Nociceptive stimulation increases NO synthase mRNA and vasopressin heteronuclearRNA in the rat paraventricular nucleus

Nociceptive stimulation causes neuroendocrine responses such as arginine vasopressin (AVP) release and activation of the hypothalamo-pituitary-adrenal (HPA) axis. We examined the effects of nociceptive stimulation on the expression levels of neuronal nitric oxide synthase (nNOS) mRNA, heteronuclear (hn)RNA for AVP and AVP mRNA in the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), using in situ hybridization histochemistry. For nociceptive stimulation, formalin (5%) or saline was injected subcutaneously (s.c.) into the bilateral hind paws of rats. The expression of the nNOS gene in the PVN was significantly increased 2 and 6 h after s.c. injection of formalin in comparison with that in untreated and saline injected rats. The expression of the nNOS gene in the SON did not change in the untreated, saline- and formalin-injected rats. The AVP hnRNA in the PVN and SON was also significantly increased 15, 30 min and 2 h after s.c. injection of formalin, though AVP mRNA did not change at any time points that we studied. Plasma concentration of AVP was significantly increased 15 min after s.c. injection of formalin. These results suggest that NO in the PVN may be involved in nociceptive stimulation-induced neuroendocrine responses.     

Response of Arginine Vasopressin-Enhanced Green Fluorescent Protein Fusion Gene in the Hypothalamus of Adjuvant-Induced Arthritic Rats

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.     

Selective forebrain fiber tract lesions implicate ventral hippocampal structures in tonic regulation of paraventricular nucleus corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA expression.

The hippocampus appears to be involved in tonic regulation of the hypothalamo-pituitary-adrenocortical axis via interactions with corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP)-containing neurons of the hypothalamic paraventricular nucleus (PVN). To further investigate the anatomical basis of such interactions, lesions were made to forebrain fiber tracts in position to communicate inhibitory information from the hippocampus to the PVN. Total fimbria-fornix transections (TFF) and lateral fimbria-fornix lesions (LFF) both significantly increased CRH mRNA levels in the medial parvocellular PVN, as assayed by semi-quantitative in situ hybridization histochemistry. Medial fimbria-fornix lesions or section of the medial corticohypothalamic tracts (MCHT) did not influence CRH mRNA levels. The LFF group showed increases in both AVP mRNA and ACTH secretion, whereas no other lesion was effective in this regard. The results suggest: (1) hippocampal efferents conferring tonic inhibition of the HPA axis probably originate in regions contributing to the lateral extent of the fornix, representing structures in the ventral subiculum and ventral extent of CA1; (2) projections from the hippocampus to the medial basal hypothalamus (travelling in the MCHT) are unlikely to affect HPA function; (3) hippocampus may influence the PVN CRH/AVP neuron at multiple levels, in that LFF and TFF lesions have differential effects on PVN AVP mRNA levels and ACTH secretion.     

The magnocellular arginine-vasopressin mRNA responds differently to food deprivation between the supraoptic and paraventricular nuclei of the hypothalamus in adrenalectomized rats with low corticosterone replacement.

We previously reported that food deprivation significantly decreased arginine-vasopressin (AVP) mRNA levels in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and also greatly stimulated the pituitary-adrenocortical system in rats. In this study, we deprived adrenalectomized rats with subcutaneously implanted low-dose corticosterone pellets (ADX + B) of food for 3 days to investigate the involvement of corticosteroid feedback regulation in the food deprivation-induced decrease in AVP mRNA in both the SON and the PVN. The plasma corticosterone levels in these animals were maintained at low levels constantly over 24 h. The ACTH concentration in the morning plasma was markedly increased in the food-deprived ADX + B rats as compared to the fed ADX + B rats. Food deprivation significantly decreased the corticotropin-releasing hormone (CRH) content in the median eminence and increased the CRH and AVP content in the neurointermediate lobe of the pituitary. Semiquantitative in situ hybridization histochemistry revealed that AVP mRNA levels were decreased in the SON but, inversely, increased in magnocellular as well as parvocellular subdivisions of the PVN following food deprivation. These results suggest that: (1) AVP mRNA responds differently to food deprivation between the SON and the PVN; (2) the glucocorticoid feedback can exert on AVP mRNA in the PVN but not in the SON in the food-deprived rats; and (3) food deprivation affects the neurohypophysial levels of CRH and AVP.     

Role of arginine vasopressin and corticotropin-releasing factor in mediating alcohol-induced adrenocorticotropin and vasopressin secretion in male rats bearing lesions of the paraventricular nuclei

In male rats, lesions of the paraventricular nucleus (PVN) of the hypothalamus attenuate, but do not abolish, adrenocorticotropin (ACTH) secretion in response to acute alcohol injection. As the PVN is the major source of corticotropin-releasing factor (CRF) in the median eminence, this observation suggests that extra-PVN brain regions, and/or ACTH secretagogues other than CRF (e.g. arginine vasopressin (AVP)), mediate ACTH stimulation by alcohol. This hypothesis was tested by examining the effect of AVP immunoneutralization in PVN-lesioned (PVNx) rats. Removal of endogenous AVP diminished alcohol-evoked ACTH secretion in both sham-operated and PVNx animals, indicating that AVP from outside the PVN partially mediates the hypothalamic-pituitary-adrenal (HPA) axis response to alcohol. This led us to determine whether alcohol might also regulate AVP steady-state gene expression in the supraoptic nucleus (SON) and PVN, and/or CRF mRNA in the PVN and the central nucleus of the amygdala (AMY). In the magnocellular portion of the PVN, sham-operated animals showed significantly increased PVN levels of both CRF and AVP mRNAs 3 h after alcohol. In the SON, alcohol administration tended to decrease AVP gene expression in PVNx rats, while the drug increased AVP mRNA levels in the SON of sham-operated rats. AMY levels of CRF mRNA were unaffected by these manipulations. Finally, since the regulation of alcohol-induced AVP mRNA levels in the SON appeared to depend on the presence of the PVN, we measured peripheral levels of AVP in both sham-operated and PVNx animals after injection of vehicle or alcohol. Although AVP decreased in all groups, alcohol depressed AVP secretion to a greater extent in PVNx animals, suggesting that AVP systems are more sensitive to inhibition in the absence of the PVN. Our results demonstrate that although AVP of PVN origin may participate in regulating the stimulatory effect to AVP on ACTH secretion, AVP from areas other than the PVN also plays a role. Additionally, regulation of both AVP gene expression in the SON and secretion in the systemic circulation are altered in rats bearing lesions of the PVN.     

Arginine vasopressin is an important regulator in antinociceptive modulation of hypothalamic paraventricular nucleus in the rat

Our previous study has proven that hypothalamic paraventricular nucleus (PVN) stimulation increases pain threshold and PVN cauterization decreases pain threshold. The studied neuropeptides in PVN were investigated to involve to pain modulation in the rat. The results showed that (1) intraventricular injection (icv) of anti-arginine vasopressin (AVP) serum completely reversed pain threshold increase induced by l-glutamate sodium (Glu) injection into the PVN, and local administration (icv) of anti-leucine-enkephalin (L-Ek) serum or anti-beta-endorphin (beta-Ep) serum partly attenuated pain threshold increase induced by Glu injection into the PVN, but pre-treatment of anti-oxytocin (OXT), dynorphinA(1-13) (DynA(1-13)), cholecystokinin-like peptide (CCK), neurotensin (NT), corticotrophin-releasing hormone (CRH), adrenocorticotrophin (ACTH), somatostatin (SST), prolactin-releasing hormone (PRH), angiotensinII (AngII), vasoactive intestinal polypeptide (VIP), melanotropin-releasing hormone (MRH), thyrotropin-releasing hormone (TRH), substance P (SP) or growth hormone-releasing hormone (GHRH) serum (icv) did not influence the analgesic effect of PVN administration with Glu; (2) PVN stimulation with Glu elevated the concentrations of AVP, OXT, CCK, NT, CRH, SST, PRH and DynA(1-13) in PVN perfusion liquid, and could not change the concentrations of L-Ek, beta-Ep, AngII, ACTH, VIP, MRH, TRH, SP and GHRH in PVN perfusion liquid; (3) Pain stimulation increased the concentrations of AVP, L-Ek, beta-Ep, DynA(1-13), CRH and ACTH in PVN perfusion liquid, and did not alter the concentrations of OXT, CCK, NT, SST, PRH, AngII, VIP, MRH, TRH, SP and GHRH in PVN perfusion liquid. The data suggested that AVP played a more important role than the other studied peptides (OXT, L-Ek, beta-Ep, DynA(1-13), CCK, NT, CRH, ACTH, SST, PRH, AngII, VIP, MRH, TRH, SP and GHRH) in PVN antinociceptive progress.     

Stimulation of CRH-Mediated ACTH Secretion by Central Administration of Neurotensin: Evidence for the Participation of the Paraventricular Nucleus

Central administration of neurotensin (NT) stimulates hypothalamic-pituitary-adrenal (HPA) activity in freely-moving rats. Increases in adrenocorticotropin hormone (ACTH) and corticosterone (B) were observed 15 rnin following central NT administration and remained elevated for up to 4 h. Of the two NT fragments tested, NT_1–8 and NT_8–13 only NT_8–13 was found to significantly elevate ACTH and B levels. Moreover, NT_8–13 activated the HPA axis with a temporal profile similar to NT_1–13, suggesting an interaction with the pharmacologically and molecularly characterized NT receptor. Animals pre-treated intravenously with the corticotropin-releasing hormone (CRH) antagonist, α-helical CRH, showed attenuated plasma ACTH and B responses to central NT administration. This indicates that CRH receptor activation is necessary for the stimulatory effects of NT on HPA function. Bilateral lesions of the paraventricular nucleus (PVN) of the hypothalamus significantly reduced NT-induced stimulation of ACTH and B release suggesting that the PVN is essential for NT's stirnulatory action. Median eminence content studies indicated that acute central NT administration stimulates CRH, but not arginine vassopressin (AVP), release in animals examined 60 min following NT injection. Taken together, these findings suggest that the stimulatory effects of NT on HPA activity occur via specific NT receptors and that one site of action of NT is likely at the level of the PVN where NT elicits the release of CRH.     

Inhibitory effects of corticosterone in the hypothalamic paraventricular nucleus (PVN) on stress-induced adrenocorticotrophic hormone secretion and gene expression in the PVN and anterior pituitary

Endogenous glucocorticoid negative-feedback influence on the hypothalamic-pituitary-adrenal (HPA) axis depends on glucocorticoid actions exerted on multiple glucocorticoid-sensitive tissues and differential glucocorticoid effects that are expressed within several distinct temporal domains. The relative contribution and underlying molecular mechanisms of action for the effects of location and timing of glucocorticoid exposure on HPA axis activity remain to be determined. In the present study, we examined the effects of acute exposure to corticosterone (CORT) at the level of the paraventricular nucleus (PVN) on the HPA axis response to a subsequent stressor in a short-term (1 h) timeframe. Intra-PVN CORT microinjection 1 h before restraint suppressed the adrenocorticotrophic hormone (ACTH) response and blunted restraint-induced corticotrophin-releasing hormone (CRH) heterogeneous nuclear (hn)RNA expression in the PVN and pro-opiomelanocortin hnRNA expression in the anterior pituitary (AP); however, it had no effect on restraint-induced plasma prolactin levels and c-fos mRNA expression (PVN and AP). This pattern of results suggests that CORT acts locally at the level of the PVN within a short-term timeframe to suppress stress-induced excitation-exocytosis coupling within CRH neurones and CRH gene induction without altering the stress-associated trans-synaptic input and intracellular signal transduction that converges on PVN c-fos gene induction. The present study is the first to demonstrate that an acute infusion of CORT into the PVN is sufficient to suppress the ACTH response to stress initiated 1 h after CORT infusion.     

Effects of leptin on fasting-induced inhibition of neuronal nitric oxide synthase mRNA in the paraventricular and supraoptic nuclei of rats

Fasting induced a reduction in neuronal nitric oxide synthase (nNOS) mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of rats. The effect of intracerebroventricular (i.c.v.) administration of leptin on the nNOS mRNA level in the PVN and SON of fasting rats was studied by in situ hybridization histochemistry. Leptin (10 microg/kg b.wt) or vehicle was administered i.c.v. at 1700 h on two successive days fasting for 2 days. Fasting for 2 days with i.c.v. administration of vehicle induced a significant reduction of nNOS mRNA in the PVN and SON. Central administration of leptin prevented the fasting-induced reduction of nNOS mRNA in the PVN and SON. Administration of leptin also prevented the fasting-induced reductions of thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH) mRNAs in the parvocellular division of the PVN. These results suggest that leptin is associated with fasting-induced reduction of nNOS mRNA in the PVN and SON as well as TRH and CRH mRNAs in the PVN.     

A single brain-derived neurotrophic factor injection modifies hypothalamo-pituitary-adrenocortical axis activity in adult male rats

Immobilization stress induces in adult male rats rapid activation of brain derived neurotrophic factor (BDNF) expression in the hypothalamic paraventricular nucleus (PVN) preceding the increases in corticotropin releasing hormone (CRH) and arginin-vasopressin (AVP) expression. The BDNF mRNA signal belatedly co-localizes with CRH and AVP mRNA signals in the PVN, as determined by in situ hybridization. Intracerebroventricular BDNF injections (5 microg/rat) in non-anesthetized adult male rats induce a gradual increase in the CRH mRNA signal whereas AVP mRNA signal progressively decreases in the parvocellular and magnocellular PVN portions. At the same time, the CRH hypothalamic content decreases while the AVP content increases. These variations are accompanied by increases in ACTH and corticosterone plasma concentrations. These results strongly suggest that BDNF could be a stress-responsive intercellular messenger since when it is exogenously administered acts as an important and early component in the activation and recruitment of hypothalamic CRH and AVP neurons.     

Hypothalamic-pituitary-adrenocortical responses to single vs. repeated endotoxin lipopolysaccharide administration in the rat

Lipopolysaccharide (LPS) is a potent stimulator of the hypothalamic-pituitary-adrenal (HPA) axis. However, the alteration in the HPA axis responsiveness and brain corticosteroid receptor levels during long-term administration of LPS has not been studied well. The present study was designed to examine the effect of single vs. repeated intraperitoneal (i.p.) LPS injection on the HPA axis and brain corticosteroid receptor levels in male Wistar rats. In addition, c-fos mRNA expression was examined in the hypothalamic paraventricular nucleus (PVN) and brainstem catecholaminergic nuclei such as the locus coeruleus (LC) and nucleus tractus solitarius (NTS), the sites known to be involved in LPS-induced HPA axis stimulation. Rats that had received i.p. LPS injection for 6 consecutive days (6-LPS group) had similar levels of plasma adrenocorticotropin (ACTH) and corticosterone (CORT) compared to animals that had received i.p. saline (6-saline group). A single injection of LPS to the 6-saline group (6-saline+challenge) resulted in a substantial increase in plasma ACTH and CORT at 2 h, whereas an additional injection of LPS to the 6-LPS group (6-LPS+challenge) showed less of an increase. As determined by in situ hybridization histochemistry, proopiomelanocortin (POMC) mRNA levels in the anterior pituitary (AP) and corticotropin-releasing hormone (CRH) mRNA levels in the PVN were higher in the 6-LPS than in the 6-saline group. A single injection of LPS to the 6-saline group resulted in a significant increase in AP POMC mRNA and PVN CRH mRNA at 2 h, while injection of LPS to the 6-LPS group showed no additional increase in these levels. C-fos mRNA expression was prominent in the PVN, LC, and NTS following a single injection of LPS, but not following repeated LPS injection. These results suggest that stimulatory input into the PVN decreased following repeated LPS injection. Furthermore, type II glucocorticoid receptor (GR) mRNA levels in the 6-LPS and 6-LPS+challenge groups were decreased in the hippocampus, but not in the PVN or AP. Adrenalectomy with 40% CORT pellet replacement restored ACTH responses following repeated LPS injections to levels similar to those following a single LPS injection. Decreased hippocampal GR mRNA may contribute to the elevated PVN CRH mRNA levels in the 6-LPS group. Nevertheless, inhibition of the pituitary ACTH response by glucocorticoids and reduced hypothalamic drive are partly responsible for decreased pituitary-adrenal responsiveness following repeated LPS injection.     

Serotonergic stimulation of corticotropin-releasing hormone and pro-opiomelanocortin gene expression

The neurotransmitter serotonin (5-HT) stimulates adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary gland via activation of central 5-HT1 and 5-HT2 receptors. The effect of 5-HT is predominantly indirect and may be mediated via release of hypothalamic corticotropin-releasing hormone (CRH). We therefore investigated the possible involvement of CRH in the serotonergic stimulation of ACTH secretion in male rats. Increased neuronal 5-HT content induced by systemic administration of the precursor 5-hydroxytryptophan (5-HTP) in combination with the 5-HT reuptake inhibitor fluoxetine raised CRH mRNA expression in the paraventricular nucleus (PVN) by 64%, increased pro-opiomelanocortin (POMC) mRNA in the anterior pituitary lobe by 17% and stimulated ACTH secretion five-fold. Central administration of 5-HT agonists specific to 5-HT1A, 5-HT1B, 5-HT2A or 5-HT2C receptors increased CRH mRNA in the PVN by 15-50%, POMC mRNA in the anterior pituitary by 15-27% and ACTH secretion three- to five-fold, whereas a specific 5-HT3 agonist had no effect. Systemic administration of a specific anti-CRH antiserum inhibited the ACTH response to 5-HTP and fluoxetine and prevented the 5-HTP and fluoxetine-induced POMC mRNA response in the anterior pituitary lobe. Central or systemic infusion of 5-HT increased ACTH secretion seven- and eight-fold, respectively. Systemic pretreatment with the anti-CRH antiserum reduced the ACTH responses to 5-HT by 80% and 64%, respectively. It is concluded that 5-HT via activation of 5-HT1A, 5-HT2A, 5-HT2C and possibly also 5-HT1B receptors increases the synthesis of CRH in the PVN and POMC in the anterior pituitary lobe, which results in increased ACTH secretion. Furthermore, the results indicate that CRH is an important mediator of the ACTH response to 5-HT.     

Glutamate agonists activate the hypothalamic-pituitary-adrenal axis through hypothalamic paraventricular nucleus but not through vasopressinerg neurons

The hypothalamic-pituitary-adrenal (HPA) axis plays a crucial role in the stress processes. The nucleus paraventricularis hypothalami (PVN) with corticotropin-releasing hormone (CRH)-containing and arginine vasopressin (AVP)-containing neurons is the main hypothalamic component of the HPA. The glutamate, a well-known excitatory neurotransmitter, can activate the HPA inducing adrenocorticotropin hormone (ACTH) elevation. The aim of our study was to examine the involvement of PVN and especially AVP in glutamate-induced HPA activation using agonists of the N-methyl-d-aspartate (NMDA) and kainate receptors. Two approaches were used: in male Wistar rats the PVN was lesioned, and AVP-deficient homozygous Brattleboro rats were also studied. Blood samples were taken through indwelling cannula and ACTH, and corticosterone (CS) levels were measured by radioimmunoassay. The i.v. administered NMDA (5 mg/kg) or kainate (2.5 mg/kg) elevated the ACTH and CS levels already at 5 min in control (sham-operated Wistar or heterozygous Brattleboro) rats. The PVN lesion had no influence on basal ACTH and CS secretion but blocked the NMDA- or kainate-induced ACTH and CS elevations. The lack of AVP in the Brattleboro animals had no significant influence on the basal or glutamate-agonists-induced ACTH and CS elevations. Our results suggest that NMDA and kainate may activate the HPA axis at central (PVN) level and not at the level of pituitary or adrenal gland and that AVP has minor role in glutamate-HPA axis interaction. The time course of the ACTH secretion was different with NMDA or kainate. If we compared the two curves, the results were not coherent with the general view that NMDA activation requires previous kainate activation. Although it has to be mentioned that the conclusion which can be drawn is limited, the bioavailability of the compounds could be different as well.     

Increased levels of hypothalamic neuronal nitric oxide synthase and vasopressin in salt-loaded Dahl rat

The plasma concentration of arginine vasopression (AVP) and the expression level of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular nucleus (PVN) and the Supraoptic nucleus (SON) of Sprague-Dawley (SD). Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats on a high salt diet were examined by radioimmunoassay for AVP and in situ hybridization histochemistry for nNOS. The high salt diet containing 8.0% NaCl was given for 4 weeks. The concentrations of AVP in hypertensive Dahl S rats were significantly increased in comparison with those in SD rats and Dahl R rats on a high salt diet. The levels of nNOS mRNA and NADPH-diaphorase activity in the PVN and SON of hypertensive Dahl S rats were greater than those in Dahl R rats on a high salt diet. The antihypertensive drugs, either nicardipine or captopril were administered to the Dahl S rats for 2 weeks beginning 2 weeks after the start of the high salt diet The nNOS mRNA in the PVN and SON of Dahl S rats given a high salt diet was not upregulated by treatment with nicardipine, while the nNOS mRNA in salt loaded Dahl S rats was greater upregulated by treatment with captopril to that greater than without the antihypertensive drug. Our results suggest that the increased NO production in the PVN and SON of hypertensive Dahl S rats may be ineffective in decreasing blood pressure or inhibiting AVP secretion.     

Acute glucocorticoid pretreatment suppresses stress-induced hypothalamic-pituitary-adrenal axis hormone secretion and expression of corticotropin-releasing hormone hnRNA but does not affect c-fos mRNA or fos protein expression in the paraventricular nucleus of the hypothalamus

Corticosterone regulates both basal and stress-induced hypothalamic-pituitary-adrenal (HPA) axis activity in a negative-feedback fashion. However, the cellular and molecular mechanisms of this negative feedback have yet to be explicitly characterized. By comparing stress-induced c-fos and corticotropin-releasing hormone (CRH) expression in the paraventricular nucleus (PVN), we may be able to determine whether acute glucocorticoid treatment affects the net neural excitatory input to the PVN (represented primarily by c-fos mRNA expression) or directly affects the ability of cells in the PVN to respond to that input (represented primarily by CRH hnRNA expression). In the following studies, we observed the effect of acute glucocorticoid (RU28362) treatment on subsequent HPA axis reactivity by measuring stress-induced plasma hormone concentration [corticosterone and adrenocorticotropic hormone (ACTH)] and gene expression (c-fos and CRH) in the PVN. First, we examined the dose-response relationship between systemically administered RU28362 (1-150 microg/kg, i.p) and suppression of the stress-induced corticosterone response. We then confirmed central nervous system access of the maximally suppressive dose of RU28362 (150 microg/kg) by an ex vivo radioligand binding assay. RU28362 selectively occupied the majority of glucocorticoid receptors in the hippocampus and hypothalamus while having no effect on mineralocorticoid receptors. In separate studies, RU28362 (150 microg/kg) and corticosterone (5 mg/kg) were injected i.p. 1 h before restraint stress. Compared to vehicle-treated controls, rats treated with RU28362 and corticosterone had substantially blunted stress-induced corticosterone and ACTH production, respectively. Furthermore, treatment with RU28362 significantly blunted stress-induced CRH hnRNA expression in the PVN. By contrast, neither RU28362 nor corticosterone treatment had an effect on stress-induced neuronal activation as measured by c-fos mRNA and its protein product in the PVN. This dissociation between c-fos and CRH gene expression suggests that glucocorticoid suppression of HPA activity within this time-frame is not a result of decreased excitatory neural input to the PVN, but instead depends on some direct effect of RU28362 on cells intrinsic to the HPA axis.     

Enhanced up-regulation of corticotropin-releasing hormone gene expression in response to restraint stress in the hypothalamic paraventricular nucleus of oxytocin gene-deficient male mice

The neuropeptide oxytocin is released not only into the blood, but also within the brain in response to various stressors. Accumulating evidence suggests that central oxytocin may play a major role in the regulation of neuroendocrine responses to stress. In the present study, using the oxytocin knockout mouse model, we tested whether oxytocin might act to attenuate stress-induced up-regulation of corticotropin-releasing hormone (CRH) mRNA expression in the brain. The expression of CRH mRNA in the paraventricular nucleus (PVN) after 4 h of restraint stress was examined in oxytocin gene-deficient (OTKO), wild-type and heterozygous male mice using in situ hybridization histochemistry. We found that basal levels of CRH mRNA were not different among the three genotypes. Although restraint stress resulted in a significant increase of CRH mRNA expression in the PVN regardless of genotype, the degree of stress induced-up-regulation was significantly higher in OTKO mice than in wild-type mice. The effects of restraint stress on the expression of the arginine vasopressin (AVP) and the oxytocin genes were also examined. Unlike CRH mRNA, basal expression (in nonstressed control groups) of AVP mRNA in OTKO mice, as well as oxytocin mRNA in heterozygous mice, was significantly lower in the PVN and the supraoptic nucleus than in wild-type mice. After restraint stress, the expression of AVP mRNA was significantly increased in the PVN of OTKO mice compared to the nonstressed control group, whereas the expression of both AVP and oxytocin mRNA were unchanged in the PVN and the supraoptic nucleus of wild-type and heterozygous mice. Finally, in a separate set of mice, restraint stress-induced Fos expression was also examined in several brain regions involved in stress response, including the lateral septum, the bed nucleus of the stria terminalis (BNST), the medial preoptic area, the PVN, the medial and central amygdala using immunohistochemistry. After 90 min of restraint stress, the number of Fos-expressing cells significantly increased in all brain regions examined regardless of genotype. However, the number of stress-induced Fos-expressing cells in the BNST and the medial amygdala of OTKO mice was significantly lower than in wild-type mice. Collectively, the findings in the present study suggest that oxytocin may regulate stress-induced CRH gene expression in the PVN. Furthermore, neuronal activity in the BNST and the medial amygdala may be involved in this neuroendocrine regulatory system.