Early-Life Stress Reprograms Stress-Coping Abilities in Male and Female Juvenile Rats.

Prenatal stress (PS) is a major risk factor for the development of emotional disorders in adulthood that may be mediated by an altered hypothalamic-pituitary-adrenal axis response to stress. Although the early onset of stress-related disorders is recognized as a major public health problem, to date, there are relatively few studies that have examined the incidence of early-life stressors in younger individuals. In this study, we assessed PS impact on the stress-coping response of juvenile offspring in behavioral tests and in the induced molecular changes in the hippocampus. Furthermore, we assessed if pregnancy stress could be driving changes in patterns of maternal behavior during early lactation. We found that PS modified stress-coping abilities of both sex offspring. In the hippocampus, PS increased the expression of bdnf-IV and crfr1 and induced sex difference changes on glucocorticoids and BDNF mRNA receptor levels. PS changed the hippocampal epigenetic landscape mainly in male offspring. Stress during pregnancy enhanced pup-directed behavior of stressed dams. Our study indicates that exposure to PS, in addition to enhanced maternal behavior, induces dynamic neurobehavioral variations at juvenile ages of the offspring that should be considered adaptive or maladaptive, depending on the characteristics of the confronting environment. Our present results highlight the importance to further explore risk factors that appear early in life that will be important to allow timely prevention strategies to later vulnerability to stress-related disorders.

Functional and Anatomical Characterization of Corticotropin-Releasing Factor Receptor Subtypes of the Rat Spinal Cord Involved in Somatic Pain Relief.

Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.

Sex differences in CRF1, CRF, and CRFBP expression in C57BL/6J mouse brain across the lifespan and in response to acute stress.

Signaling pathways mediated by corticotropin-releasing factor and its receptor 1 (CRF1) play a central role in stress responses. Dysfunction of the CRF system has been associated with neuropsychiatric disorders. However, dynamic changes in the CRF system during brain development and aging are not well investigated. In this study, we characterized CRF1, CRF, and corticotropin-releasing factor binding protein (CRFBP) expression in different brain regions in both male and female C57BL/6J mice from 1 to 18 months of age under basal conditions as well as after an acute 2-hr-restraint stress. We found that CRF and CRF1 levels tended to increase in the hippocampus and hypothalamus, and to decrease in the prefrontal cortex with aging, especially at 18 months of age, whereas CRFBP expression followed an opposite direction in these brain areas. We also observed area-specific sex differences in the expression of these three proteins. For example, CRF expression was lower in females than in males in all the brain regions examined except the prefrontal cortex. After acute stress, CRF and CRF1 were up-regulated at 1, 6, and 12 months of age, and down-regulated at 18 months of age. Females showed more robust changes compared to males of the same age. CRFBP expression either decreased or remained unchanged in most of the brain areas following acute stress. Our findings suggest that brain CRF1, CRF, and CRFBP expression changes dynamically across the lifespan and under stress condition in a sex- and regional-specific manner. Sex differences in the CRF system in response to stress may contribute to the etiology of stress-related neuropsychiatric disorders.

Chronic stress impairs male spermatogenesis function and Nectin-3 protein expression in the testis.

Chronic stress is a crucial public issue that occurs when a person is repetitively stimulated by various stressors. Previous researches have reported that chronic stress induces spermatogenesis dysfunction in the reproductive system, but its molecular mechanisms remain unclear. The nectin protein family, including nectin-1 to nectin-4, is Ca(2+)-independent immunoglobulin-like cell adhesion molecules, that are widely expressed in the hippocampus, testicular tissue, epithelial cells and other sites. Nectin-3 contributes to the sperm development at the late stage, and the abnormal expression of nectin-3 impairs spermatogenesis. Some recent studies have demonstrated that stress induces a decrease in nectin-3 expression in the hippocampus via corticotropin-releasing hormone (CRH) to corticotropin-releasing hormone receptor 1 (CRHR1) pathway. Here, we tested whether chronic stress also caused a reduction in nectin-3 expression in the testis. We established a chronic social defeat stress paradigm, which provides naturalistic and complex chronic stress inmale C57BL/6 mice. After 25 days of chronic social defeat stress, the mice showed weight loss, thymic atrophy and some other typical symptoms of chronic stress (e.g.anxiety-like behavior and social avoidance behavior). We found gonad atrophy, testicular histological structure changes and semen quality reductions in the stressed mice. The stressed male mice significantly spent more time to impregnate the female mice than the control male mice. Moreover, nectin-3 protein levels in stressed mice were significantly decreased in the testes compared with those in control mice. In addition, we found that the CRHR1 expression level was increased in the testes of stressed mice. Therefore, we demonstrated a decreased level of nectin-3 expression and an increase in CRHR1 expression in the testis after exposure to chronic stress, which may provide a potential therapeutic target for the spermatogenesis dysfunction induced by chronic stress.

Exercise before pregnancy attenuates the effects of prenatal stress in adult mice in a sex-dependent manner.

The present study aimed to investigate the long-term effects of exercise before pregnancy on changes induced by prenatal stress. Female and male Balb/c mice were divided into three groups: control (CON), prenatal restraint stress (PNS), and exercise before the gestational period plus PNS (EX + PNS). As adult, fear/anxiety behavior, corticosterone secretion, expression of hypothalamic-pituitary-adrenal (HPA)-related genes, as well as epigenetic modifications were evaluated. Exercise before gestation did not prevent the increased fear/anxiety behavior in PNS mice. A nearly significant (p = .06) basal corticosterone increase was observed in PNS males and the exercise before pregnancy reduced the stress-induced corticosterone increase in PNS females. In addition, an increase on prefrontal cortex (PFC) CRHR1 gene expression was observed in PNS females, which was attenuated by the exercise before gestation. We have also found a glucocorticoid receptor (GR) gene expression decrease in the prefrontal cortex in PNS males, as well as a histone H3 acetylation decrease (p = .06) close to the significance level. In conclusion, pregestational exercise may attenuate developmental changes induced by prenatal stress in a sex-dependent manner.

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.

Enhanced CRFR1-Dependent Regulation of a Ventral Tegmental Area to Prelimbic Cortex Projection Establishes Susceptibility to Stress-Induced Cocaine Seeking.

The ability of stress to trigger cocaine seeking in humans and rodents is variable and is determined by the amount and pattern of prior drug use. This study examined the role of a corticotropin releasing factor (CRF)-regulated dopaminergic projection from the ventral tegmental area (VTA) to the prelimbic cortex in shock-induced cocaine seeking and its recruitment under self-administration conditions that establish relapse vulnerability. Male rats with a history of daily long-access (LgA; 14 × 6 h/d) but not short-access (ShA; 14 × 2 h/d) self-administration showed robust shock-induced cocaine seeking. This was associated with a heightened shock-induced prelimbic cortex Fos response and activation of cholera toxin b retro-labeled VTA neurons that project to the prelimbic cortex. Chemogenetic inhibition of this pathway using a dual virus intersectional hM4Di DREADD (designer receptor exclusively activated by designer drug) based approach prevented shock-induced cocaine seeking. Both shock-induced reinstatement and the prelimbic cortex Fos response were prevented by bilateral intra-VTA injections of the CRF receptor 1 (CRFR1) antagonist, antalarmin. Moreover, pharmacological disconnection of the CRF-regulated dopaminergic projection to the prelimbic cortex by injection of antalarmin into the VTA in one hemisphere and the D1 receptor antagonist, SCH23390, into the prelimbic cortex of the contralateral hemisphere prevented shock-induced cocaine seeking. Finally, LgA, but not ShA, cocaine self-administration resulted in increased VTA CRFR1 mRNA levels as measured using in situ hybridization. Altogether, these findings suggest that excessive cocaine use may establish susceptibility to stress-induced relapse by recruiting CRF regulation of a stressor-responsive mesocortical dopaminergic pathway.SIGNIFICANCE STATEMENT Understanding the neural pathways and mechanisms through which stress triggers relapse to cocaine use is critical for the development of more effective treatment approaches. Prior work has demonstrated a critical role for the neuropeptide corticotropin releasing factor (CRF) in stress-induced cocaine seeking. Here we provide evidence that stress-induced reinstatement in a rat model of relapse is mediated by a CRF-regulated dopaminergic projection from the ventral tegmental area (VTA) that activates dopamine D1 receptors in the prelimbic cortex. Moreover, we report that this pathway may be recruited as a result of daily cocaine self-administration under conditions of extended drug access/heightened drug intake, likely as a result of increased CRFR1 expression in the VTA, thereby promoting susceptibility to stress-induced cocaine seeking.

Increased expression of CRF and CRF-receptors in dorsal striatum, hippocampus, and prefrontal cortex after the development of nicotine sensitization in rats.

BACKGROUND: Nicotine addiction supports tobacco smoking, a main preventable cause of disease and death in Western countries. It develops through long-term neuroadaptations in the brain reward circuit by modulating intracellular pathways and regulating gene expression. This study assesses the regional expression of the transcripts of the CRF transmission in a nicotine sensitization model, since it is hypothesised that the molecular neuroadaptations that mediate the development of sensitization contribute to the development of addiction. METHODS: Rats received intraperitoneal nicotine administrations (0.4 mg/kg) once daily for either 1 day or over 5 days. Locomotor activity was assessed to evaluate the development of sensitization. The mRNA expression of CRF and CRF1 and CRF2 receptors was measured by qPCR in the ventral mesencephalon, ventral striatum, dorsal striatum (DS), prefrontal cortex (PFCx), and hippocampus (Hip). RESULTS: Acute nicotine administration increased locomotor activity in rats. In the sub-chronic group, locomotor activity progressively increased and reached a clear sensitization. Significant effects of sensitization on CRF mRNA levels were detected in the DS (increasing effect). Significantly higher CRF1 and CRF2 receptor levels after sensitization were detected in the Hip. Additionally, CRF2 receptor levels were augmented by sensitization in the PFCx, and treatment and time-induced increases were detected in the DS. Nicotine treatment effects were observed on CRF1R levels in the DS. CONCLUSIONS: This study suggests that the CRF transmission, in addition to its role in increasing withdrawal-related anxiety, may be involved in the development of nicotine-habituated behaviours through reduced control of impulses and the aberrant memory plasticity characterising addiction.

Regulation of anxiety-like behavior and Crhr1 expression in the basolateral amygdala by LMO3.

The LIM domain only protein LMO3 is a transcriptional regulator that has been shown to regulate several behavioral responses to alcohol. Specifically, Lmo3 null (Lmo3Z) mice consume more ethanol in a binge-drinking test and show enhanced ethanol-induced sedation. Due to the high comorbidity of alcohol use and anxiety, we investigated anxiety-like behavior in Lmo3Z mice. Lmo3Z mice spent more time in the open arms of the elevated plus maze compared with their wild-type littermates, but the effect was confounded by reduced locomotor activity. To verify the anxiety phenotype in the Lmo3Z mice, we tested them for novelty-induced hypophagia and found that they also showed reduced anxiety in this test. We next explored the mechanism by which LMO3 might regulate anxiety by measuring mRNA and protein levels of corticotropin releasing factor (encoded by the Crh gene) and its receptor type 1 (Crhr1) in Lmo3Z mice. Reduced Crhr1 mRNA and protein was evident in the basolateral amygdala (BLA) of Lmo3Z mice. To examine whether Lmo3 in the amygdala is important for anxiety-like behavior, we locally reduced Lmo3 expression in the BLA of wild type mice using a lentiviral vector expressing a short hairpin RNA targeting the Lmo3 transcript. Mice with Lmo3 knockdown in the BLA exhibited decreased anxiety-like behavior relative to control mice. These results suggest that Lmo3 promotes anxiety-like behavior specifically in the BLA, possibly by altering Crhr1 expression. This study is the first to support a role for Lmo3 in anxiety-like behavior.

Targeted overexpression of CRH receptor subtype 1 in central amygdala neurons: effect on alcohol-seeking behavior.

RATIONALE: The corticotropin-releasing hormone (CRH) system is a key mediator of stress-induced responses in alcohol-seeking behavior. Recent research has identified the central nucleus of the amygdala (CeA), a brain region involved in the regulation of fear and stress-induced responses that is especially rich in CRH-positive neurons, as a key player in mediating excessive alcohol seeking. However, detailed characterization of the specific influences that local neuronal populations exert in mediating alcohol responses is hampered by current limitations in pharmacological and immunohistochemical tools for targeting CRH receptor subtype 1 (CRHR1). OBJECTIVE: In this study, we investigated the effect of cell- and region-specific overexpression of CRHR1 in the CeA using a novel transgenic tool. METHODS: Co-expression of CRHR1 in calcium-calmodulin-dependent kinase II (αCaMKII) neurons of the amygdala was demonstrated by double immunohistochemistry using a Crhr1-GFP reporter mouse line. A Cre-inducible Crhr1-expressing adeno-associated virus (AAV) was site-specifically injected into the CeA of αCaMKII-CreERT2 transgenic rats to analyze the role of CRHR1 in αCaMKII neurons on alcohol self-administration and reinstatement behavior. RESULTS: Forty-eight percent of CRHR1-containing cells showed co-expression of αCaMKII in the CeA. AAV-mediated gene transfer in αCaMKII neurons induced a 24-fold increase of Crhr1 mRNA in the CeA which had no effect on locomotor activity, alcohol self-administration, or cue-induced reinstatement. However, rats overexpressing Crhr1 in the CeA increased responding in the stress-induced reinstatement task with yohimbine serving as a pharmacological stressor. CONCLUSION: We demonstrate that CRHR1 overexpression in CeA-αCaMKII neurons is sufficient to mediate increased vulnerability to stress-triggered relapse into alcohol seeking.

The Effect of Acute and Repeated Stress on CRH-R1 and CRH-R2 mRNA Expression in Pituitaries of Wild Type and CRH Knock-Out Mice.

The activation of the HPA axis is the endocrine measure of stress responsiveness that is initiated by corticotropin-releasing hormone (CRH). CRH exerts its effects via CRHR1 and CRH-R2 receptors coupled to the cAMP signaling system and this process involves transcription factor cAMP-responsive element-binding protein (CREB).This study investigated the role of CRH and the possible involvement of CREB in gene regulation of CRH receptor, under basal conditions and after stress application in the pituitary. We used wild type (wt +/+) controls and CRH knock-out (CRH-KO -/-) male mice. Using CRH-deficient mice, we were able to investigate the consequences of the lack of the CRH on the expression of CRH receptors and transcriptional regulation mediated by CREB. We estimated the effect of acute (IMO 1×) and repeated (IMO 7×) restraint stressors lasting 30 and 120 min on the expression of mRNA CREB, CRH-R1, and CRH-R2 by qPCR. We found very significant difference in the expression of these peptides under the effect of single and repeated stress in control and CRH-KO mice. Our results indicate that both CRH receptors and CREB might be involved in the regulation of stress response in the pituitary of mice. We propose that regulation of the stress response may be better understood if more were known about the mechanisms of CRH receptor signal transduction and involvement of CREB system.

Inhibition of corticotropin-releasing hormone receptor 1 and activation of receptor 2 protect against colonic injury and promote epithelium repair.

Maternal separation (MS) in neonates can lead to intestinal injury. MS in neonatal mice disrupts mucosal morphology, induces colonic inflammation and increases trans-cellular permeability. Several studies indicate that intestinal epithelial stem cells are capable of initiating gut repair in a variety of injury models but have not been reported in MS. The pathophysiology of MS-induced gut injury and subsequent repair remains unclear, but communication between the brain and gut contribute to MS-induced colonic injury. Corticotropin-releasing hormone (CRH) is one of the mediators involved in the brain-gut axis response to MS-induced damage. We investigated the roles of the CRH receptors, CRHR1 and CRHR2, in MS-induced intestinal injury and subsequent repair. To distinguish their specific roles in mucosal injury, we selectively blocked CRHR1 and CRHR2 with pharmacological antagonists. Our results show that in response to MS, CRHR1 mediates gut injury by promoting intestinal inflammation, increasing gut permeability, altering intestinal morphology, and modulating the intestinal microbiota. In contrast, CRHR2 activates intestinal stem cells and is important for gut repair. Thus, selectively blocking CRHR1 and promoting CRHR2 activity could prevent the development of intestinal injuries and enhance repair in the neonatal period when there is increased risk of intestinal injury such as necrotizing enterocolitis.

miR-34b attenuates trauma-induced anxiety-like behavior by targeting CRHR1.

Exposure to trauma is a potential contributor to anxiety; however, the molecular mechanisms responsible for trauma-induced anxiety require further clarification. In this study, in an aim to explore these mechanisms, we observed the changes in the hypothalamic pituitary adrenal (HPA) axis using a radioimmunoassay and the changes in anxiety-like behavior using the open field test and elevated plus maze test in a rat model following intervention with NBI­27914, a specific corticotropin­releasing hormone receptor 1 (CRHR1) antagonist. CRHR1 was found to be involved in trauma­induced anxiety. We then applied bioinformatic analysis to screen microRNAs (miRNAs or miRs) that target CRHR1, and miR­34b was determined to negatively regulate CRHR1 mRNA in primary hypothalamic neurons. The overexpression of miR­34b in the paraventricular nucleus (PVN) by a miRNA agomir using a drug delivery system decreased the hyperactivity of the HPA axis and anxiety­like behavior. Overall, the involvement of the HPA axis in trauma­induced anxiety was demonstrated, and trauma-induced anxiety was attenuated by decreasing the hyperactivity of the HPA axis via miR­34b by targeting CRHR1.

Adolescent Chronic Unpredictable Stress Exposure Is a Sensitive Window for Long-Term Changes in Adult Behavior in Mice.

Adolescence is a time period in development when the brain undergoes substantial remodeling in response to the environment. To determine whether a stressful experience during adolescence affects adult behavior, we exposed adolescent male and female C57BL/6J mice to chronic unpredictable stress (CUS) for 12 days starting at postnatal day 28 (PND28). We also exposed adult male and female mice to CUS for 12 days beginning at PND70 to determine whether adolescence is a sensitive time period when stress can have long-lasting effects on behavior. Regardless of when mice were exposed to stress, they were all tested exactly 30 days later in the marble burying task, elevated zero maze, acoustic startle response, and forced swim test. Adolescent stress exposure increased anxiety-like behaviors in adult male and female mice and decreased acoustic startle response in a sex-dependent manner. However, adult stress exposure did not change anxiety or response to an acoustic tone in adult male or female mice as compared with nonstressed animals. Of interest, increased depression-like behavior in the forced swim test was observed in all mice, regardless of when the stress occurred. Gene expression analysis showed significant upregulation of corticotropin releasing factor receptor 2 (CrfR2) in the amygdala of males subjected to CUS during adolescence, but not in males that experienced CUS during adulthood. In contrast, females, regardless of when they were exposed to CUS, were not affected. These data support clinical evidence suggesting that early-life stress may predispose individuals to increased anxiety and depression later in life.

Lateral hypothalamus orexinergic system modulates the stress effect on pentylenetetrazol induced seizures through corticotropin releasing hormone receptor type 1.

Stress is a trigger factor for seizure initiation which activates hypothalamic pituitary adrenal (HPA) axis as well other brain areas. In this respect, corticotropin releasing hormone (CRH) and lateral hypothalamus (LH) orexinergic system are involved in seizure occurrence. In this study, we investigated the role of LH area and orexin expression in (mediation of) stress effect on pentylenetetrazol (PTZ) -induced seizures with hippocampal involvement. Two mild foot shock stresses were applied to intact and adrenalectomized animals; with or without CRHr1 blocking (NBI 27914) in the LH area. Then, changes in orexin production were evaluated by RT-PCR. Intravenous PTZ infusion (25 mg/ml) -induced convulsions were scored upon modified Racine scale. Finally, hippocampal glutamate and GABA were evaluated to study excitability changes. We demonstrated that the duration and severity of convulsions in stress-induced as well as adrenalectomized group were increased. Plasma corticosterone (CRT) level and orexin mRNA expression were built up in the stress and/or seizure groups. Furthermore, glutamate and GABA content was increased and decreased respectively due to stress and seizures. In contrast, rats receiving CRHr1 inhibitor showed reduced severity and duration of seizures, increased GABA, decreased glutamate and corticosterone and also orexin mRNA compared to the inhibitor free rats. Stress and adrenalectomy induced augmenting effect on seizure severity and duration and the subsequent reduction due to CRHr1 blocking with parallel orexin mRNA changes, indicated the likely involvement of CRH1r induced orexin expression of the LH in gating stress effect on convulsions.

Binge Drinking Decreases Corticotropin-Releasing Factor-Binding Protein Expression in the Medial Prefrontal Cortex of Mice.

BACKGROUND: Dysregulation of the corticotropin-releasing factor (CRF) system has been observed in rodent models of binge drinking, with a large focus on CRF receptor 1 (CRF-R1). The role of CRF-binding protein (CRF-BP), a key regulator of CRF activity, in binge drinking is less well understood. In humans, single-nucleotide polymorphisms in CRHBP are associated with alcohol use disorder and stress-induced alcohol craving, suggesting a role for CRF-BP in vulnerability to alcohol addiction. METHODS: The role and regulation of CRF-BP in binge drinking were examined in mice exposed to the drinking in the dark (DID) paradigm. Using in situ hybridization, the regulation of CRF-BP, CRF-R1, and CRF mRNA expression was determined in the stress and reward systems of C57BL/6J mice after repeated cycles of DID. To determine the functional role of CRF-BP in binge drinking, CRF-BP knockout (CRF-BP KO) mice were exposed to 6 cycles of DID, during which alcohol consumption was measured and compared to wild-type mice. RESULTS: CRF-BP mRNA expression was significantly decreased in the prelimbic (PL) and infralimbic medial prefrontal cortex (mPFC) of C57BL/6J mice after 3 cycles and in the PL mPFC after 6 cycles of DID. No significant changes in CRF or CRF-R1 mRNA levels were observed in mPFC, ventral tegmental area, bed nucleus of the stria terminalis, or amygdala after 3 cycles of DID. CRF-BP KO mice do not show significant alterations in drinking compared to wild-type mice across 6 cycles of DID. CONCLUSIONS: These results reveal that repeated cycles of binge drinking alter CRF-BP mRNA expression in the mPFC, a region responsible for executive function and regulation of emotion and behavior, including responses to stress. We observed a persistent decrease in CRF-BP mRNA expression in the mPFC after 3 and 6 DID cycles, which may allow for increased CRF signaling at CRF-R1 and contribute to excessive binge-like ethanol consumption.

Urocortins and CRF receptor type 2 variants in the male rat colon: gene expression and regulation by endotoxin and anti-inflammatory effect.

Urocortins (Ucns) 1, 2, and 3 and corticotropin-releasing factor receptor 2 (CRF2) mRNA are prominently expressed in various layers of the upper gut. We tested whether Ucns and CRF2 variants are also expressed in the different layers of the rat colon, regulated by LPS (100 µg/kg ip) and play a modulatory role in the colonic immune response to LPS. Transcripts of Ucns and CRF2b, the most common isoform in the periphery, were detected in all laser microdissected layers, including myenteric neurons. LPS increased the mRNA level of Ucn 1, Ucn 2, and Ucn 3 and decreased that of CRF2b in both the colonic mucosa and submucosa + muscle (S+M) layers at 2, 6, and 9 h after injection with a return to basal at 24 h. In addition, CRF2a, another variant more prominent in the brain, and a novel truncated splice variant CRF2a-3 mRNA were detected in all segments of the large intestine. LPS reciprocally regulated the colonic expression of these CRF2 variants by decreasing both CRF2a and CRF2b, while increasing CRF2a-3 in the mucosa and S+M. The CRF2 antagonist astressin2-B further enhanced LPS-induced increase of mRNA level of interleukin (IL)-1ß, TNF-α, and inducible nitric oxide synthase in S+M layers and IL-1ß in the mucosa and evoked TNF-α expression in the mucosa. These data indicate that Ucns/CRF2 variants are widely expressed in all colonic layers and reciprocally regulated by LPS. CRF2 signaling dampens the CD14/TLR4-mediated acute inflammatory response to Gram-negative bacteria in the colon.

CRF-R1 activation in the anterior-dorsal BNST induces maternal neglect in lactating rats via an HPA axis-independent central mechanism.

Adequate maternal behavior in rats requires minimal corticotropin-releasing factor receptor (CRF-R) activation in the medial-posterior bed nucleus of the stria terminalis (mpBNST). Based on the architectural heterogeneity of the BNST and its distinct inter-neural connectivity, we tested whether CRF-R manipulation in another functional part, the anterior-dorsal BNST (adBNST), differentially modulates maternal behavior. We demonstrate that in the adBNST, activation of CRF-R1 reduced arched back nursing (ABN) and nursing, whereas activation of CRF-R2 resulted in an initial reduction in nursing but significantly increased the incidence of ABN 5h after the treatment. Following stressor exposure, which is detrimental to maternal care, ABN tended to be protected by CRF-R1 blockade. Maternal motivation, maternal aggression, and anxiety were unaffected by any manipulation. Furthermore, under basal and stress conditions, activation of adBNST CRF-R1 increased plasma ACTH and corticosterone concentrations, whereas stimulation of adBNST CRF-R2 increased basal plasma ACTH and corticosterone concentrations, but blocked the stress-induced increase in plasma corticosterone secretion. Moreover, both the CRF-R1 and -R2 antagonists prevented the stress-induced increase in plasma corticosterone secretion. Importantly, elevated levels of circulating corticosterone induced by intra-adBNST administration of CRF-R1 or -R2 agonist did not impact maternal care. Finally, Crf mRNA expression in the adBNST was increased during lactation; however, Crfr1 mRNA expression was similar between lactating and virgin rats. In conclusion, maternal care is impaired by adBNST CRF-R1 activation, and this appears to be the result of a central action, rather than an effect of elevated circulating levels of CORT. These data provide new insights into potential causes of disturbed maternal behavior postpartum.

PKR deficiency alters E. coli-induced sickness behaviors but does not exacerbate neuroimmune responses or bacterial load.

BACKGROUND: Systemic inflammation induces neuroimmune activation, ultimately leading to sickness (e.g., fever, anorexia, motor impairments, exploratory deficits, and social withdrawal). In this study, we evaluated the role of protein kinase R (PKR), a serine-threonine kinase that can control systemic inflammation, on neuroimmune responses and sickness. METHODS: Wild-type (WT) PKR+/+ mice and PKR-/- mice were subcutaneously injected with live Escherichia coli (E. coli) or vehicle. Food consumption, rotarod test performance, burrowing, open field activity, object investigation, and social interaction were monitored. Plasma TNF-α and corticosterone were measured by ELISA. The percentage of neutrophils in blood was deduced from blood smears. Inflammatory gene expression (IL-1ß, TNF-α, IL-6, cyclooxygenase (COX)-2, iNOS) in the liver and the brain (hypothalamus and hippocampus) were quantified by real-time PCR. Blood and lavage fluid (injection site) were collected for microbiological plate count and for real-time PCR of bacterial 16S ribosomal DNA (rDNA). Corticotrophin-releasing hormone (CRH) expression in the hypothalamus was also determined by real-time PCR. RESULTS: Deficiency of PKR diminished peripheral inflammatory responses following E. coli challenge. However, while the core components of sickness (anorexia and motor impairments) were similar between both strains of mice, the behavioral components of sickness (reduced burrowing, exploratory activity deficits, and social withdrawal) were only observable in PKR-/- mice but not in WT mice. Such alteration of behavioral components was unlikely to be caused by exaggerated neuroimmune activation, by an impaired host defense to the infection, or due to a dysregulated corticosterone response, because both strains of mice displayed similar neuroimmune responses, bacterial titers, and plasma corticosterone profiles throughout the course of infection. Nevertheless, the induction of hypothalamic corticotrophin-releasing hormone (CRH) by E. coli was delayed in PKR-/- mice relative to WT mice, suggesting that PKR deficiency may postpone the CRH response during systemic inflammation. CONCLUSIONS: Taken together, our findings show that (1) loss of PKR could alter E. coli-induced sickness behaviors and (2) this was unlikely to be due to exacerbated neuroimmune activation, (3) elevated bacterial load, or (4) dysregulation in the corticosterone response. Further studies can address the role of PKR in the CRH response together with its consequence on sickness.

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.