Corticotropin-releasing Hormone System Research Articles

Response of CRH system in brain and gill of marine medaka to seawater acidification.

Corticotropin-releasing hormone (CRH) is mainly secreted by the hypothalamus to regulate stress when environmental factors change. Gills contact with water directly and may also secrete CRH to maintain local homeostasis. Ocean acidification changes water chemical parameters and is becoming an important environmental stressor for marine fish. The response of brain and gill CRH systems to ocean acidification remains unclear. In this study, marine medaka were exposed to CO2-acidified seawater (440ppm, 1000ppm, and 1800ppm CO2) for 2h, 4h, 24h, and 7 d, respectively. At 2h and 4h, the expression of crh mRNA in gills increased with increasing CO2 concentration. Crh protein is expressed mainly in the lamellae cells. crhbp and crhr1 expression also increased significantly. However, at 2h and 4h, acidification caused little changes in these genes and Crh protein expression in the brain. At 7 d, Crh-positive cells were detected in the hypothalamus; moreover, Crh protein expression in the whole brain increased. It is suggested that CRH autocrine secretion in gills is responsible for local acid-base regulation rather than systemic mobilization after short-term acidification stress, which may help the rapid regulation of body damage caused by environmental stress.

Two Adverse Early Life Events Induce Differential Changes in Brain CRH and Serotonin Systems in Rats along with Hyperphagia and Depression.

Different types of stress inflicted in early stages of life elevate the risk, among adult animals and humans, to develop disturbed emotional-associated behaviors, such as hyperphagia or depression. Early-life stressed (ELS) adults present hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis, which is a risk factor associated with mood disorders. However, the prevalence of hyperphagia (17%) and depression (50%) is variable among adults that experienced ELS, suggesting that the nature, intensity, and chronicity of the stress determines the specific behavioral alteration that those individuals develop. We analyzed corticosterone serum levels, Crh, GR, Crhr1 genes expression in the hypothalamic paraventricular nucleus, amygdala, and hippocampus due to their regulatory role on HPA axis in adult rats that experienced maternal separation (MS) or limited nesting material (LNM) stress; as well as the serotonergic system activity in the same regions given its association with the corticotropin-releasing hormone (CRH) pathway functioning and with the hyperphagia and depression development. Alterations in dams' maternal care provoked an unresponsive or hyper-responsive HPA axis function to an acute stress in MS and LNM adults, respectively. The differential changes in amygdala and hippocampal CRH system seemed compensating alterations to the hypothalamic desensitized glucocorticoids receptor (GR) in MS or hypersensitive in LNM. However, both adult animals developed hyperphagia and depression-like behavior when subjected to the forced-swimming test, which helps to understand that both hypo and hypercortisolemic patients present those disorders. Different ELS types induce neuroendocrine, brain CRH and 5-hydroxytriptamine (5-HT) systems' alterations that may interact converging to develop similar maladaptive behaviors.

Electroacupuncture intervention on stress-induced cardiac autonomic imbalance in rats involves corticotropin-releasing hormone system activity.

Autonomic imbalance is a core aspect of stress response that strongly correlates to cardiovascular diseases. Enhanced activity of the central corticotropin-releasing hormone (CRH) system may result in autonomic imbalance to cause cardiovascular responses in a stress state. Electroacupuncture at PC6 acupoints has been demonstrated to prevent and treat cardiovascular diseases. In this study, we aim to demonstrate the protective role of electroacupuncture at PC6 in ameliorating cardiac autonomic imbalance and investigate the underlying mechanisms in immobilization stress rats. Four groups were subjected. Immobilization stress was applied to three groups. And the rats in two electroacupuncture-intervened groups exerted electroacupuncture at PC6 or tail respectively. Then, we performed ECG recording for heart rate variability (HRV) analysis, and rats were sacrificed after experiments for biological analysis. HRV analysis indicated that electroacupuncture at PC6 improved the enhanced low-frequency band of the power spectrum (LF), the reduced high-frequency band of the power spectrum (HF), and the enhanced LF/HF ratio caused by immobilization stress. Besides, electroacupuncture at PC6 significantly decreased phosphorylated tyrosine hydroxylase expression and increased acetylcholine esterase expression in heart of immobilization stress rats. Furthermore, electroacupuncture at PC6 significantly decreased CRH level and CRH 1 type receptor and CRH 2 type receptor (CRHR2) expressions in the rostral ventrolateral medulla (RVLM), and CRH level and CRHR2 expression in the nucleus of the solitary tract (NTS) of immobilization stress rats. Our findings suggest that electroacupuncture at PC6 can ameliorate stress-induced cardiac autonomic imbalance by modulating the CRHergic input in the RVLM and NTS.

Sleep-Disturbance-Induced Microglial Activation Involves CRH-Mediated Galectin 3 and Autophagy Dysregulation.

Chronic sleep disturbances (CSDs) including insomnia, insufficient sleep time, and poor sleep quality are major public health concerns around the world, especially in developed countries. CSDs are major health risk factors linked to multiple neurodegenerative and neuropsychological diseases. It has been suggested that CSDs could activate microglia (Mg) leading to increased neuroinflammation levels, which ultimately lead to neuronal dysfunction. However, the detailed mechanisms underlying CSD-mediated microglial activation remain mostly unexplored. In this study, we used mice with three-weeks of sleep fragmentation (SF) to explore the underlying pathways responsible for Mg activation. Our results revealed that SF activates Mg in the hippocampus (HP) but not in the striatum and prefrontal cortex (PFc). SF increased the levels of corticotropin-releasing hormone (CRH) in the HP. In vitro mechanism studies revealed that CRH activation of Mg involves galectin 3 (Gal3) upregulation and autophagy dysregulation. CRH could disrupt lysosome membrane integrity resulting in lysosomal cathepsins leakage. CRHR2 blockage mitigated CRH-mediated effects on microglia in vitro. SF mice also show increased Gal3 levels and autophagy dysregulation in the HP compared to controls. Taken together, our results show that SF-mediated hippocampal Mg activation involves CRH mediated galectin 3 and autophagy dysregulation. These findings suggest that targeting the hippocampal CRH system might be a novel therapeutic approach to ameliorate CSD-mediated neuroinflammation and neurodegenerative diseases.

Different oxytocin and corticotropin-releasing hormone system changes in bipolar disorder and major depressive disorder patients.

SummaryBackgroundOxytocin (OXT) and corticotropin-releasing hormone (CRH) are both produced in hypothalamic paraventricular nucleus (PVN). Central CRH may cause depression-like symptoms, while peripheral higher OXT plasma levels were proposed to be a trait marker for bipolar disorder (BD). We aimed to investigate differential OXT and CRH expression in the PVN and their receptors in prefrontal cortex of major depressive disorder (MDD) and BD patients. In addition, we investigated mood-related changes by stimulating PVN-OXT in mice.MethodsQuantitative immunocytochemistry and in situ hybridization were performed in the PVN for OXT and CRH on 6 BD and 6 BD-controls, 9 MDD and 9 MDD-controls. mRNA expressions of their receptors (OXTR, CRHR1 and CRHR2) were determined in anterior cingulate cortex and dorsolateral prefrontal cortex (DLPFC) of 30 BD and 34 BD-controls, and 24 MDD and 12 MDD-controls. PVN of 41 OXT-cre mice was short- or long-term activated by chemogenetics, and mood-related behavior was compared with 26 controls.FindingsSignificantly increased OXT-immunoreactivity (ir), OXT-mRNA in PVN and increased OXTR-mRNA in DLPFC, together with increased ratios of OXT-ir/CRH-ir and OXTR-mRNA/CRHR-mRNA were observed in BD, at least in male BD patients, but not in MDD patients. PVN-OXT stimulation induced depression-like behaviors in male mice, and mixed depression/mania-like behaviors in female mice in a time-dependent way.InterpretationIncreased PVN-OXT and DLPFC-OXTR expression are characteristic for BD, at least for male BD patients. Stimulation of PVN-OXT neurons induced mood changes in mice, in a pattern different from BD.Funding10.13039/501100001809National Natural Science Foundation of China (81971268, 82101592).

Interactions between the κ opioid system, corticotropin-releasing hormone and oxytocin in partner loss.

Selective adult social attachments, or ‘pair bonds’, represent central relationships for individuals in a number of social species, including humans. Loss of a pair mate has emotional consequences that may or may not diminish over time, and that often translate into impaired psychological and physical health. In this paper, we review the literature on the neuroendocrine mechanisms for the emotional consequences of partner loss, with a special focus on hypothesized interactions between oxytocin, corticotropin-releasing hormone and the κ opioid system.This article is part of the theme issue ‘Interplays between oxytocin and other neuromodulators in shaping complex social behaviours’.

Amphetamine exposure alters behaviors, and neuronal and neurochemical activation in the brain of female prairie voles

Previous studies have shown that 3-day d-amphetamine (AMPH) treatment effectively induced conditioned place preferences (CPP) and impaired pair bonding behaviors in prairie voles (Microtus ochrogaster). Using this established animal model and treatment regimen, we examined the effects of the demonstrated threshold rewarding dose of AMPH on various behaviors and their potential underlying neurochemical systems in the brain of female prairie voles. Our data show that 3-day AMPH injections (0.2 mg/kg/day) impaired social recognition and decreased depressive-like behavior in females without affecting their locomotion and anxiety-like behaviors. AMPH treatment also decreased neuronal activation indicated by the labeling of the early growth response protein 1 (Egr-1) as well as the number of neurons double-labeled for Egr-1 and corticotrophin-releasing hormone (CRH) in the dentate gyrus (DG) of the hippocampus and paraventricular nucleus of the hypothalamus (PVN) in the brain. Further, AMPH treatment decreased the number of neurons double-labeled for Egr-1 and tyrosine hydroxylase (TH) but did not affect oxytocinergic neurons in the PVN or cell proliferation and neurogenesis markers in the DG. These data not only demonstrate potential roles of the brain CRH and dopamine systems in mediating disrupted social recognition and depressive-like behaviors by AMPH in female prairie voles, but also further confirm the utility of the prairie vole model for studying interactions between psychostimulants and social behaviors.

Corticotropin-Releasing Hormone Receptor Alters the Tumor Development and Growth in Apcmin/+ Mice and in a Chemically-Induced Model of Colon Cancer.

The neuroendocrine circuit of the corticotropin-releasing hormone (CRH) family peptides, via their cognate receptors CRHR1 and CRHR2, copes with psychological stress. However, peripheral effects of the CRH system in colon cancer remains elusive. Thus, we investigate the role of CRHR1 and CRHR2 in colon cancer. Human colon cancer biopsies were used to measure the mRNA levels of the CRH family by quantitative real-time PCR. Two animal models of colon cancer were used: Apcmin/+ mice and azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice. The mRNA levels of CRHR2 and UCN III are reduced in human colon cancer tissues compared to those of normal tissues. Crhr1 deletion suppresses the tumor development and growth in Apcmin/+ mice, while Crhr2 deficiency exacerbates the tumorigenicity. Crhr1 deficiency not only inhibits the expression of tumor-promoting cyclooxygenase 2, but also upregulates tumor-suppressing phospholipase A2 in Apcmin/+ mice; however, Crhr2 deficiency does not change these expressions. In the AOM/DSS model, Crhr2 deficiency worsens the tumorigenesis. In conclusion, Crhr1 deficiency confers tumor-suppressing effects in Apcmin/+ mice, but Crhr2 deficiency worsens the tumorigenicity in both Apcmin/+ and AOM/DSS-treated mice. Therefore, pharmacological inhibitors of CRHR1 or activators of CRHR2 could be of significance as anti-colon cancer drugs.

Brain corticotropin releasing hormone and stress reactivity

The hypothalamic-pituitary-adrenocortical axis is one of the main components of stress adaptation. Corticotropin-releasing hormone (CRH) coming from the nucleus paraventricularis hypothalami (PVN) is the canonical central regulator of the axis. This CRH acts on the CRH-R1 receptors of the pituitary, and, through adrenocorticotropin, stimulates glucocorticoid release from the adrenal cortex. However, it may be synthetized in other parts of the brain as well, and may act both on CRH-R1 and CRH-R2 receptors. These areas form the central CRH network. Many of them are also stress reactive and participate in physical and psychological stress response. The central nucleus of the amygdala and bed nucleus of stria terminalis are two areas best known for their role in emotions, while hippocampus is mostly involved in glucocorticoid feedback as well as memory formation, all heavily connected to stress adaptation. Among others, the brainstem raphe nuclei get dense CRHergic innervation that, through CRH-R1 receptors, may influence the serotoninergic tone of the brain. Both stress and serotonin are strongly implicated in depression, therefore, it is not surprising that CRH-R1 antagonists were developed as therapeutic tools that extensively act on the brain CRH system. Our review suggests a general role of brain CRH network in stress adaptation which is not restricted to PVN.

Chapter 9 - The hypothalamus in anxiety disorders

Of all mental disorders, anxiety disorders are currently the strongest contributors to the global burden of disease, with 7.3% of the general population affected worldwide. The hypothalamus is crucial hub of a network of neural structures modulating fear conditioning and extinction and, as such, highly relevant to the pathophysiology of these conditions. Three hypothalamic systems have emerged as particularly relevant in this context. First, the oxytocin system is highly likely to be involved anxiety disorders and in particular in the cognitive and behavioral deficits pertaining to social anxiety disorder. Second, peripheral markers of the hypothalamic-pituitary-adrenal axis appear altered in patients with panic disorder and generalized anxiety disorder, which may denote aberrant functioning of their central corticotropin-releasing hormone system. Furthermore, cortisol seems to augment the effects of exposure therapy in patients with specific phobia. Third, the integrity of the hypothalamic-pituitary-thyroid axis is likely compromised in panic disorder. Further, cross-disciplinary research efforts are required to shed more light on how, exactly, these hypothalamic systems interact with the neural structures involved in fear conditioning and extinction, which should ultimately open up new avenues for the prevention and treatment of anxiety disorders.

Intracerebroventricularly injected nesfatin-1 activates central cyclooxygenase and lipoxygenase pathways.

Nesfatin-1 is a multifunctional neuropeptide having crucial autonomic roles. It is well known that nesfatin-1 collaborates with other central neuromodulatory systems, such as central corticotropin-releasing hormone, melanocortin, oxytocin, and cholinergic systems to show its autonomic effects. Central arachidonic acid cascade plays an important role to provide the homeostasis by exhibiting similar autonomic effects to nesfatin-1. Based on these similarities, the current study was designed to show the effects of intracerebroventricularly (ICV) injected nesfatin-1 on the hypothalamic arachidonic acid (AA) cascade. Immunochemistry and western blot approaches demonstrated that ICV administration of nesfatin-1 provokes an increase in the hypothalamic cyclooxygenase (COX) -1, -2 and lipoxygenase (LOX) protein expression. Moreover, the microdialysis study demonstrated that centrally injected nesfatin-1 increased the posterior hypothalamic extracellular AA products. In conclusion, these findings report that while nesfatin-1 is generating its autonomic effects, it also might be using central prostaglandins and leukotrienes by activating central COX and LOX pathways.

Activation of corticotropin-releasing hormone system in the locus ceruleus induces the visceral sensitivity of irritable bowel syndrome rats

Objective To study the role of corticotropin-releasing hormone (CRH) system in locus ceruleus (LC) in irritable bowel syndrome (IBS) and to explore its molecular mechanism. Methods The IBS rat was established by maternal separation following with postnatal stress. The tissues sample of LC was obtained by micropunched nuclei. The expression of c-Fos, CRH and its receptors including corticotropin-releasing hormone receptor (CRHR) 1 and CRHR2 of rats’ LC tissues of control group and IBS group was detected by quantitative polymerase chain reaction(PCR). The expression of DNA methyltransferase (DNTM) 1, DNMT3a and DNMT3b at the mRNA level were also measured. In addition, the expression of histone methyltransferase ASH2-like protein (ASH2L) and SET and MYND domain containing 2 (SMYD2) was determined by Western blotting. T test was used for statistical analysis. Results The rectal pneumatic pressure of IBS group was lower than that of control group ((69.82±5.47) mmHg vs. (86.86±5.98) mmHg; 1 mmHg=0.133 kPa), however compared with that of control group, the expression of c-Fos at the mRNA level increased (2.11±0.44 vs.1.00±0.19), and the differences were statistically significant (t=6.215 and 2.321, P 0.05). Conclusions Maternal separation combined with postnatal stress affect the transcription of Crh gene in LC and cause the activation of the stress regulation network CRH and norepinephrine system, resulting in the increase of the visceral sensitivity of rats. The abnormal transcription of Crh gene may be related with SMYD2-mediated histone H3K36 methylation, but not related with the modification of DNA methylation. Key words: Irritable bowel syndrome; Locus ceruleus; Maternal separation stress; Corticotropin-releasing hormone system; Epigenetics

Imaging Brain Mechanisms of Functional Somatic Syndromes: Potential as a Biomarker?

When patients present with persistent bodily complaints that cannot be explained by a symptom-linked organic pathology (medically unexplained symptoms), they are diagnosed with 'functional' somatic syndromes (FSS). Despite their prevalence, the management of FSS is notoriously challenging in clinical practice. This may be because FSS are heterogeneous disorders in terms of etiopathogenesis. They include patients with primarily peripheral dysfunction, primarily centrally driven somatic symptoms, and a mix of both. Brain-imaging studies, particularly data-driven pattern recognition methods using machine learning algorithms, could provide brain-based biomarkers for these clinical conditions. In this review, we provide an overview of our brain imaging data on brain-body interactions in one of the most well-known FSS, irritable bowel syndrome (IBS), and discuss the possible development of a brain-based biomarker for FSS. Anticipation of unpredictable pain, which commonly elicits fear in FSS patients, induced increased activity in brain areas associated with hypervigilance during rectal distention and non-distention conditions in IBS. This was coupled with dysfunctional inhibitory influence of the medial prefrontal cortex (mPFC) and pregenual anterior cingulate cortex (pACC) on stress regulation systems, resulting in the activated autonomic nervous system (ANS) and neuroendocrine system stimulated by corticotropin-releasing hormone (CRH). IBS subjects with higher alexithymia, a risk factor for FSS, showed stronger activity in the insula during rectal distention but reduced subjective sensitivity. Reduced top-down regulation of the ANS and CRH system by mPFC and pACC, discordance between the insula response to stimulation and subjective sensation of pain, and stronger threat responses in hypervigilance-related areas may be a candidate brain-based biomarker.

Cancer-Specific Loss of Urocortin3 in Human Renal Cancer.

The corticotropin-releasing hormone (CRH) system, its receptors corticotropin-releasing hormone receptor 1 (CRHR1) and 2 (CRHR2), and its corresponding binding protein corticotropin-releasing hormone-binding protein (CRHBP) as well as the urocortin proteins-structural homologues to CRH, which are included in this peptide family-have become interesting oncological targets recently. Carcinogenesis of various human tumors has been reported with an altered presence of members of this system. The aim of the present study was to examine the role of urocortin3 (UCN3) in renal cell carcinoma (RCC). Therefore, tumoral tissues of 106 patients with RCC and available corresponding normal tissues were analyzed using qPCR for quantitative mRNA expression analysis. Tissue localization and protein signals of UCN3 in normal and tumoral renal specimens were evaluated using western blot and immunohistochemistry. In addition, correlation studies of UCN3 mRNA expression with clinicopathological parameters of patients with RCC and different histological subtypes were evaluated. UCN3 mRNA was significantly downregulated in nearly all tumoral tissues (p = 7.92 × 10-13). The same effect was observed at protein level using immunohistochemistry. Level of UCN3 mRNA expression was not directly correlated with clinicopathological parameters. We report for the first time the significant downregulation of UCN3 in RCC. These results demonstrate a possible involvement of the CRH system and its significance in carcinogenesis of RCC.

Deletion of CRH From GABAergic Forebrain Neurons Promotes Stress Resilience and Dampens Stress-Induced Changes in Neuronal Activity.

Dysregulation of the corticotropin-releasing hormone (CRH) system has been implicated in stress-related psychopathologies such as depression and anxiety. Although most studies have linked CRH/CRH receptor 1 signaling to aversive, stress-like behavior, recent work has revealed a crucial role for distinct CRH circuits in maintaining positive emotional valence and appetitive responses under baseline conditions. Here we addressed whether deletion of CRH, specifically from GABAergic forebrain neurons (CrhCKO–GABA mice) differentially affects general behavior under baseline and chronic stress conditions. Expression mapping in CrhCKO–GABA mice revealed absence of Crh in GABAergic neurons of the cortex and limbic regions including the hippocampus, central nucleus of the amygdala and the bed nucleus of the stria terminals, but not in the paraventricular nucleus of hypothalamus. Consequently, conditional CRH knockout animals exhibited no alterations in circadian and stress-induced corticosterone release compared to controls. Under baseline conditions, absence of Crh from forebrain GABAergic neurons resulted in social interaction deficits but had no effect on other behavioral measures including locomotion, anxiety, immobility in the forced swim test, acoustic startle response and fear conditioning. Interestingly, following exposure to chronic social defeat stress, CrhCKO–GABA mice displayed a resilient phenotype, which was accompanied by a dampened, stress-induced expression of immediate early genes c-fos and zif268 in several brain regions. Collectively our data reveals the requirement of GABAergic CRH circuits in maintaining appropriate social behavior in naïve animals and further supports the ability of CRH to promote divergent behavioral states under baseline and severe stress conditions.

Molecular evolution of CRH and CRHR subfamily before the evolutionary origin of vertebrate

Corticotropin-releasing hormone (CRH) is well-cited for its important role in governing the stress responses via neuroendocrine system in vertebrates. After the identification of homologs of CRH receptor (CRHR) in both deuterostome and arthropod lineages, it was suggested that the ancestral homolog of CRH-CRHR molecular system is present in the bilaterian. However, homolog sequences from arthropods differ considerably from vertebrate CRH-like peptide sequences. Due to the significant difference between the biological system, as well as the gene regulatory network, of protostome and that of vertebrate, physiological studies on the protostomes may not provide important insight into the evolutionary history of vertebrate CRH system, while tunicate and amphioxus, two close relatives to vertebrate, which have diverged before two rounds of whole genome duplication (2WGDs) do. Given the identification of amphioxus CRH-like peptide by our group, this review aims to reexamine the current hypotheses on the evolution of CRH subfamily. It is generally accepted that paralogs of CRH and CRHR have been produced through 2WGDs, which occurred during the early vertebrate evolution. The identification of a single crh-like gene in amphioxi and tunicates by in silico search and the presence of two paralogons with a total of 5 crh-like genes in gnathostomes has shown that an additional duplication event might have happened to the ancestral crh-like gene before 2WGDs. On the other hand, the evolution of crhr gene subfamily appears to be mainly influenced by 2WGDs and only two receptor genes have been retained in the genomes of jawed vertebrates.

Microbiota affects the expression of genes involved in HPA axis regulation and local metabolism of glucocorticoids in chronic psychosocial stress

The commensal microbiota affects brain functioning, emotional behavior and ACTH and corticosterone responses to acute stress. However, little is known about the role of the microbiota in shaping the chronic stress response in the peripheral components of the hypothalamus-pituitary-adrenocortical (HPA) axis and in the colon. Here, we studied the effects of the chronic stress-microbiota interaction on HPA axis activity and on the expression of colonic corticotropin-releasing hormone (CRH) system, cytokines and 11β-hydroxysteroid dehydrogenase type 1 (11HSD1), an enzyme that determines locally produced glucocorticoids. Using specific pathogen-free (SPF) and germ-free (GF) BALB/c mice, we showed that the microbiota modulates emotional behavior in social conflicts and the response of the HPA axis, colon and mesenteric lymph nodes (MLN) to chronic psychosocial stress. In the pituitary gland, microbiota attenuated the expression of Fkbp5, a gene regulating glucocorticoid receptor sensitivity, while in the adrenal gland, it attenuated the expression of genes encoding steroidogenesis (MC2R, StaR, Cyp11a1) and catecholamine synthesis (TH, PNMT). The pituitary expression of CRH receptor type 1 (CRHR1) and of proopiomelanocortin was not influenced by microbiota. In the colon, the microbiota attenuated the expression of 11HSD1, CRH, urocortin UCN2 and its receptor, CRHR2, but potentiated the expression of cytokines TNFα, IFNγ, IL-4, IL-5, IL-6, IL-10, IL-13 and IL-17, with the exception of IL-1β. Compared to GF mice, chronic stress upregulated in SPF animals the expression of pituitary Fkbp5 and colonic CRH and UCN2 and downregulated the expression of colonic cytokines. Differences in the stress responses of both GF and SPF animals were also observed when immunophenotype of MLN cells and their secretion of cytokines were analyzed. The data suggest that the presence of microbiota/intestinal commensals plays an important role in shaping the response of peripheral tissues to stress and indicates possible pathways by which the environment can interact with glucocorticoid signaling.

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

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). In this study, we investigated the effect of cell- and region-specific overexpression of CRHR1 in the CeA using a novel transgenic tool. 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. 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. We demonstrate that CRHR1 overexpression in CeA-αCaMKII neurons is sufficient to mediate increased vulnerability to stress-triggered relapse into alcohol seeking.

Dissociable Role of Corticotropin Releasing Hormone Receptor Subtype 1 on Dopaminergic and D1 Dopaminoceptive Neurons in Cocaine Seeking Behavior.

The ability of many drugs of abuse, including cocaine, to mediate reinforcement and drug-seeking behaviors is in part mediated by the corticotropin-releasing hormone (CRH) system, in which CRH exerts its effects partly via the CRH receptor subtype 1 (CRHR1) in extra-hypothalamic areas. In fact, CRHR1 expressed in regions of the mesolimbic dopamine (DA) system have been demonstrated to modify cocaine-induced DA release and alter cocaine-mediated behaviors. Here we examined the role of neuronal selectivity of CRHR1 within the mesolimbic system on cocaine-induced behaviors. First we used a transgenic mouse line expressing GFP under the control of the Crhr1 promoter for double fluorescence immunohistochemistry to demonstrate the cellular location of CRHR1 in both dopaminergic and D1 dopaminoceptive neurons. We then studied cocaine sensitization, self-administration, and reinstatement in inducible CRHR1 knockouts using the CreERT2/loxP in either dopamine transporter (DAT)-containing neurons (DAT-Crhr1) or dopamine receptor 1 (D1)-containing neurons (D1-Crhr1). For sensitization testing, mice received five daily injections of cocaine (15 mg/kg IP). For self-administration, mice received eight daily 2 h cocaine (0.5 mg/kg per infusion) self-administration sessions followed by extinction and reinstatement testing. There were no differences in the acute or sensitized locomotor response to cocaine in DAT-Crhr1 or D1-Crhr1 mice and their respective controls. Furthermore, both DAT-Crhr1 and D1-Crhr1 mice reliably self-administered cocaine at the level of controls. However, DAT-Crhr1 mice demonstrated a significant increase in cue-induced reinstatement relative to controls, whereas D1-Crhr1 mice demonstrated a significant decrease in cue-induced reinstatement relative to controls. These data demonstrate the involvement of CRHR1 in cue-induced reinstatement following cocaine self-administration, and implicate a bi-directional role of CRHR1 for cocaine craving.

Changes in Gene Expression in the Locus Coeruleus-Amygdala Circuitry in Inhibitory Avoidance PTSD Model.

The locus coeruleus (LC)-amygdala circuit is implicated in playing a key role in responses to emotionally arousing stimuli and in the manifestation of post-traumatic stress disorder (PTSD). Here, we examined changes in gene expression of a number of important mediators of the LC-amygdala circuitry in the inhibition avoidance model of PTSD. After testing for basal acoustic startle response (ASR), rats were exposed to a severe footshock (1.5mA for 10s) in the inhibitory avoidance apparatus. They were given contextual situational reminders every 5day for 25days. Controls were treated identically but with the footshock inactivated. Animals were re-tested on second ASR and decapitated 1h later. The shock group had enhanced hyperarousal and several changes in gene expression compared to controls. In the LC, mRNA levels of norepinephrine (NE) biosynthetic enzymes (TH, DBH), NE transporter (NET), NPY receptors (Y1R, Y2R), and CB1 receptor of endocannabinoid system were elevated. In the basolateral amygdala (BLA), there were marked reductions in gene expression for CB1, and especially Y1R, with rise for corticotropin-releasing hormone (CRH) system (CRH, CRH receptor 1), and no significant changes in the central amygdala. Our results suggest a fast forward mechanism in the LC-amygdala circuitry in the shock group.