Single prolonged stress PTSD model triggers progressive severity of anxiety, altered gene expression in locus coeruleus and hypothalamus and effected sensitivity to NPY.

PTSD is heterogeneous disorder that can be long lasting and often has delayed onset following exposure to a traumatic event. Therefore, it is important to take a staging approach to evaluate progression of biological mechanisms of the disease. Here, we begin to evaluate the temporal trajectory of changes following exposure to traumatic stressors in the SPS rat PTSD model. The percent of animals displaying severe anxiety on EPM increased from 17.5% at one week to 57.1% two weeks after SPS stressors, indicating delayed onset or progressive worsening of the symptoms. The LC displayed prolonged activation, and dysbalance of the CRH/NPY systems, with enhanced CRHR1 gene expression, coupled with reduced mRNAs for NPY and Y2R. In the mediobasal hypothalamus, increased CRH mRNA levels were sustained, but there was a flip in alterations of HPA regulatory molecules, GR and FKBP5 and Y5 receptor at two weeks compared to one week. Two weeks after SPS, intranasal NPY at 300 µg/rat, but not 150 µg which was effective after one week, reversed SPS triggered elevated anxiety. It also reversed SPS elicited depressive/despair symptoms and hyperarousal. Overall, the results reveal time-dependent progression in development of anxiety symptoms and molecular impairments in gene expression for CRH and NPY systems in LC and mediobasal hypothalamus by SPS. With longer time afterwards only a higher dose of NPY was effective in reversing behavioral impairments triggered by SPS, indicating that therapeutic approaches should be adjusted according to the degree of biological progression of the disorder.

Noise Stress-Induced Changes in mRNA Levels of Corticotropin-Releasing Hormone Family Molecules and Glucocorticoid Receptors in the Rat Brain.

Noise is a widespread stress resource that may lead to detrimental effects on the health. However, the molecular basis of the stress response caused by noise remains elusive. We have studied the effects of acute and chronic noise stress on stress-related molecules in the hypothalamus and hippocampus and also corticosterone responses. Sprague Dawley rats were randomized into control, acute and chronic noise stress groups. While the chronic noise stress group animals were exposed to 100 dB white noise for 4 h/a day during 30 days, the acute noise stress group of animals was exposed to the same level of stress once for 4 h. The expression profiles of corticotropin releasing hormone (CRH), CRH1, CRH2 receptors and glucocorticoid receptor (GR) mRNAs were analysed by RT-PCR. Chronic noise stress upregulated CRH mRNA levels in the hypothalamus. Both acute and chronic noise increased CRH-R1 mRNA in the hypothalamus but decreased it in the hippocampus. GR mRNA levels were decreased by chronic noise stress in the hippocampus. The present results suggest that while corticosterone responses have habituated to continuous noise stress, the involvement of CRH family molecules and glucocorticoid receptors in the noise stress responses are different and structure specific.

Dynamics of appetite-mediated gene expression in daidzein-fed female rats in the meal-feeding method.

We previously found that daidzein decreased food intake in female rats. The present study aimed to elucidate the relationship between dynamics of appetite-mediated neuropeptides and the anorectic effect of daidzein. We examined appetite-mediated gene expression in the hypothalamus and small intestine during the 3 meals per day feeding method. Daidzein had an anorectic effect specifically at the second feeding. Neuropeptide-Y (NPY) and galanin mRNA levels in the hypothalamus were significantly higher after feeding in the control but not in the daidzein group, suggesting that daidzein attenuated the postprandial increase in NPY and galanin expression. The daidzein group had higher corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamus after feeding, and increased cholelcystokinin (CCK) mRNA levels in the small intestine, suggesting that CCK is involved in the hypothalamic regulation of this　anorectic effect. Therefore, daidzein may induce anorexia by suppressing expression of NPY and galanin and increasing expression of CRH in the hypothalamus.

Catalpol regulates function of hypothalamic-pituitary-adrenocortical-axis in an Alzheimer's disease rat model.

AIMS: To investigate the regulating effects of catalpol on the hypothalamic-pituitary- adrenocortical-axis (HPA) in an Alzheimer's disease (AD) rat model. METHODS: Healthy male Wistar Rats were selected. The AD model was generated by orthotopic injection of beta-amyloid 25-35 (Abeta25-35) into the right lateral ventricle. The animals were divided into five study groups: Catalpol at low dose (5 mg/kg), Catalpol at high dose (10 mg/kg), model control group and sham surgery control group, n = 9 respectively. The serum concentration of hydrocortisone (HYD), adrenocorticotropin (ACTH) and corticotropin releasing hormone (CRH) determined by Enzyme-Linked Immunosorbent Assay (ELISA). Structural alterations of the hypothalamus were examined by H&E stain and electron microscope. The CRH receptor 1 (CRHR1) positive neurons were detected with immunohistochemistry. RESULTS: Serum HYD level was significantly increased (p < 0.01), and both ACTH and CRH were dramatically decreased (p < 0.01) in the AD model group rats compared with normal control rats at day 7. Catalpol treatment was able to improve the hormone secretion disorder in AD model group rats compared with the model group (p < 0.01 or p < 0.05) in particular at 21 days. Structure damage of hypothalamus in the AD rat as evidenced less CRHR1 positive neurons, rough endoplasmic reticulum dilation and degranulation, and mitochondrial swelling under electron microscope. Catalpol treatment at both high and low doses was able to alleviate the structure damage of the hypothalamus in the AD rats. CONCLUSIONS: Catalpol could improve the endocrine function of the HPA and alleviate the structural damage of hypothalamus in AD rats.

Effects of social isolation on mRNA expression for corticotrophin-releasing hormone receptors in prairie voles.

Previous studies have demonstrated that various type of stressors modulate messenger ribonucleic acid (mRNA) for type 1 corticotropin-releasing hormone (CRH) receptor (CRH-R1 mRNA) and type 2 CRH receptor (CRH-R2 mRNA). The purpose of this study was to explore the effect of social isolation stress of varying durations on the CRH, CRH-R1 and CRH-R2 mRNAs expression in the hypothalamus, hippocampus and pituitary of socially monogamous female and male prairie voles (Microtus ochrogaster). Isolation for 1h (single isolation) or 1h of isolation every day for 4 weeks (repeated isolation) was followed by a significant increase in plasma corticosterone levels. Single or repeated isolation increased hypothalamic CRH mRNA expression, but no changes in CRH-R1 mRNA in the hypothalamus were observed. Continuous isolation for 4 weeks (chronic isolation) showed no effect on hypothalamic CRH or CRH-R1 mRNAs in female or male animals. However, hypothalamic CRH-R2 mRNA was significantly reduced in voles exposed to chronic isolation. Single or repeated isolation, but not chronic isolation, significantly increased CRH-R1 mRNA and decreased CRH-R2 mRNA in the pituitary. Despite elevated CRH mRNA expression, CRH-R1 and CRH-R2 mRNAs were not modulated in the hippocampus following single or repeated isolation. Although, chronic isolation did not affect hippocampal CRH or CRH-R1 mRNAs, it did increase CRH-R2 mRNA expression in females and males. The results of the present study in prairie voles suggest that social isolation has receptor subtype and species-specific consequences for the modulation of gene expression for CRH and its receptors in brain and pituitary. Previous studies have revealed a female-biased increase in oxytocin in response to chronic isolation; however, we did not find a sex difference in CRH or its receptors following single, repeated or chronic social isolation, suggesting that sexually dimorphic processes beyond the CRH system, possibly involving vasopressin, might explain this difference.

[¹²5I]YP20: a novel radioligand specific for the extracellular domain of the CRF1 receptor.

The peptide corticotropin-releasing factor (CRF) binds to the CRF1 receptor via a two-domain mechanism such that the extracellular domain (ECD) of the receptor captures the CRF's C-terminus to facilitate the binding of CRF's N-terminus to the juxta-membrane or "J"-site. Known small molecule antagonists bind to the J-site while known CRF1 receptor peptide radioligands bind to both sites. We report here the in vitro binding properties of the first radioligand that binds exclusively to the ECD of the CRF1 receptor. This ligand, which we named [¹²5I]Yamada peptide 20 ([¹²5I]YP20), is a radiolabeled analog of a synthetic peptide first reported by Yamada et al. (2004). We confirmed its high affinity for the [¹²5I]CRF binding site on the hCRF1 receptor and also found it to potently antagonize CRF-stimulated cAMP production in hCRF1-CHO cells. Under optimized conditions, 20 pM [¹²5I]YP20 reproducibly bound to hCRF1-CHO membranes with a pharmacology consistent with binding specific to the ECD of the CRF1 receptor. Saturation binding studies revealed the presence of a high affinity site with an estimated K(d) of ≈0.9 nM. The kinetic association of 20 pM [¹²5I]YP20 binding best fit to a rapid component (t(1/2)=0.69 min) and a sluggish component (t(1/2)=42 min). [¹²5I]YP20's specific binding was rapidly reversible with dissociation kinetics also best described by two phases (t(1/2)=0.92 min and t(1/2)=11.7 min). While [¹²5I]YP20's binding kinetics are complex, its high affinity and pharmacological specificity indicate that it is an excellent radioligand for probing the ECD site of the CRF1 receptor.

The effect of restraint stress on prepulse inhibition and on corticotropin-releasing factor (CRF) and CRF receptor gene expression in Wistar-Kyoto and Brown Norway rats.

Stress plays a role in many psychiatric disorders that are characterized by deficits in prepulse inhibition (PPI), a form of sensorimotor gating. Corticotropin-releasing factor (CRF) is one of the most important neurotransmitters involved in behavioral components of the stress response, and central infusion of CRF decreases PPI in rodents. We recently demonstrated that restraint stress decreases PPI and attenuates the increase in PPI caused by repeated testing. To broaden our investigation into how restraint affects PPI, we subjected Wistar-Kyoto (WKY) and Brown Norway (BN) rats to 10 consecutive days of 2-hour restraint, or to brief handling, prior to assessing PPI. We next examined the effects of 1 or 10days of 2-hour restraint on plasma corticosterone levels in order to determine whether the endocrine response to stress parallels the behavioral effect of stress. Finally, we examined the effects of 1 or 10days of 2-hour restraint on CRF and CRF receptor gene expression in the amygdala, hippocampus, frontal cortex, and hypothalamus in order to determine whether a temporal pattern of gene expression parallels the change in the behavioral response to stress. The major findings of the present study are that 1) restraint stress attenuates the increase in PPI caused by repeated testing in both WKY and BN rats, and BN rats are more sensitive to the effects of restraint on PPI than WKY rats, 2) restraint-induced increases in corticosterone levels mirror the effect of restraint on PPI in WKY rats but not in BN rats, 3) laterality effects on gene expression were observed for the amygdala, whereby restraint increases CRF gene expression in the left, but not right, amygdala, and 4) some restraint-induced changes in CRF and CRF receptor gene expression precede changes in PPI while other changes coincide with altered PPI in a rat strain- and brain region-dependent manner.

Long-term consequences of neonatal rearing on central corticotropin-releasing factor systems in adult male rat offspring.

In a series of studies on the long-term consequences of neonatal rearing, we compared hypothalamic and extrahypothalamic central corticotropin-releasing factor (CRF) systems in male rats reared under conditions of animal facility rearing, nonhandling (HMS0), handling with brief maternal separation for 15 min (HMS15), or handling with moderate maternal separation for 180 min (HMS180) daily from postnatal days 2-14. CRF-like immunoreactivity (CRFir) was elevated in lumbar cerebrospinal fluid of adult HMS180 and HMS0 rats relative to the other groups. In the paraventricular nucleus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and locus coeruleus, CRFir and CRF mRNA levels were significantly elevated in HMS0 and HMS180 rats. Neonatal maternal separation was associated with regionally specific alterations in CRF receptor type 1 (CRF1) mRNA density in HMS180 rats. No rearing-associated differences in CRF2alpha binding were apparent in either the lateral septum or the ventromedial hypothalamus. These findings indicate that early rearing conditions can permanently alter the developmental set-point of central CRF systems, and potentially influence the expression of behavioral and endocrine responses to stress throughout life, thereby providing a possible neurobiological substrate for the relationship between early life events and increased vulnerability for hypothalamic-pituitary-adrenal axis and coping skill alterations and the frequency of mood disorders in patients with a history of such experiences.

Reduced hypothalamic vasopressin secretion underlies attenuated adrenocorticotropin stress responses in pregnant rats.

We sought to explain decreased ACTH secretory responses to stress in pregnant rats by investigating hypothalamic CRH and vasopressin secretion and actions on anterior pituitary corticotrophs. In late pregnancy median eminence, CRH content was reduced (by 12%). Anterior pituitary proopiomelanocortin mRNA expression, measured by in situ hybridization but not radioimmunoassayed ACTH content, was also reduced (by 45% on d 21); CRH receptor (CRHR)1 mRNA expression was unaltered in pregnancy, but V1b receptor mRNA expression was reduced (by 19%). ACTH secretory responses, measured in jugular blood, to CRH (200 ng/kg iv) or vasopressin (1.7 microg/kg, iv) were reduced on d 21 vs. virgins (49% and 44%), but the response to combined CRH and vasopressin injection was intact. Either antalarmin (CRHR1 antagonist; 20 mg/kg ip) or dP(Tyr(Me)2),Arg-NH2(9))AVP (V1a/b antagonist; 10 microg/kg, iv) pretreatment reduced the ACTH secretory response to forced swimming (90 sec) in virgin rats (by 57% and 40%), but only antalarmin was effective in pregnant rats (53% decrease). In vitro, measuring ACTH secretion from acutely dispersed anterior pituitary cells showed increased corticotroph sensitivity in pregnancy to CRH and to CRH augmentation by vasopressin, attributable to increased intracellular cAMP action. Hence, in late pregnancy, reduced anterior pituitary CRHR1 or V1b receptor expression did not impair corticotroph responses to CRH or vasopressin. Rather, diminished secretagogue secretion in vivo accounts for reduced action of stress levels of exogenous CRH or vasopressin alone; the late pregnancy attenuated ACTH secretory response to swim stress is deduced to be due to reduced vasopressin release by parvocellular paraventricular nuclei neurones.

Mouse corticotropin-releasing factor receptor type 2alpha gene: isolation, distribution, pharmacological characterization and regulation by stress and glucocorticoids.

Effects of the corticotropin-releasing factor (CRF) family of peptides are mediated through activation of two receptors, CRF receptor (CRFR) 1 and CRFR2. Based on the homology between known mammalian CRFR genes, we have isolated a cDNA encoding the mouse CRFR2alpha (mCRFR2alpha) ortholog from brain. The isolated cDNA encodes a 411-amino acid protein with high identity to the rat (approximately 97%) and human (approximately 93%) receptors. Central and peripheral expression of mCRFR2alpha, determined by RT-PCR followed by Southern hybridization, revealed that mCRFR2alpha is restricted mainly to brain structures, with highest levels in the hypothalamus and olfactory bulb. In situ hybridization showed mCRFR2alpha localization in discrete brain regions, including the lateral septum and the ventromedial hypothalamus, whereas mCRFR2beta is found only in the choroid plexus. Binding and signaling of CRF-related ligands was studied using COS-M6 or HEK293T cells transiently transfected with mCRFR2alpha. Urocortins (Ucns) show different affinities for binding to mCRFR2alpha: Ucn 3 binds mCRFR2alpha with approximately 11-fold lower affinity than Ucn 2, which displays an affinity similar to Ucn 1 (approximately 1 nm). Cyclase activation, determined by intracellular cAMP accumulation and cAMP response element-luciferase activity, showed no differences between CRFR2alpha and CRFR2beta in response to stimulation by Ucn 1, Ucn 2, and Ucn 3. Interestingly, Ucn 3 was less efficacious than Ucn 1 or Ucn 2 in activating MAPK (ERK1/2-p44/p42) via CRFR2alpha, but all three Ucns showed equivalent efficacy for activating MAPK through mCRFR2beta. We found a significant reduction in hypothalamic mCRFR2alpha mRNA levels after acute and chronic restraint stress in mice. Hypothalamic mCRFR2alpha gene transcription in mice was inhibited by glucocorticoid administration and elevated by adrenalectomy. In addition, we demonstrated that the mCRFR2alpha gene is increased in the hypothalamus of the CRFR1-null compared with wild type mice. The predicted mCRFR2alpha promoter region was isolated and fused to a luciferase reporter gene and found to be decreased by glucocorticoids in a dose and time-dependent manner when transfected into CATH.a cells. Computer analysis revealed the presence of 23 putative half-palindromic glucocorticoid response element sequences within 2.4 kb of the mCRFR2alpha 5' flanking region. Elucidation of the structure and processing of the mCRFR2 gene and examination of the mCRFR2alpha gene regulation in various conditions will enable better understanding of the involvement of this receptor in the central response to stress in normal and transgenic mice models.

Urocortin III-immunoreactive projections in rat brain: partial overlap with sites of type 2 corticotrophin-releasing factor receptor expression.

Urocortin (Ucn) III, or stresscopin, is a new member of the corticotropin-releasing factor (CRF) peptide family identified in mouse and human. Pharmacological studies showed that Ucn III is a high-affinity ligand for the type 2 CRF receptor (CRF-R2). To further understand physiological functions the peptide may serve in the brain, the distribution of Ucn III neurons and fibers was examined by in situ hybridization and immunohistochemistry in the rat brain. Ucn III-positive neurons were found predominately within the hypothalamus and medial amygdala. In the hypothalamus, Ucn III neurons were observed in the median preoptic nucleus and in the rostral perifornical area lateral to the paraventricular nucleus. The Ucn III fibers were distributed mainly in the hypothalamus and limbic structures. Hypothalamic regions that were innervated prominently by Ucn III fibers included the ventromedial nucleus, medial preoptic nucleus, and ventral premammillary nucleus. Outside the hypothalamus, the densest projections were found in the intermediate part of the lateral septum, posterior division of the bed nucleus stria terminalis, and the medial nucleus of the amygdala. Several major Ucn III terminal fields identified in the present study, including the lateral septum and the ventromedial hypothalamus, are known to express high levels of CRF-R2. Thus, these anatomical data strongly support the notion that Ucn III is an endogenous ligand for CRF-R2 in these areas. These results also suggest that Ucn III is positioned to play a role in mediating physiological functions, including food intake and neuroendocrine regulation.

Fluoxetine induces the transcription of genes encoding c-fos, corticotropin-releasing factor and its type 1 receptor in rat brain.

Fluoxetine is a serotonin re-uptake blocker commonly used to treat endogenous depression. The present experiments were carried out to assess the effects of fluoxetine on c-fos induction throughout the rat brain. In addition, intron-directed in situ hybridization analysis was used to examine fluoxetine regulation of corticotropin-releasing factor heteronuclear gene transcription in the paraventricular nucleus of the hypothalamus. Because the actions of corticotropin-releasing factor are mediated by membrane-bound corticotropin-releasing factor type 1 receptors, we also evaluated the stimulation of such receptors after acute fluoxetine exposure. The immediate-early gene, c-fos, was markedly induced in several telencephalic and diencephalic brain structures. For instance, a strong hybridized signal was apparent 30 min after fluoxetine (10 mg/kg; intraperitoneal) administration in the caudate putamen, septal nucleus, bed nucleus of stria terminalis, anterodorsal preoptic area, paraventricular nucleus, supraoptic nucleus, ventromedial hypothalamus and posterior hypothalamic nucleus. In addition, c-fos-expressing neurons were also evident in discrete amygdaloid nuclei. This nuclear induction was brief in duration, as levels of the immediate-early gene were mostly undetectable 90 min after drug administration. In contrast to the extensive induction of c-fos by fluoxetine throughout the brain parenchyma, elevation of corticotropin-releasing factor heteronuclear RNA levels were confined exclusively to neurosecretory nerve cells of the paraventricular nucleus, with peak levels detected 30 min after fluoxetine exposure. Therefore, the time-course of corticotropin-releasing factor heteronuclear RNA closely paralleled that of c-fos. Significant changes in corticotropin-releasing factor type 1 receptor messenger RNA levels were also observed in the paraventricular nucleus but with a slow incremental biosynthesis of the receptor messenger RNA, as high levels were discernible only 360 min after fluoxetine treatment. Finally, we failed to detect sex-related differences in the acute response to fluoxetine, as both female and male rat brains showed a comparable induction of c-fos, corticotropin-releasing factor heteronuclear RNA and corticotropin-releasing factor type 1 receptor expression within parvocellular neurosecretory nerve cells that govern the stress response. All of these findings are discussed in terms of specific sequences of nuclear events that couple fluoxetine-based serotonin input with changes in gene expression in selective neurons.

Modulation of CRF-R1 mRNA in rat anterior pituitary by dexamethasone: correlation with POMC mRNA.

Glucocorticoids have been shown to decrease CRF receptor binding in the anterior pituitary. To determine whether glucocorticoids or CRF peptide modulate CRF-R1 gene expression, CRF-R1 mRNA levels in rat pituitary and brain were measured after administration of a synthetic glucocorticoid, dexamethasone (DEX) or rat/human CRF (r/hCRF), using a sensitive solution hybridization RNase protection assay. DEX (400 micrograms/day) or r/hCRF (100 micrograms/kg/day) was administered twice daily for 5 days to male rats. DEX treatment caused a significant decrease in CRF-R1 mRNA levels in the anterior pituitary. Also, a significant positive correlation was found between CRF-R1 and POMC mRNA levels in the anterior pituitary of the individual animals: levels of both mRNAs were reduced by DEX. However, r/hCRF treatment had no significant effect on CRF-R1 mRNA levels in the anterior pituitary. In extrahypothalamic brain regions, mRNA levels of CRF-R1 did not change following either DEX or r/hCRF administration. Our data suggest that in addition to POMC and CRF genes, CRF-R1 gene may also be subject to negative feedback control by glucocorticoids.

Signaling properties of mouse and human corticotropin-releasing factor (CRF) receptors: decreased coupling efficiency of human type II CRF receptor.

CRF is the primary neuroregulator of the function of the hypothalamic-pituitary-adrenal axis. We have recently cloned a mouse CRF receptor (mCRF-R) complementary DNA (cDNA) from an AtT-20 cell cDNA library by polymerase chain reaction. To compare the functions of mCRF-R to those of the human type I and type II CRF receptors (hCRF-RI and hCRF-RII), cDNAs were cloned into the expression vector pcDNA1 and transfected into COS-7 cells. CRF binding and CRF-stimulated cAMP accumulation as well as phosphoinositide hydrolysis were measured. Scatchard analysis of the binding of 125I-labeled [Tyr0]r/hCRF ([125I]CRF) to COS-7 cells expressing mCRF-R and hCRF-RI cDNAs revealed the same apparent Kd (9 nM). In contrast, the apparent binding Kd for hCRF-RII was 20 nM CRF. Maximal stimulatory concentrations (1 microM) of rat/human CRF-(1-41) (r/hCRF) increased cAMP accumulation in COS-7 cells transfected with mCRF-R, hCRF-RI, and hCRF-RII cDNA plasmid (10 micrograms each) from basal values of 8-19 pmol/10(5) cells.15 min to 84 +/- 10, 87 +/- 16, and 45 +/- 16 pmol/10(5) cells.15 min, respectively. The EC50 values of r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing mCRF-R and hCRF-RI cDNAs were similar at 0.4 +/- 0.2 and 0.7 +/- 0.2 nM, respectively. Conversely, the EC50 of r/hCRF-stimulated cAMP accumulation in hCRF-RII-transfected COS-7 cells was 47.5 +/- 18.9 nM. As the level of expression of hCRF-RII was lower than that of hCRF-RI, we compared r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing low and high levels of hCRF-RI. The EC50 for r/hCRF-stimulated cAMP accumulation in COS-7 cells transfected with hCRF-RI did not change when receptor expression was varied by a factor of 1- to 8.4-fold. In contrast, the EC50 for r/hCRF-stimulated cAMP accumulation mediated by hCRF-RII was at least 100-fold higher than that mediated by the hCRF-RI in COS-7 cells, which suggests poor coupling between hCRF-RII and adenylate cyclase. Inositol phosphate (IP) levels were also determined in mCRF-R, hCRF-RI, and hCRF-RII cDNA-transfected COS-7 cells stimulated with increasing concentrations of r/hCRF. r/hCRF-stimulated IPs accumulation was dose dependent in COS-7 cells expressing mCRF-R and hCRF-RI using 100 and 1000 nM r/hCRF. Concentrations of 10 (or less) nM r/hCRF had no effect on IP generation. hCRF-RII did not mediate stimulation of IP even at 1000 nM r/hCRF.(ABSTRACT TRUNCATED AT 400 WORDS)

Immune challenge and immobilization stress induce transcription of the gene encoding the CRF receptor in selective nuclei of the rat hypothalamus.

The present study investigated the effect of intraperitoneal (i.p.) administration of endotoxin lipopolysaccharide (LPS) and immobilization stress on the genetic expression of corticotropin-releasing factor receptor (CRF-R) in the brains of conscious male Sprague-Dawley rats. One group of rats was killed at 1, 3, 6, 9, and 12 hr after a single intraperitoneal injection of either the LPS (250 micrograms/100 gm of body weight) or the vehicle solution; the other group was killed before, immediately after, 1.5, 3, 6, and 12 hr after a 90 min acute session of immobilization stress. Rats were deeply anesthetized and rapidly perfused with a solution of 4% paraformaldehyde-borax. Frozen brains were mounted on a microtome and cut from the olfactory bulb to the medulla in 30 microns coronal sections. mRNA encoding the rat CRF-R was assayed by in situ hybridization histochemistry using a 35S-labeled riboprobe, and CRF-R localization within CRF-immunoreactive neurons in the PVN was determined using a combination of immunocytochemistry and in situ hybridization techniques. Strong basal levels of CRF-R transcripts were observed in several regions of the brain (piriform cortex, medial and basolateral nuclei of the amygdala, red nucleus, pontine gray, cerebellum, laterodorsal tegmental nucleus, caudal division of the zona incerta, nucleus incertus, spinal and principal sensory nuclei of the trigeminal nerve, and various layers of the cortex). A low to moderate signal was also detected in multiple sites (medial septal nucleus, nucleus of the diagonal band, supraoptic nucleus, arcuate nucleus of the hypothalamus, interpeduncular nucleus, and nucleus prepositus). Whereas vehicle-treated and control rats displayed hardly detectable signals of CRF-R mRNA in the paraventricular nucleus (PVN), CRF-R gene transcription was highly stimulated by LPS administration and immobilization stress in this hypothalamic structure. Indeed, the CRF-R mRNA signal was positive in the dorsomedial parvocellular PVN 3 hr after LPS injection, strong and maximum in both parvo- and magno-PVN at 6 hr postinjection, and declined 9 and 12 hr after treatment. Similarly, 90 min and 3 hr after the immobilization session, mRNA encoding the CRF-R was highly expressed in the parvo-PVN and totally vanished 12 hr after the stress. A lower but significant increase in the CRF-R transcript signal was also observed in the supraoptic nucleus 6 hr after the LPS treatment.(ABSTRACT TRUNCATED AT 400 WORDS)