Corticotropin-releasing Factor mRNA Levels Research Articles

Effects of repeated tianeptine treatment on CRF mRNA expression in non-stressed and chronic mild stress-exposed rats

Accumulating evidence suggests that dysregulation of corticotropin-releasing factor (CRF) may play a role in depression and that this dysregulation may be corrected by antidepressant drug treatment. Here, we examined whether chronic mild stress (CMS) alters CRF mRNA levels in stress-related brain areas including the bed nucleus of the stria terminalis (BNST) and the central nucleus of amygdala (CeA), and whether repeated tianeptine treatment can attenuate CMS-induced changes in CRF mRNA levels. Male rats were exposed to CMS for 19 days, and control animals were subjected to brief handling. Both groups were injected daily with tianeptine or saline. CMS significantly increased CRF mRNA levels in the dorsal BNST (dBNST), but not in other areas. Repeated tianeptine treatment prevented the CMS-induced increase in CRF mRNA levels in the dBNST, and reduced CRF mRNA levels in dBNST in non-stressed controls. Moreover, repeated tianeptine treatment significantly decreased CRF mRNA levels in the ventral BNST and CeA of non-stressed controls as well as CMS-exposed rats. These results show that CMS induces a rather selective increase of CRF mRNA in the dBNST. In addition, these results suggest that repeated tianeptine treatment diminishes the basal activity of CRF neurons and reduces their sensitivity to stress.

Corticotropin-releasing factor and neuropeptide Y mRNA levels are modified by glucocorticoids in rainbow trout, Oncorhynchus mykiss

The primary stress response involves neuronal activation that ultimately leads to the release of glucocorticoids. Circulating glucocorticoids are thought to influence their own synthesis and release through a negative feedback mechanism that inhibits the activity of the hypothalamic and pituitary components of the stress axis. This study was designed to address the hypothesis that glucocorticoids modify corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) mRNA levels in the rainbow trout ( Oncorhynchus mykiss) brain. Cortisol implantation significantly reduced CRF1 and NPY mRNA levels in fish exposed to an isolation stress. In contrast, cortisol implantation did not prevent the stress-induced elevation of CRF1 and NPY mRNA levels during confinement. Treatment with the glucocorticoid receptor antagonist RU-486 reduced CRF1 mRNA levels in both isolated and confined fish, but had no effect on NPY mRNA. Although the cytochrome P450 inhibitor metyrapone reduced ACTH-induced cortisol secretion in vitro, plasma cortisol levels were elevated in isolated trout treated with metyrapone. Nevertheless, metyrapone implantation increased CRF1 and NPY mRNA levels in confined fish. Together, these results implicate cortisol as a modulator of CRF and NPY mRNA levels in the preoptic area of the trout brain, but that cortisol is only one such regulating mechanism.

Stress elevates corticotropin-releasing factor (CRF) and CRF-binding protein mRNA levels in rainbow trout (Oncorhynchus mykiss)

The objectives of this study were to characterize rainbow trout (Oncorhynchus mykiss) corticotropin-releasing factor (CRF)-binding protein (CRF-BP) cDNA and to examine the variations in CRF-BP and CRF mRNA levels in response to different intensities of stress. Trout were physically disturbed by a single or three consecutive periods of chasing until exhaustion followed by 2 h of recovery. The pituitary CRF-BP and preoptic area CRF1 mRNA contents were significantly increased only after repeated chasing events. Physical disturbance increased plasma cortisol levels with the largest change occurring in the group of trout that were exposed to repeated chasing events. Trout were also individually isolated in 120 l tanks or confined to 1.5 l boxes for 4, 24 or 72 h. CRF-BP mRNA levels in confined fish were greater than those of isolated fish at 72 h although there were no differences compared with the control group. CRF1 mRNA levels in the preoptic area were greater and remained elevated for a longer period in confined compared with isolated trout. Isolation led to a transient increase in plasma cortisol levels, but the higher cortisol values developed in the confined fish suggest that this treatment was more stressful than isolation. These results demonstrate that the intensity and duration of stress are important factors regulating CRF and CRF-BP mRNA levels in rainbow trout. We hypothesize that pituitary CRF-BP is involved in regulating the activity of the stress axis, possibly by reducing access to CRF1 receptors in the corticotropes.

Differential Increase in Forebrain and Caudal Neurosecretory System Corticotropin-Releasing Factor and Urotensin I Gene Expression Associated with Seawater Transfer in Rainbow Trout

Transfer to seawater (SW) in rainbow trout elicits an increase in plasma cortisol and a bout of anorexia. Although the corticotropin-releasing factor (CRF) system has known hypophysiotropic and anorexigenic properties, it is not known whether CRF-related peptides originating from either the forebrain or the caudal neurosecretory system (CNSS) play a role during SW acclimation. Therefore, we examined the effects of SW transfer on food intake, plasma osmolality, hypothalamic-pituitary-interrenal axis activity, and the expression of CRF and urotensin I (UI) in the forebrain and the CNSS. While SW transfer chronically suppressed food intake over a 2-wk period, it transiently increased plasma osmolality, ACTH, and cortisol. Similarly, 24 h after SW transfer, hypothalamic and preoptic area CRF mRNA levels were significantly increased but recovered to pretransfer levels within 7 d. Conversely, SW transfer elicited a delayed increase in hypothalamic UI mRNA levels and had no effect on preoptic area UI expression. In the CNSS, SW exposure was associated with parallel increases in CRF and UI mRNA levels from 24 h post transfer through 7 d. Finally, in situ hybridization demonstrated an extensive and overlapping pattern of CNSS CRF and UI expression. These results differentially implicate specific neuronal populations of the CRF system in the acute and chronic responses to a hyperosmotic stress and suggest that forebrain and CNSS CRF-related peptides have different roles in the coordinated response to fluid balance disturbances.

Enduring, Handling-Evoked Enhancement of Hippocampal Memory Function and Glucocorticoid Receptor Expression Involves Activation of the Corticotropin-Releasing Factor Type 1 Receptor

Early-life experience, including maternal care, influences hippocampus-dependent learning and memory throughout life. Handling of pups during postnatal d 2-9 (P2-9) stimulates maternal care and leads to improved memory function and stress-coping. The underlying molecular mechanisms may involve early (by P9) and enduring reduction of hypothalamic corticotropin-releasing factor (CRF) expression and subsequent (by P45) increase in hippocampal glucocorticoid receptor (GR) expression. However, whether hypothalamic CRF levels influence changes in hippocampal GR expression (and memory function), via reduced CRF receptor activation and consequent lower plasma glucocorticoid levels, is unclear. In this study we administered selective antagonist for the type 1 CRF receptor, NBI 30775, to nonhandled rats post hoc from P10-17 and examined hippocampus-dependent learning and memory later (on P50-70), using two independent paradigms, compared with naive and vehicle-treated nonhandled, and naive and antagonist-treated handled rats. Hippocampal GR and hypothalamic CRF mRNA levels and stress-induced plasma corticosterone levels were also examined. Transient, partial selective blockade of CRF1 in nonhandled rats improved memory functions on both the Morris watermaze and object recognition tests to levels significantly better than in naive and vehicle-treated controls and were indistinguishable from those in handled (naive, vehicle-treated, and antagonist-treated) rats. GR mRNA expression was increased in hippocampal CA1 and the dentate gyrus of CRF1-antagonist treated nonhandled rats to levels commensurate with those in handled cohorts. Thus, the extent of CRF1 activation, probably involving changes in hypothalamic CRF levels and release, contributes to the changes in hippocampal GR expression and learning and memory functions.

Chronic ethanol ingestion modulates proanxiety factors expressed in rat central amygdala

Withdrawal anxiety following chronic ethanol exposure is often associated with relapse in recovering alcoholics. It is likely that brain regions regulating anxiety-like behaviors adapt during chronic ethanol exposure to ultimately regulate such behaviors. The central amygdala contains numerous neurotransmitter systems that have been implicated in the regulation of anxiety-like behavior, including corticotropin releasing factor (CRF) and NMDA-type glutamate receptors. Chronic ethanol exposure causes functional adaptations in both CRF and NMDA receptors that are likely to regulate anxiety-like behaviors expressed during withdrawal. However, the molecular mechanisms governing these adaptations remain unexplored. We therefore evaluated these neurotransmitter systems in Sprague–Dawley rats during chronic ingestion of an ethanol-containing liquid diet. Quantitative real-time reverse transcription–PCR demonstrated that preproCRF mRNA was significantly upregulated by chronic ethanol exposure, whereas mRNA expression of CRF binding protein did not change. There were also no significant changes observed in any of the NMDA subunit mRNAs, although there was a trend toward greater NR2A mRNA expression during chronic ethanol exposure. Using Western blotting analysis we measured NMDA receptor subunit protein expression. Chronic ethanol exposure did not affect protein levels of the NR1 and NR2B subunits. Like the mRNA measures, chronic ethanol exposure did influence NR2A protein levels but the effects were modest. Our results demonstrate that NMDA receptor subunit mRNA and protein expressions are not strongly influenced by exposure to chronic ethanol. This suggests that the functional NMDA receptor adaptations identified in previous studies [Roberto, M., Schweitzer, P., Madamba, S. G., Stouffer, D. G., Parsons, L. H., & Siggins, G. R. (2004). Acute and chronic ethanol exposure alter glutamatergic transmission in rat central amygdala: an in vitro and in vivo analysis. J Neurosci 24, 1594–1603] are likely to be mediated by post-translational events. In contrast, enhanced levels of CRF during/after chronic ethanol exposure are likely to be mediated by increased levels of prepro CRF mRNA. Together, our findings suggest that adaptations to chronic ethanol exposure by proanxiety factors expressed in the central nucleus appear to be mediated by distinct cellular and molecular mechanisms.

Effect of ethanol on the regulation of corticotropin-releasing factor (CRF) gene expression

Ethanol stimulates hypothalamic–pituitary–adrenal axis activity in vivo. To determine the cellular and molecular mechanisms through which ethanol regulates corticotropin-releasing factor (CRF) gene expression, we compared the effect of ethanol and forskolin on CRF peptide secretion and messenger RNA levels in hypothalamic primary cell cultures, and on CRF promoter activity in the NG108-15 cell line. CRF secretion, mRNA levels, and gene transcription significantly increased in response to ethanol or forskolin. Mutation of the cAMP-response element (CRE) reduced luciferase activity under basal conditions as well as in response to forskolin or ethanol. On the other hand, plasmid with five CRE repeats yielded dramatically elevated basal luciferase activity and significantly increased upregulation by ethanol. Inclusion of adenosine deaminase reduced the promoter response to ethanol. Finally a PKA inhibitor and a cAMP antagonist both decreased ethanol-induced CRF peptide secretion, gene expression, and transcription. These results suggest that ethanol upregulates CRF expression through cAMP/PKA-dependent pathways.

Chronic stress reduces body fat content in both obesity-prone and obesity-resistant strains of mice

Unpredictable stressors have been used to assess the effect of stress on energy metabolism in obesity-prone (C57BL6J) and obesity-resistant (AJ) mice. Mice were exposed for 25 days to a stress protocol. Both strains of mice were divided into groups of control and stressed mice, which had access to either a high-fat or a high-carbohydrate diet. Twenty-four hours after the last session of stress, mice were sacrificed for blood and brain collections. Insulin, corticosterone, and glucose concentrations in plasma were measured, and expressions of corticotropin-releasing factor (CRF) in the paraventricular hypothalamic nucleus (PVH) and the central amygdala (CeA) were determined by in situ hybridization. Stressed mice in all groups had lower body fat contents than control mice, and all mice fed with the high-fat diet had heavier retroperitoneal and inguinal fat pads than mice fed with carbohydrate. CRF mRNA level in the CeA was lower in B6 mice than in AJ mice. Stressed mice had a lower expression of CRF in the CeA than control mice. In conclusion, chronic stress reduces body fat content in obesity-prone as well as in obesity-resistant mice.

Postnatal handling does not normalize hypothalamic corticotropin-releasing factor mRNA levels in animals prenatally exposed to ethanol

Postnatal handling has been shown to attenuate some of the deficits in developmental outcome observed following prenatal ethanol exposure (E) although it appears to be ineffective at ameliorating the hypothalamic–pituitary–adrenal (HPA) hyperresponsiveness to stressors that has been observed in adult E animals. However, the effects of postnatal handling on central regulation of HPA activity in E animals, particularly with regard to alterations in steady-state hypothalamic corticotropin-releasing factor (CRF) activity, have not been examined. In the present study, offspring from E, pair-fed (PF), and ad-libitum-fed control (C) groups were exposed to daily handling during the first 2 weeks of life (H) or were left entirely undisturbed until weaning (NH). Basal CRF and arginine vasopressin (AVP) mRNA in the parvocellular portion of the paraventricular nucleus (pPVN) of the hypothalamus were assessed at 90–110 days of age. Prenatal ethanol exposure resulted in elevated basal pPVN CRF mRNA levels compared to those in ad-libitum-fed controls. Handling altered CRF mRNA levels in a sex-specific and prenatal treatment-specific manner. Females showed no significant effects of handling. In contrast, handling decreased CRF mRNA levels in PF and C but not E males compared to their NH counterparts. There were no effects of prenatal ethanol or postnatal handling on AVP mRNA levels. These findings indicate that prenatal ethanol exposure results in elevated basal CRF mRNA levels in adulthood and that handling appears to be ineffective in normalizing those elevations, supporting the suggestion that altered basal HPA regulation in E animals may, at least in part, underlie their HPA hyperresponsiveness to stressors.

Organizational Role for Testosterone and Estrogen on Adult Hypothalamic-Pituitary-Adrenal Axis Activity in the Male Rat

Organizational effects of testosterone during a critical period of neonatal life have major irreversible effects on adult sexual behavior. We have investigated whether perinatal androgen changes also affect another major sexually differentiated system, the hypothalamo-pituitary-adrenal axis. This was assessed in male rats who had been exposed to perinatal flutamide or 1,4,6-androstatriene-3,17-dione (ATD). Once the animals reached adulthood, an automated sampling system was used to collect blood from freely moving animals at 10-min intervals over 24 h, followed by a noise stress and then the administration of lipopolysaccharide (LPS). Perinatal flutamide- and ATD-treated rats not only had higher mean corticosterone levels and increased frequency and amplitude of corticosterone pulses over the 24 h compared with vehicle-injected controls, but they also showed markedly increased corticosterone responses to both noise and LPS. All parameters of increased hypothalamo-pituitary-adrenal activity resembled the normal physiological state of the intact adult female rather than that of the intact adult male rat. Furthermore, 3 h after LPS administration, both flutamide- and ATD-treated animals had markedly higher levels of corticotropin-releasing factor mRNA in the parvocellular paraventricular nucleus (PVN) and proopiomelanocortin mRNA in the adenohypophysis. Flutamide-treated rats also had a greater level of PVN arginine vasopressin mRNA. PVN glucocorticoid receptor mRNA levels were significantly lower in both the flutamide- and the ATD-treated male rats. These data highlight the importance of perinatal exposure to both testosterone and estrogen(s) on the development of a masculinized circadian corticosterone profile and stress-induced hypothalamo-pituitary-adrenal axis activity in the adult male rat.

Age-specific threats induce CRF expression in the paraventricular nucleus of the hypothalamus and hippocampus of young rats

Young animals respond to threatening stimuli in an age-specific way. Their endocrine and behavioral responses reflect the potential threat of the situation at a given age. The aim of the present study was to determine whether corticotropin-releasing factor (CRF) is involved in the endocrine and behavioral responses to threat and their developmental changes in young rats. Preweaning 14-day-old and postweaning 26-day-old rats were exposed to two age-specific threats, cat odor and an adult male rat. The acute behavioral response was determined during exposure. After exposure, the time courses of the corticosterone response and of CRF expression in the paraventricular nucleus of the hypothalamus (PVN) and in extrahypothalamic areas were assessed. Preweaning rats became immobile when exposed to cat odor or the male rat, whereas postweaning rats became immobile to cat odor only. Male exposure increased serum corticosterone levels in 14-day-old rats, but cat odor failed to increase levels at either age. Exposure induced elevation of CRF mRNA levels in the PVN that paralleled changes in corticosterone levels. CRF may thus play a role in endocrine regulation and its developmental changes during early life. Neither cat odor nor the adult male altered CRF mRNA levels in the bed nucleus of the stria terminalis (BNST) or the amygdala, but both stimuli increased levels in the hippocampus. Hippocampal CRF mRNA expression levels did not parallel cat odor or male-induced immobility, indicating that CRF is not involved in this response in young rats but may be involved in aspects of learning and memory.

Cocaine- and amphetamine-regulated transcript activates the hypothalamic-pituitary-adrenal axis through a corticotropin-releasing factor receptor-dependent mechanism.

Cocaine- and amphetamine-regulated transcript (CART) is a highly expressed hypothalamic transcript that is concentrated in areas associated with the stress response. There is evidence for a role of CART in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. However, it is not clear whether CART regulates activity of the HPA axis by directly stimulating ACTH release from pituitary corticotropes or through interaction with hypothalamic factors. To address this issue, the effects of central and peripheral administration of CART on the HPA axis were compared. Central administration of CART(55-102) (1 microg) significantly increased circulating levels of ACTH (481 +/- 122 vs. 93 +/- 14 pg/ml; CART vs. vehicle) and corticosterone (460 +/- 29 vs. 179 +/- 62 ng/ml; CART vs. vehicle). In contrast, iv injection of CART(55-102) (0.09-9.0 nmol/kg) did not significantly affect circulating levels of ACTH or corticosterone. The corticotropin-releasing factor (CRF) receptor antagonist Astressin B was used to determine whether CART(55-102) elicits ACTH secretion via a CRF receptor-dependent mechanism. Injection of Astressin B (50 microg/kg, iv) inhibited CART(55-102)-induced ACTH and corticosterone responses. The effects of CART(55-102) on CRF and arginine vasopressin (AVP) expression were also examined in static hypothalamic explants. RT-PCR analysis revealed a significant up-regulation of CRF and AVP mRNA levels after CART(55-102) (10 nm and 1 microm) treatment. Last, the effects of CART(55-102) on CRF- and AVP-mediated ACTH release was investigated in dispersed rat anterior pituitary cells. Incubation of CART(55-102) (10-100 nm) did not significantly affect ACTH release from anterior pituitary cells. Findings from the present study suggest that CART regulates activity of the HPA axis through a CRF-dependent central mechanism and not by means of direct interaction with pituitary corticotropes.

Region-specific onset of handling-induced changes in corticotropin-releasing factor and glucocorticoid receptor expression.

Early-life experience including maternal care profoundly influences hormonal stress responses during adulthood. Daily handling on postnatal day (P) 2-9, eliciting augmented maternal care upon returning pups to their cage, permanently modifies the expression of the stress neuromodulators corticotropin-releasing factor (CRF) and glucocorticoid receptor (GR). We have previously demonstrated reduced hypothalamic CRF expression already at the end of the handling period, followed by enhanced hippocampal GR mRNA levels (by P45). However, the initial site(s) and time of onset of these enduring changes have remained unclear. Therefore, we used semiquantitative in situ hybridization to delineate the spatiotemporal evolution of CRF and GR expression throughout stress-regulatory brain regions in handled (compared with undisturbed) pups. Enhanced CRF mRNA expression was apparent in the amygdaloid central nucleus (ACe) of handled pups already by P6. By P9, the augmented CRF mRNA levels persisted in ACe, accompanied by increased peptide expression in the bed nucleus of the stria terminalis and reduced expression in the paraventricular nucleus. The earliest change in GR consisted of reduced expression in the ACe of handled pups on P9, a time point when hippocampal GR expression was not yet affected. Thus, altered gene expression in ACe, bed nucleus of the stria terminalis as well as paraventricular nucleus may contribute to the molecular cascade by which handling (and increased maternal care) influences the stress response long term.

The hypothalamic–pituitary–adrenal and glucose responses to daily repeated immobilisation stress in rats: individual differences

It is accepted that there are important individual differences in the vulnerability to stress-induced pathologies, most of them associated to the hypothalamic-pituitary and sympatho–medullo–adrenal axes, the two prototypical stress-responsive systems. However, there are few studies specifically aimed at characterising individual differences in the physiological response to daily repeated stress in rats. In the present work, male rats were submitted to repeated immobilisation (IMO) stress (1 h daily for 13 days) and several samples were taken at specific days and time points. Animals only subjected to blood sampling procedure served as controls. Daily adrenocorticotropic-hormone (ACTH), corticosterone and glucose responses to immobilisation (that included the post-immobilisation period) progressively declined over the days. In addition, repeated immobilisation resulted in decreased relative thymus weight, increased relative adrenal weight, elevated corticotropin-releasing factor (CRF) mRNA levels in the hypothalamic paraventricular nucleus (PVN), and down-regulation of glucocorticoid receptor gene transcription in hippocampus CA1. However, only CRF mRNA levels in the paraventricular nucleus correlated with the ACTH (on day 1) and corticosterone responses (from day 4–13) to immobilisation. When the animals were classified in three groups on the basis of their plasma ACTH levels immediately after the first immobilisation, individual differences in the ACTH response progressively disappeared on successive exposures to the stressor, whereas those in corticosterone and glucose were more sustained. The present results suggest that there are individual differences in the physiological response to stress that tend to be reduced rather than accentuated by repeated exposure to the stressor. Nevertheless, this buffering effect of repeated stress was dependent on the particular variable studied.

Effects of cortisol on food intake, growth, and forebrain neuropeptide Y and corticotropin-releasing factor gene expression in goldfish

Although elevated plasma cortisol levels and a reduction in food intake are common features of the response to stress in fish, the potential role of cortisol in the regulation of food intake in these animals is poorly understood. In this study, goldfish ( Carassius auratus) were fed ad libitum for 21 days diets prepared to contain 0 (Control), 50 (Low) or 500 (High) μg cortisol/g of food. While feeding remained unchanged in controls and in fish fed the High cortisol diet, daily food intake gradually increased in the Low cortisol diet group and was significantly elevated between days 9 and 21. At the end of the feeding trial, specific growth rate was lowest in fish fed the High cortisol diet, intermediate in those fed the Low cortisol diet, and highest in the controls. Feed conversion efficiency, on the other hand, was significantly reduced in both groups of fish fed the cortisol diets. After 3 weeks on the diets and relative to controls, the Low cortisol diet group was characterized by a 34% increase in neuropeptide Y (NPY) and a 22% decrease in corticotropin-releasing factor (CRF) mRNA levels in the telencephalon-preoptic brain region. In contrast, the High cortisol diet group was characterized by a 46% decrease in CRF mRNA levels and no significant change in NPY gene expression. In a separate experiment, intraperitoneal implants of cortisol (150 and 300 μg cortisol/g body weight) elicited a dose-dependent increase in NPY and decrease in CRF mRNA levels in the telencephalon-preoptic region at 72 h post-treatment. These results show that while moderate increases in plasma cortisol can stimulate food intake slowly over days, larger catabolic doses of glucocorticoids may mask the appetite-stimulatory effects of cortisol. Therefore, excess cortisol in goldfish can be associated with poor growth despite normal food intake. Furthermore, our results indicate that forebrain NPY and CRF may play a role in mediating the effects of cortisol on food intake in goldfish.

Corticotropin-releasing factor type-1 receptor mRNA is not induced in mouse hypothalamus by either stress or osmotic stimulation.

In rats, acute stress substantially increases corticotropin-releasing factor (CRF) type 1 receptor (CRFR-1) mRNA expression in the paraventricular nucleus (PVN) and osmotic stimulation induces both CRF and CRFR-1 mRNA in magnocellular PVN and supraoptic nucleus (SON). However, these phenomena have not been analysed in other species. We compared CRF and CRFR-1 expression in rat and mouse hypothalamus. Male C57BL/6 mice and Wistar rats were exposed to acute restraint stress for 3 h, or to hypertonic saline ingestion for 7 days. Restraint stress increased CRF and c-fos mRNA expression in both rat and mouse PVN. CRFR-1 mRNA was barely detectable in controls, whereas restraint stress substantially increased CRFR-1 mRNA in rat PVN, but not in mouse. Hypertonic saline ingestion induced CRF mRNA in magnocellular PVN and SON of the rat, but did not alter CRF mRNA levels in mouse hypothalamus. CRFR-1 mRNA was also induced in magnocellular PVN and SON of the rat in response to osmotic stimulation, but not in mouse. Immunohistochemistry demonstrated that CRFR-1-like immunoreactivity (ir) was distributed within parvocellular and magnocellular PVN of mouse and rat. CRFR-1-ir in rat PVN was increased by acute stress and osmotic stimulation. By contrast, these treatments did not alter CRFR-1-ir in mouse PVN. Combined immunohistochemistry and in situ hybridization revealed that CRFR-1-ir was most frequently colocalized to CRF in mouse PVN, whereas only a small percentage of oxytocin and vasopressin-producing cells coexpressed CRFR-1-ir. These results indicate that (i) by contrast to rats, neither acute stress nor osmotic stimulation induces CRFR-1 mRNA expression in the mouse PVN; (ii) osmotic stimulation does not alter CRF mRNA expression in parvocellular and magnocellular neurones of mouse PVN; and (iii) acute stress increases c-fos and CRF mRNA to a similar degree in mouse and rat PVN. Thus, differences may exist between mouse and rat in the regulation of CRF and CRFR-1 gene expression in hypothalamus following stress and osmotic stimulation.

Extinction of cocaine self-administration produces alterations in corticotropin releasing factor gene expression in the paraventricular nucleus of the hypothalamus

The long-term effect of cocaine self-administration on corticotropin releasing factor (CRF) mRNA content in the hypothalamic CRF-containing neurons has not yet been established. The purpose of this study was to examine the time course effects of the extinction of cocaine self-administration behavior on CRF gene expression in the paraventricular nucleus of the hypothalamus (PVN) using in situ hybridization histochemistry (IHHS). Seventy-two littermate male Lewis rats were randomly assigned in triads to one of three conditions: (a) contingent intravenous self-administration of 1 mg/kg/injection of cocaine (CONT), (b) non-contingent injections of either 1 mg/kg/injection of cocaine (NONCONT) or (c) saline yoked (SALINE) to the intake of the self-administering subject. The self-administering rats were trained to self-administer cocaine under a fixed ratio 5 (FR5) schedule of reinforcement for a minimum of 3 weeks. After stable baseline levels of drug intake had been reached, saline was substituted for drug. Following this first extinction period, cocaine self-administration was reinstated for an additional period of 2 weeks. Immediately after cessation of the last session of cocaine self-administration (Day 0) and 1, 5 and 10 days after the second extinction period, animal brains in each triad were removed to be processed for IHHS. CRF mRNA levels in the PVN were significantly lower in the NONCONT cocaine group at Day 0 compared to CONT or SALINE groups. On Day 1, hypothalamic CRF gene expression significantly decreased in the CONT cocaine group with respect to the SALINE group, but there were no differences between the cocaine groups or among the NONCONT cocaine and SALINE groups. After 5 and 10 days of extinction, no differences were found in CRF mRNA content in the PVN between the three conditions of this study. These results suggest that, after the extinction of cocaine self-administration, changes in hypothalamic CRF gene expression are differentially affected depending upon the type of cocaine administration, and that the stages of cocaine withdrawal might not be associated with enduring changes in hypothalamic CRF mRNA levels.

Effects of long-term voluntary exercise on the mouse hypothalamic-pituitary-adrenocortical axis.

We studied the effects of long-term (i.e. 4 wk) voluntary exercise on the hypothalamic-pituitary-adrenocortical (HPA) axis in male mice. Voluntary exercise was provided by giving mice access to a running wheel, in which they indeed ran for about 4 km/d. Exercising mice showed similar body weights as control animals but presented less abdominal fat, lighter thymuses, and heavier adrenal glands. Exercise resulted in asymmetric structural changes in the adrenal glands. Whereas control mice had larger left than right adrenals, this condition was abolished in exercising animals, mainly because of enlargement of the right adrenal cortex. Tyrosine hydroxylase mRNA expression in the adrenal medullas of exercising mice was increased. In exercising mice, early-morning baseline plasma ACTH levels were decreased, whereas plasma corticosterone levels at the start of the dark phase were twice as high as those in control animals. To forced swimming and restraint stress, exercising mice responded with higher corticosterone levels than those of the control animals but with similar ACTH levels. However, if exposed to a novel environment, then exercising mice presented decreased ACTH responses. Interestingly, exercising mice showed a decreased corticosterone response to novelty only when the novel environment contained a functioning running wheel. Glucocorticoid receptor levels were unchanged, whereas mineralocorticoid receptor levels were decreased, in hippocampus of exercising animals. Corticotropin-releasing factor mRNA levels in the paraventricular nucleus were lower in exercising mice. Thus, voluntary exercise results in complex, adaptive changes at various levels within the HPA axis as well as in sympathoadrenomedullary and limbic/neocortical afferent control mechanisms. These changes seem to underlie the differential responsiveness of the HPA axis to physical vs. emotional challenges.

Corticotropin-releasing factor and neuropeptide Y mRNA levels are elevated in the preoptic area of socially subordinate rainbow trout

The objectives of this study were to characterize rainbow trout ( Oncorhynchus mykiss) corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) cDNAs and to determine their mRNA levels in response to social stress. Standard cloning techniques were used to obtain cDNAs, sequences for trout NPY and two CRF isoforms. At the predicted amino acid level, our NPY sequence differs from the trout amino acid sequence reported by Jensen and Conlon (1992). A phylogenetic analysis suggests that the two CRF isoforms result from a gene duplication that occurred in a common ancestor of salmonids. A tissue distribution demonstrated that the mRNAs of both CRF isoforms are predominantly present in the preoptic area of the trout brain, whereas NPY mRNA is more abundant in the telencephalon. Pairs of sized-matched juvenile female trout were allowed to interact for 72 h and social ranks were assigned on the basis of behavioural observations. Mean plasma cortisol levels were 13-fold higher in subordinate than in dominant trout. As measured by ribonuclease protection assay, CRF1 and NPY mRNA levels were respectively 51 and 32% higher in the preoptic area of subordinate trout; in addition, CRF1 and NPY mRNA levels were positively correlated ( R 2=0.44). These results suggest that subordinate rainbow trout chronically maintain high levels of CRF mRNA during social stress and that NPY may be involved in the control of the stress axis in trout.

Foster litters prevent hypothalamic-pituitary-adrenal axis sensitization mediated by neonatal maternal separation

Neonatal maternal separation of rat pups has been shown to produce long-term increases in hypothalamic-pituitary-adrenal (HPA) axis responsiveness, elevated levels of hypothalamic corticotropin releasing factor (CRF) mRNA in the hypothalamic paraventricular nucleus (PVN), and enhanced anxiety-like behavior. These effects appear to be at least partially mediated by subtle disruptions in the quality of maternal-pup interactions. This hypothesis was tested by providing half the dams with foster litters during the maternal separation paradigm, so that in those litters, only the pups and not the dams were experiencing a period of separation. The separation protocol took place daily from PND2-14 for either 15 min (HMS15, handled) or 180 min (HMS180, maternal separation). During the period of separation dams were either transferred to adjacent cages without any pups present (HMS15, HMS180) or to cages containing an age-matched foster litter (HMS15F, HMS180F). As adults, the HMS180 progeny exhibited the expected increased expression of CRF mRNA in the PVN, stress hyper-responsiveness to airpuff startle and evidence of impaired feedback both in the CORT response, as well as in response to the dexamethasone suppression test. The HMS180F rats, however, appeared to be resistant to these effects of maternal separation as they demonstrated CRF mRNA levels intermediate between HMS15 and HMS180 rats. Their stress responses and feedback regulation of the HPA axis was comparable to that of the HMS15 rats. GR mRNA was elevated in the cortex of HMS180F rats. Overall, these studies support the thesis that the long-term effects of neonatal maternal separation may largely result from alterations in the quality of maternal care rather than from direct effects of the separation per se on the pups.