1 on activities of the ERK1/2 cascade and of the transcriptional factor CREB in hypothalamic vasopressinergic neurons, we formulated hypothesis about the mediated action of Bcl-2 on vasopressin biosynthesis by transcription factor CREB and/or the ERK1/2 signal cascade. In the presented work we studied effects of Bcl-2 inactivation on vasopressin expression and activity of the ERK1/2 cascade and CREB in experiments on briefly incubated hypothalamus slides containing supraoptic nuclei. We have shown that inactivation of Bcl-2 in vitro by the blocker HA14-1 led to a decrease in the CREB activity, reduced significantly the vasopressin mRNA level, and increased the vasopressin content in the supraoptic nucleus neurons, with the ERK1/2 cascade activity being increased. Effect of Bcl-2 on the vasopressin synthesis activity did not depend on activity of the ERK1/2 signal cascade. Thus, Bcl-2 has been shown to play an activating role in control of vasopressin neurons of the hypothalamus supraoptic nucleus. The presented data confirm that effect of Bcl-2 on vasopressin synthesis by the supraoptic nucleus neurons is mediated by its ability to control activity of the transcriptional factor CREB.

Brain-Targeted (Pro)renin Receptor Knockdown Attenuates Angiotensin II–Dependent Hypertension

The (pro)renin receptor is a newly discovered member of the brain renin-angiotensin system. To investigate the role of brain (pro)renin receptor in hypertension, adeno-associated virus-mediated (pro)renin receptor short hairpin RNA was used to knockdown (pro)renin receptor expression in the brain of nontransgenic normotensive and human renin-angiotensinogen double-transgenic hypertensive mice. Blood pressure was monitored using implanted telemetric probes in conscious animals. Real-time PCR and immunostaining were performed to determine (pro)renin receptor, angiotensin II type 1 receptor, and vasopressin mRNA levels. Plasma vasopressin levels were determined by ELISA. Double-transgenic mice exhibited higher blood pressure, elevated cardiac and vascular sympathetic tone, and impaired spontaneous baroreflex sensitivity. Intracerebroventricular delivery of (pro)renin receptor short-hairpin RNA significantly reduced blood pressure, cardiac and vasomotor sympathetic tone, and improved baroreflex sensitivity compared with the control virus treatment in double-transgenic mice. (Pro)renin receptor knockdown significantly reduced angiotensin II type 1 receptor and vasopressin levels in double-transgenic mice. These data indicate that (pro)renin receptor knockdown in the brain attenuates angiotensin II-dependent hypertension and is associated with a decrease in sympathetic tone and an improvement of the baroreflex sensitivity. In addition, brain-targeted (pro)renin receptor knockdown is associated with downregulation of angiotensin II type 1 receptor and vasopressin levels. We conclude that central (pro)renin receptor contributes to the pathogenesis of hypertension in human renin-angiotensinogen transgenic mice.

Galanin and vasopressin response to hyperosmotic stimulation: in vitro study.

Galanin (Gal)--a neuropeptide present in the nervous system and peripheral tissues--may be involved in the regulation of hypothalamo-neurohypophysial system function. It was shown that centrally injected galanin inhibits osmotically stimulated vasopressin (VP) secretion into the blood and reduces VP mRNA level in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. The aim of the present study in vitro was to investigate the influence of Gal on vasopressin release from isolated rat hypothalamus (Hth), neurohypophysis (NH) or hypothalamo-neurohypophysial explants (Hth-NH). The effect of Gal on VP secretion was studied under conditions of direct osmotic (i.e., Na⁺-evoked) (series 1) as well as nonosmotic (i.e., K⁺-evoked) (series 2) stimulation. In series 3, vasopressin response to Gal was studied using the neural tissues obtained from animals drinking 2 percent NaCl solution for eight days (indirect osmotic stimulation). Gal in a concentration of 10⁻¹⁰ M and 10⁻⁸ M inhibited basal VP release from Hth, NH and Hth-NH explants isolated from euhydrated rats as well as from Hth-NH complex of osmotically challenged animals. When the neural tissues obtained from previously salt-loaded rats were incubated in K⁺-enriched medium the inhibitory effect of Gal was completely blocked. It may be concluded that the effect of Gal is depending on the current functional status of the hypothalamo-neurohypophysial system.

Androgen Receptors in the Posterior Bed Nucleus of the Stria Terminalis Increase Neuropeptide Expression and the Stress-Induced Activation of the Paraventricular Nucleus of the Hypothalamus

The posterior bed nuclei of the stria terminalis (BST) are important neural substrate for relaying limbic influences to the paraventricular nucleus (PVN) of the hypothalamus to inhibit hypothalamic-pituitary-adrenal (HPA) axis responses to emotional stress. Androgen receptor-expressing cells within the posterior BST have been identified as projecting to the PVN region. To test a role for androgen receptors in the posterior BST to inhibit PVN motor neurons, we compared the effects of the non-aromatizable androgen dihydrotestosterone (DHT), the androgen receptor antagonist hydroxyflutamide (HF), or a combination of both drugs implanted unilaterally within the posterior BST. Rats bearing unilateral implants were analyzed for PVN Fos induction in response to acute-restraint stress and relative levels of corticotrophin-releasing hormone and arginine vasopressin (AVP) mRNA. Glutamic acid decarboxylase (GAD) 65 and GAD 67 mRNA were analyzed in the posterior BST to test a local involvement of GABA. There were no changes in GAD expression to support a GABA-related mechanism in the BST. For PVN neuropeptide expression and Fos responses, basic effects were lateralized to the sides of the PVN ipsilateral to the implants. However, opposite to our expectations of an inhibitory influence of androgen receptors in the posterior BST, PVN AVP mRNA and stress-induced Fos were augmented in response to DHT and attenuated in response to HF. These results suggest that a subset of androgen receptor-expressing cells within the posterior BST region may be responsible for increasing the biosynthetic capacity and stress-induced drive of PVN motor neurons.

Vasopressinergic neurons of the supraoptic nucleus in perinatal rats: reaction to osmotic stimulation and its regulation

Osmotic stimulation (OS) of vasopressin (VP) neurons of the supraoptic nucleus (SON) promotes VP secretion and tyrosine hydroxylase (TH) expression in adult mammals. VP secretion is under a noradrenaline control, whereas the regulation of TH expression remains uncertain. This study was aimed to determine at what period of ontogenesis: (1) VP neurons begin to react to OS by modifying simultaneously VP and TH gene expression and synthesis, (2) the noradrenergic control of VP neurons is established. Rats on the 21st embryonic day (E), third postnatal day (P), P13 were salt loaded or salt loaded and treated with an antagonist (prazosin) or agonist (phenylephrine) of α1-adrenoreceptors. According to our immunocytochemical and in situ hybridization data, OS resulted in an increased amount of VP mRNA in each age group and of VP on E21 and P3. TH gene and synthesis was initially expressed under OS on P3. The number of TH-expressing neurons diminished by threefold in salt loaded rats from P3 to P13. OS combined with prazosin administration resulted in an increased level of VP mRNA on P3 and P13, but not on E21 suggesting the onset of the noradrenaline inhibitory control after birth. OS together with prazosin treatment stimulated TH expression on P3 and P13, whereas phenylephrine provided an opposite effect. Thus, VP neurons begin to react to OS by an increased VP synthesis at the end of fetal life and by the onset of TH expression shortly after birth; the expression of both substances appears to be under the inhibitory control of noradrenaline.

Naturally occurring variations in defensive burying behavior are associated with differences in vasopressin, oxytocin, and androgen receptors in the male rat

Largely ignored in tests of defensive burying is the capacity for individual animals to display marked variations in active coping behaviors. To expose the neurobiological correlates of this behavioral differentiation rats were exposed to a mousetrap that was remotely triggered upon approach to remove the quality of pain. Relative to animals showing no significant levels of defensive burying activity, rats showing sustained elevations in defensive burying displayed higher levels of arginine vasopressin (AVP) mRNA and increased numbers of androgen receptor positive cells in the medial amygdala and posterior bed nuclei of the stria terminalis, brain regions that integrate emotional appraisal and sensory information. In contrast, animals showing little to no defensive burying responses displayed relatively higher levels of AVP and oxytocin (OT) mRNA within the supraoptic nucleus and subregions of the paraventricular nucleus of the hypothalamus responsible for neuroendocrine and autonomic function. Finally, animals showing sustained levels of burying also displayed increased levels of testosterone and pituitary-adrenal hormones under stress conditions. These findings implicate roles for central AVP and OT in mediating differential avoidance behaviors and demonstrate the utility of using a pain-free test of defensive burying as a framework for exploring naturally occurring differences in coping style and neuroendocrine capacity.

Impact of recurring intermediate insulin-induced hypoglycemia on hypothalamic paraventricular corticotropin-releasing hormone, oxytocin, vasopressin and glucokinase gene profiles: role of type II glucocorticoid receptors

Activation of central type II glucocorticoid receptors (GR) during neutral protamine Hagedorn insulin (NPH) administration exacerbates recurring hypoglycemia. The hypothalamic paraventricular nucleus (PVN) integrates metabolic sensory input, controls autonomic and neuroendocrine motor outflow, and is characterized by abundant GR expression. The present studies investigated the hypothesis that PVN GR mediate intensification of hypoglycemia by serial NPH dosing, and that PVN glucokinase (GCK) and glucoregulatory neuropeptide genes acclimate to this treatment paradigm through GR-dependent mechanisms. Groups of adult male rats were injected subcutaneously with one or four doses of NPH, on as many days, while controls received vehicle. Bilateral administration of the selective GR antagonist, CP-472555, into the PVN prior to the first three NPH injections prevented amplification of hypoglycemia in response to the final insulin dose, while intra-PVN delivery of the GR agonist, dexamethasone, to euglycemic rats did not modify ensuing NPH-induced hypoglycemia. Quantitative real-time RT-PCR analysis of microdissected PVN tissue revealed that GCK, corticotropin-releasing hormone (CRH), oxytocin (OT), and vasopressin (VP) mRNA levels were unchanged in response to acute NPH, and baseline gene profiles measured 24 h after antecedent injections were similar to vehicle controls. In contrast, serial dosing with NPH elevated CRH and GCK, diminished OT, but did not alter VP gene transcripts. Intracerebroventricular CP-472555 delivery in conjunction with antecedent NPH dosing prevented transcriptional habituation of GCK and OT genes, but did not modify CRH or VP mRNA profiles. The present data show that activation of PVN GR during antecedent intermediate insulin-induced hypoglycemia is required for exacerbation of recurring hypoglycemia, and receptor stimulation in the absence of hypoglycemia and/or its sequelae does not intensify the effects of subsequent NPH administration. The results also provide evidence for acclimation of PVN CRH, GCK, and OT gene profiles to serial NPH dosing, and demonstrate that GR may be involved in GCK and OT transcriptional adaptation to ongoing intermediate insulin-induced hypoglycemia.

Novel Leptin-Regulated Genes Revealed by Transcriptional Profiling of the Hypothalamic Paraventricular Nucleus

Leptin plays a major role in coordinating the integrated response of the CNS to changes in nutritional state. Neurons within the paraventricular nucleus (PVN) of the hypothalamus express leptin receptors and receive dense innervation from leptin receptor-expressing neurons in the arcuate nucleus. To obtain new insights into the effects of circulating leptin on PVN function, we compared global transcriptional profiles of laser-captured PVN from ad libitum fed mice versus 48 h fasted mice receiving either sham or leptin treatment intraperitoneally. Five hundred twenty-seven PVN-expressed genes were altered by fasting in a manner that was at least partially reversible by leptin. Consistent with previous reports, thyrotrophin releasing hormone mRNA levels were decreased by fasting but restored to fed levels with leptin treatment. mRNA levels of oxytocin, vasopressin, and somatostatin were also reduced by fasting and restored by leptin. Given the known effects of leptin on synaptic remodeling, it is notable that, among the top 15 genes that were positively regulated by leptin, five have been implicated in synaptic function and/or plasticity (basigin, apolipoprotein E, Gap43, GABA(A) receptor-associated protein, and synuclein-gamma). Pathway analysis identified oxidative phosphorylation, in particular, genes encoding complex 1 proteins that play a role in ubiquinone biosynthesis, to be the predominant gene set that was significantly regulated in a leptin-dependent manner. Thus, in addition to its effects on the expression of a broad range of neuropeptides, leptin may also exert more general influences on synaptic function in, and the bioenergetic state of, the PVN.

Oxytocin and vasopressin gene expression and RNA splicing patterns in the rat supraoptic nucleus.

In this study, we test the hypothesis that there are differential splicing patterns between the expressed oxytocin (OT) and vasopressin (VP) genes in the rat supraoptic nucleus (SON). We quantify the low abundance, intron-containing heteronuclear RNAs (hnRNAs) and the higher abundance mRNAs in the SON using two-step, quantitative SYBR Green real-time reverse transcription (RT)-PCR and external standard curves constructed using synthetic 90 nt sense-strand oligonucleotides. The levels of OT and VP mRNA in the SON were found to be similar, approximately 10(8) copies/SON pair, whereas the copy numbers of VP hnRNAs containing intron 1 or 2 and the OT hnRNA containing intron 1 are much lower, i.e., approximately 10(2)-10(3) copies/rat SON pair. However, the estimated copy number of the intron 2-containing OT hnRNA is much larger, approximately 10(6) copies/SON pair. The relative distributions of all the OT and VP RNA species were invariant and independent of the physiological status of the rats (e.g., osmotically stimulated or lactating rats). Using intron-specific riboprobes against hnRNAs, we demonstrate by fluorescence in situ hybridization strong signals of OT hnRNA containing intron 2 predominantly in the cytoplasm, in contrast to the localization of the VP hnRNA found only in the nuclei. Taken together, these data support the view that the splicing patterns between OT and VP gene transcripts are different and show that there is a selective cytoplasmic retention of OT intron 2.

Selective contributions of the medial preoptic nucleus to testosterone-dependant regulation of the paraventricular nucleus of the hypothalamus and the HPA axis

Previous data have consistently demonstrated an inhibitory effect of androgens on stress-induced hypothalamic-pituitary-adrenal (HPA) responses. Several brain regions may influence androgen-mediated inhibition of the HPA axis, including the medial preoptic area. To test the role of the medial preoptic nucleus (MPN) specifically, we examined in high- and low-testosterone-replaced gonadectomized rats bearing discrete bilateral lesions of the MPN basal and stress-induced indexes of HPA function, and the relative levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA in the amygdala. High testosterone replacement decreased plasma adrenocorticotropin hormone (ACTH) and paraventricular nucleus (PVN) Fos responses to restraint exposure in sham- but not in MPN-lesioned animals. AVP-, but not CRH-immunoreactivity staining in the external zone of the median eminence was increased by testosterone in sham animals, and MPN lesions blocked this increment in AVP. A similar interaction between MPN lesions and testosterone occurred on AVP mRNA levels in the medial nucleus of the amygdala. These findings support an involvement of MPN projections in mediating the AVP response to testosterone in both the medial parvocellular PVN and medial amygdala. We conclude that the MPN forms part of an integral circuit that mediates the central effects of gonadal status on neuroendocrine and central stress responses.

A Novel Mouse Model for Acute and Long-Lasting Consequences of Early Life Stress

Chronic early-life stress (ES) exerts profound acute and long-lasting effects on the hypothalamic-pituitary-adrenal system, with relevance to cognitive function and affective disorders. Our ability to determine the molecular mechanisms underlying these effects should benefit greatly from appropriate mouse models because these would enable use of powerful transgenic methods. Therefore, we have characterized a mouse model of chronic ES, which was provoked in mouse pups by abnormal, fragmented interactions with the dam. Dam-pup interaction was disrupted by limiting the nesting and bedding material in the cages, a manipulation that affected this parameter in a dose-dependent manner. At the end of their week-long rearing in the limited-nesting cages, mouse pups were stressed, as apparent from elevated basal plasma corticosterone levels. In addition, steady-state mRNA levels of CRH in the hypothalamic paraventricular nucleus of ES-experiencing pups were reduced, without significant change in mRNA levels of arginine vasopressin. Rearing mouse pups in this stress-provoking cage environment resulted in enduring effects: basal plasma corticosterone levels were still increased, and CRH mRNA levels in paraventricular nucleus remained reduced in adult ES mice, compared with those of controls. In addition, hippocampus-dependent learning and memory functions were impaired in 4- to 8-month-old ES mice. In summary, this novel, robust model of chronic early life stress in the mouse results in acute and enduring neuroendocrine and cognitive abnormalities. This model should facilitate the examination of the specific genes and molecules involved in the generation of this stress as well as in its consequences.

Neuronal nitric oxide synthase gene inactivation reduces the expression of vasopressin in the hypothalamic paraventricular nucleus and of catecholamine biosynthetic enzymes in the adrenal gland of the mouse

The impact of a lifelong absence of the neuronal nitric oxide synthase (nNOS) in the neuroendocrine stress response was investigated in nNOS knockout (KO) and wild type (WT) mice under basal conditions and in response to forced swimming. In the hypothalamic paraventricular nucleus oxytocin and corticotropin-releasing-hormone mRNA levels did not differ between these genotypes under resting conditions, whereas vasopressin mRNA levels were significantly lower in nNOS KO than in WT animals. Also, in the adrenal glands basal levels of tyrosine hydroxylase protein, the rate-limiting enzyme for catecholamine biosynthesis, and of phenylethanolamine N-methyltransferase, which converts norepinephrine to epinephrine, were significantly reduced in nNOS KO mice. Plasma adrenocorticotropin, corticosterone, norepinephrine and epinephrine levels were similar in the KO and WT genotypes under resting conditions. In response to forced swimming, a similar increase in plasma adrenocorticotropin and corticosterone was observed in KO and WT animals. Stressor exposure triggered also an increased epinephrine release in WT animals, but did not significantly alter plasma epinephrine levels in KO mice. These data suggest that the chronic absence of nNOS reduces the capacity of epinephrine synthesising enzymes in the adrenal gland to respond to acute stressor exposure with an adequate epinephrine release.