Corticotropin-releasing Factor Receptor Research Articles

Hypothalamic mechanisms associated with corticotropin-releasing factor-induced anorexia in chicks

Central administration of corticotropin-releasing factor (CRF), a 41-amino acid peptide, is associated with potent anorexigenic effects in rodents and chickens. However, the mechanism underlying this effect remains unclear. Hence, the objective of the current study was to elucidate the hypothalamic mechanisms that mediate CRF-induced anorexia in 4 day-old Cobb-500 chicks. After intracerebroventricular (ICV) injection of 0.02 nmol of CRF, CRF-injected chicks ate less than vehicle chicks while no effect on water intake was observed at 30 min post-injection. In subsequent experiments, the hypothalamus samples were processed at 60 min post-injection. The CRF-injected chicks had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), dorsomedial nucleus (DMN), ventromedial hypothalamus (VMH), and paraventricular nucleus (PVN) of the hypothalamus than vehicle-treated chicks. CRF injection was associated with decreased whole hypothalamic mRNA abundance of neuropeptide Y receptor sub-type 1 (NPYR1). In the ARC, CRF-injected chicks expressed more CRF and CRF receptor sub-type 2 (CRFR2) mRNA but less agouti-related peptide (AgRP), NPY, and NPYR1 mRNA than vehicle-injected chicks. CRF-treated chicks expressed greater amounts of CRFR2 and mesotocin mRNA than vehicle chicks in the PVN and VMH, respectively. In the DMN, CRF injection was associated with reduced NPYR1 mRNA. In conclusion, the results provide insights into understanding CRF-induced hypothalamic actions and suggest that the anorexigenic effect of CRF involves increased CRFR2-mediated signaling in the ARC and PVN that overrides the effects of NPY and other orexigenic factors.

Probing the Interactome of Corticotropin-Releasing Factor Receptor Heteromers Using Mass Spectrometry.

Mass spectrometry is a sensitive technique used in the field of proteomics that allows for simultaneous detection and characterization of several proteins in a sample. It is also a powerful methodology to elucidate protein-protein interactions in a sequence-dependent and unbiased manner. G protein-coupled receptors (GPCRs) seldom function in isolation and characterization of proteins present in the receptor complex (or its interactome) is critical for understanding the vast spectrum of functions these receptors perform in a context-dependent manner. Here, we describe a mass spectrometry-based method to sequence and characterize proteins present in heteromeric complexes formed by corticotropin-releasing factor (CRF) receptors that belong to class B GPCRs. CRF receptor heteromeric complexes were identified in HEK293 cells co-transfected with tagged CRF receptors 1 and 2. CRF receptors were immunoprecipitated using antibodies against the tags from transfected HEK293 cells and proteins in their interactome were identified using liquid chromatography mass spectrometry method (LC-MS/MS). Both CRF receptors were identified in this interactome. A few of the proteins identified in the CRF receptor interactome using MS were confirmed to be true interactions using traditional co-immunoprecipitation and Western blotting methods.

Small-Molecule Ligands as Challenge for Positron Emission Tomography of Peptide Receptors in Neurons and Microglia of the Brain

Neuropeptide and chemokine receptors of the G protein-coupled receptor (GPCR) family belong to different classes and subgroups providing different docking sites and special binding behavior at extracellular and also transmembrane domains for small molecules potentially suitable for positron emission tomography (PET). The contribution gives an overview updating developments of small-molecule, nonpeptide ligands at a selection of peptide and chemokine receptors, expressed in neurons and microglia of the brain, regarding the last five years. Orexin 1 and orexin 2 receptors (OX1R; OX2R) and neuropeptide Y1 and Y2 receptors (NPY1R, NPY2R) were chosen as representatives of Class A neuropeptide receptors, chemokine receptor CX3C (CX3CR1) as Class A, protein-activated receptor, highly expressed in activated microglia, and corticotropin releasing factor receptor 1 (CRFR1) as representative Class B1 receptor. Structural differences between binding domains and their endogenous ligands as well as parallel expression in different types of cells and generally low density of these receptors in brain tissue are factors making the search for selective and sensitive ligands more difficult than for classical GPCR receptors. Main progress in ligand development is observed for NPY receptor antagonists and orexin receptor antagonists. For orexin receptors, search for suitable ligands can be supported with modelling approaches, as recently the complete molecular structure of these receptors is available. Small molecules, binding at CRFR1, as for other Class B1 receptor ligands, in PET and investigations of pharmacodynamics revealed rather allosteric binding modes, although, the complete crystal structure of CRFR1 as prototype of Class B1 provides, hitherto, improved possibilities for understanding binding mechanisms. Highly specific as a marker of microglia among the GPCRs, CX3CR1 is focused as target of PET during inflammation of brain and spinal cord.

CRF1/CRF2 and MC3/MC4 Receptors Affect Glutamate- Induced Food Intake in Neonatal Meat-Type Chicken

ABSTRACT Central glutamate, melanocortin and corticotropin systems have mediatory role on several physiologic functions in the brain, but their interactions on appetite regulation are not fully elicited. So, the aim of the current study was to determine interaction of the glutamate with melanocortin and corticotropin systems on food intake in 3-h food-deprived (FD3) neonatal meat-type chicken. In experiment 1, chicken intracerebroventricular (ICV) injected (A) phosphate-buffered saline (PBS), (B) glutamate (75 nmol), (C) glutamate (150 nmol) and (D) glutamate (300 nmol). In experiment 2, (A) PBS, (B) astressin-B (CRF1/CRF2 receptors antagonist, 30 µg), (C) glutamate (300 nmol) and (D) astressin-B+glutamate were ICV injected. Experiments 3-5 were similar to experiment 2, except birds were injected with astressin2-B (CRF2 receptor antagonist, 30 µg), SHU9119 (MC3/MC4 receptor antagonist, 0.5 nmol) and MCL0020 (MC4 receptor antagonist, 0.5 nmol) instead of the astressin-B. In experiment 6, the injections were (A) PBS, (B) MTII (MC3/MC4 receptor agonist, 2.5ng), (C) glutamate (75nmol) and (D) MTII+glutamate. Then, cumulative feed intake was recorded at 30, 60 and 120 minutes after injection. According to the results, dose dependent hypophagia observed by ICV injection of the glutamate (75, 150 and 300nmol) compared to control group in neonatal broiler chicken (p<0.05). Co-injection of the astressin-B+glutamate and astressin2-B+glutamate decreased glutamate-induced hypophagia in neonatal broiler chicken (p<0.05). Co-injection of the glutamate+MC3/MC4 receptors antagonist decreased hypophagic effect of the glutamate (p<0.05). These results suggested hypophagic effect of the glutamate mediates via CRF1/CRF2 and MC3/MC4 receptors in chickens.

Characterization and gonadal hormone regulation of a sexually dimorphic corticotropin‐releasing factor receptor 1 cell group

Corticotropin-releasing factor binds with high affinity to CRF receptor 1 (CRFR1) and is implicated in stress-related mood disorders such as anxiety and depression. Using a validated CRFR1-green fluorescent protein (GFP) reporter mouse, our laboratory recently discovered a nucleus of CRFR1 expressing cells that is prominent in the female rostral anteroventral periventricular nucleus (AVPV/PeN), but largely absent in males. This sex difference is present in the early postnatal period and remains dimorphic into adulthood. The present investigation sought to characterize the chemical composition and gonadal hormone regulation of these sexually dimorphic CRFR1 cells using immunohistochemical procedures. We report that CRFR1-GFP-ir cells within the female AVPV/PeN are largely distinct from other dimorphic cell populations (kisspeptin, tyrosine hydroxylase). However, CRFR1-GFP-ir cells within the AVPV/PeN highly co-express estrogen receptor alpha as well as glucocorticoid receptor. A single injection of testosterone propionate or estradiol benzoate on the day of birth completely eliminates the AVPV/PeN sex difference, whereas adult gonadectomy has no effect on CRFR1-GFP cell number. These results indicate that the AVPV/PeN CRFR1 is regulated by perinatal but not adult gonadal hormones. Finally, female AVPV/PeN CRFR1-GFP-ir cells are activated following an acute 30-min restraint stress, as assessed by co-localization of CRFR1-GFP cells with phosphorylated (p) CREB. CRFR1-GFP/pCREB cells were largely absent in the male AVPV/PeN. Together, these data indicate a stress and gonadal hormone responsive nucleus that is unique to females and may contribute to sex-specific stress responses.

Computational investigation of binding mechanism of substituted pyrazinones targeting corticotropin releasing factor-1 receptor deliberated for anti-depressant drug design

In spite of various research investigations towards anti-depressant drug discovery program, no one drug has not yet launched last 20 years. Corticotropin-releasing factor-1 (CRF-1) is one of the most validated targets for the development of antagonists against depression, anxiety and post-traumatic stress disorders. Various research studies suggest that pyrazinone based CRF-1 receptor antagonists were found to be highly potent and efficacious. In this research investigation, we identified the pharmacophore and binding pattern through 2D and 3D-QSAR and molecular docking respectively. Molecular dynamics studies were also performed to explore the binding pattern recognition. We establish the relationship between activity and pharmacophoric features to design new potent compounds. The best 2D-QSAR model was generated through multiple linear regression method with r2 value of 0.97 and q2 value of 0.89. Also 3D-QSAR model was obtained through k-nearest neighbor molecular field analysis method with q2 value of 0.52 and q2_se value of 0.36. Molecular docking and binding energy were also evaluated to define binding patterns and pharmacophoric groups, including (i) hydrogen bond with residue Asp284, Glu305 and (ii) π–π stacking with residue Trp9. Compound 11i has the highest binding affinity compared to reference compounds, so this compound could be a potent drug for stress related disorders. Most of the compounds, including reference compounds were found within acceptable range of physicochemical parameters. These observations could be provided the leads for the design and optimization of novel CRF-1 receptor antagonists.Communicated by Ramaswamy H. Sarma

Interaction Between Stress and Addiction: Contributions From Latin-American Neuroscience.

Drug addiction is a chronic neuropsychiatric disorder that escalates from an initial exposure to drugs of abuse, such as cocaine, cannabis, or heroin, to compulsive drug-seeking and intake, reduced ability to inhibit craving-induced behaviors, and repeated cycles of abstinence and relapse. It is well-known that chronic changes in the brain’s reward system play an important role in the neurobiology of addiction. Notably, environmental factors such as acute or chronic stress affect this system, and increase the risk for drug consumption and relapse. Indeed, the HPA axis, the autonomic nervous system, and the extended amygdala, among other brain stress systems, interact with the brain’s reward circuit involved in addictive behaviors. There has been a growing interest in studying the molecular, cellular, and behavioral mechanisms of stress and addiction in Latin-America over the last decade. Nonetheless, these contributions may not be as strongly acknowledged by the broad scientific audience as studies coming from developed countries. In this review, we compile for the first time a series of studies conducted by Latin American-based neuroscientists, who have devoted their careers to studying the interaction between stress and addiction, from a neurobiological and clinical perspective. Specific contributions about this interaction include the study of CRF receptors in the lateral septum, investigations on the neural mechanisms of cross-sensitization for psychostimulants and ethanol, the identification of the Wnt/β-catenin pathway as a critical neural substrate for stress and addiction, and the emergence of the cannabinoid system as a promising therapeutic target. We highlight animal and human studies, including for instance, reports coming from Latin American laboratories on single nucleotide polymorphisms in stress-related genes and potential biomarkers of vulnerability to addiction, that aim to bridge the knowledge from basic science to clinical research.

A Corticotropin Releasing Factor Network in the Extended Amygdala for Anxiety.

The central amygdala (CeA) is important for fear responses to discrete cues. Recent findings indicate that the CeA also contributes to states of sustained apprehension that characterize anxiety, although little is known about the neural circuitry involved. The stress neuropeptide corticotropin releasing factor (CRF) is anxiogenic and is produced by subpopulations of neurons in the lateral CeA and the dorsolateral bed nucleus of the stria terminalis (dlBST). Here we investigated the function of these CRF neurons in stress-induced anxiety using chemogenetics in male rats that express Cre recombinase from a Crh promoter. Anxiety-like behavior was mediated by CRF projections from the CeA to the dlBST and depended on activation of CRF1 receptors and CRF neurons within the dlBST. Our findings identify a CRFCeA→CRFdlBST circuit for generating anxiety-like behavior and provide mechanistic support for recent human and primate data suggesting that the CeA and BST act together to generate states of anxiety.SIGNIFICANCE STATEMENT Anxiety is a negative emotional state critical to survival, but persistent, exaggerated apprehension causes substantial morbidity. Identifying brain regions and neurotransmitter systems that drive anxiety can help in developing effective treatment. Much evidence in rodents indicates that neurons in the bed nucleus of the stria terminalis (BST) generate anxiety-like behaviors, but more recent findings also implicate neurons of the CeA. The neuronal subpopulations and circuitry that generate anxiety are currently subjects of intense investigation. Here we show that CeA neurons that release the stress neuropeptide corticotropin-releasing factor (CRF) drive anxiety-like behaviors in rats via a pathway to dorsal BST that activates local BST CRF neurons. Thus, our findings identify a CeA→BST CRF neuropeptide circuit that generates anxiety-like behavior.

Morphine exposure before conception affects anxiety-like behavior and CRF level (in the CSF and plasma) in the adult male offspring

It has been proven that exposure to some drugs even before gestation had transgenerational effects. To investigate the changes which induced by parental morphine exposure before gestation; mainly the anxiety-like behavior, Corticotropin Releasing Factor (CRF) level in the CSF and plasma, CRF Receptor 1 (CRFR1), and the level of protein kinase C (PKC-α) were evaluated in the male offspring.Male and female Wistar rats were exposed to morphine for 21 following days. Ten days after last drug exposure, animals were prepared for mating in 4 distinct groups as follow: drug-naïve female and male (used as control), drug-naïve female and morphine-abstinent male, drug-naïve male and morphine-abstinent female, and morphine abstinent male and female. Offspring were subjected to assess anxiety-like behavior (using elevated plus maze test). CSF and plasma were gathered, and the CRF level was evaluated by ELISA. Using real-time PCR, the CRFR1 level in the brain was evaluated.Results showed that anxiety-like behavior increased in the offspring of morphine-abstinent parent(s) compared with the control group. CRF level in the plasma and CSF also increased in the litter of morphine-abstinent parent(s). CRFR1 mRNA level was upregulated in the brain of offspring with one and/or two morphine-abstinent parent(s). Furthermore, the level of PKC-α was decreased in the brain of offspring which had one and/or two morphine-abstinent parent(s).Taken together, our findings indicated that morphine exposure even before gestation induced transgenerational effects via dysregulation of HPA axis which results in anxiety in the adult male offspring.

Predator odor increases avoidance and glutamatergic synaptic transmission in the prelimbic cortex via corticotropin-releasing factor receptor 1 signaling.

Acute exposure to a salient stressor, such as in post-traumatic stress disorder, can have lasting impacts upon an individual and society. To study stress in rodents, some naturalistic methods have included acute exposure to a predator odor, such as the synthetic fox odor 2,4,5, trimethyl-3-thiazoline (TMT). These experiments explore the stress-related behaviors and cortical activity induced by TMT exposure in adult male C57BL/6J mice and the influence of the stress neuropeptide corticotropin-releasing factor (CRF) on these responses. Compared to H2O and a novel odorant, vanilla, mice exposed to TMT in the home cage showed increased avoidance and defensive burying indicative of evident stress responses. Consistent with stress-induced activation of the medial prefrontal cortex (mPFC), we found that the prelimbic (PL) and infralimbic (IL) subregions of the mPFC had elevated c-Fos immunolabeling after TMT and vanilla compared to H2O. Slice physiology recordings were performed in layers 2/3 and 5 of the PL and IL, following TMT, vanilla, or H2O exposure. In TMTmice, but not vanilla or H2O mice, PL layers 2/3 showed heightened spontaneous excitatory post-synaptic currents and synaptic drive, suggesting TMT enhanced excitatory transmission. Synaptic drive in PL was increased in both TMT and H2O mice following bath application of 300 nM CRF, but only H2O mice increased excitatory currents with 100 nM CRF, suggesting dose-effect curve shifts in TMT mice. Further, systemic pretreatment with the CRF-R1 antagonist CP154526 and bath application with the CRF-R1 antagonist NBI27914 reduced excitatory transmission in TMT mice, but not H2O mice. CP154526 also reduced stress-reactive behaviors induced by TMT. Taken together, these findings suggest that exposure to TMT leads to CRF-R1 driven changes in behavior and changes in synaptic function in layer 2/3 neurons in the PL, which areconsistent with previous findings that CRF-R1 in the mPFC plays an important role in predator odor-related behaviors.

Differential effect of amphetamine over the corticotropin-releasing factor CRF2 receptor, the orexin OX1 receptor and the CRF2-OX1 heteroreceptor complex

Stress is one of the factors underlying drug seeking behavior that often goes in parallel with loss of appetite. We here demonstrate that orexin 1 receptors (OX1R) may form complexes with the corticotropin releasing factor CRF2 receptor. Two specific features of the heteromer were a cross-antagonism and a blockade by CRF2 of OX1R signaling. In cells expressing one of the receptors, agonist-mediated signal transduction mechanisms were potentiated by amphetamine. Sigma 1 (σ1) and 2 (σ2) receptors are targets of drugs of abuse and, despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is not known. We here show that σ1 receptors interact with CRF2 receptors and that σ2 receptors interact with OX1R. Moreover, we show that amphetamine effect on CRF2 receptors was mediated by σ1R whereas the effect on OX1 receptors was mediated by σ2R. Amphetamine did potentiate the negative cross-talk occurring within the CRF2-OX1 receptor heteromer context, likely by a macromolecular complex involving the two sigma receptors and the two GPCRs. Finally, in vivo microdialysis experiments showed that amphetamine potentiated orexin A-induced dopamine and glutamate release in the ventral tegmental area (VTA). Remarkably, the in vivo orexin A effects were blocked by a selective CRF2R antagonist. These results show that amphetamine impacts on the OX1R-, CRF2R- and OX1R/CRF2R-mediated signaling and that cross-antagonism is instrumental for in vivo detection of GPCR heteromers.This article is part of the Special Issue entitled ‘Receptor heteromers and their allosteric receptor-receptor interactions’.

Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability

Psychological stress and heightened mast cell (MC) activation are linked with important immunologic disorders, including allergy, anaphylaxis, asthma, and functional bowel diseases, but the mechanisms remain poorly defined. We have previously demonstrated that activation of the corticotropin-releasing factor (CRF) system potentiates MC degranulation responses during IgE-mediated anaphylaxis and psychological stress through corticotropin-releasing factor receptor subtype 1 (CRF1) expressed on MCs. In this study we investigated the role of corticotropin-releasing factor receptor subtype 2 (CRF2) as a modulator of stress-induced MC degranulation and associated disease pathophysiology. Invitro MC degranulation assays were performed with bone marrow-derived mast cells (BMMCs) derived from wild-type (WT) and CRF2-deficient (CRF2-/-) mice and RBL-2H3 MCs transfected with CRF2-overexpressing plasmid or CRF2 small interfering RNA. Invivo MC responses and associated pathophysiology in IgE-mediated passive systemic anaphylaxis and acute psychological restraint stress were measured in WT, CRF2-/-, and MC-deficient KitW-sh/W-sh knock-in mice. Compared with WT mice, CRF2-/- mice exhibited greater serum histamine levels and exacerbated IgE-mediated anaphylaxis and colonic permeability. In addition, CRF2-/- mice exhibited increased serum histamine levels and colonic permeability after acute restraint stress. Experiments with BMMCs and RBL-2H3 MCs demonstrated that CRF2 expressed on MCs suppresses store-operated Ca2+ entry signaling and MC degranulation induced by diverse MC stimuli. Experiments with MC-deficient KitW-sh/W-sh mice systemically engrafted with WT and CRF2-/- BMMCs demonstrated the functional importance of MC CRF2 in modulating stress-induced pathophysiology. MC CRF2 is a negative global modulator of stimuli-induced MC degranulation and limits the severity of IgE-mediated anaphylaxis and stress-related disease pathogenesis.

Sexually dimorphic metabolic responses mediated by CRF2 receptor during nutritional stress in mice

BackgroundChronic stress is a major contributor in the development of metabolic syndrome and associated diseases, such as diabetes. High-fat diet (HFD) and sex are known modifiers of metabolic parameters. Peptide hormones corticotropin-releasing factor (CRF) and urocortins (UCN) mediate stress responses via activation and feedback to the hypothalamic-pituitary-adrenal (HPA) axis. UCN3 is a marker of pancreatic β-cell differentiation, and UCN2 is known to ameliorate glucose levels in mice rendered diabetic with HFD. CRF receptor 2 (CRF2) is the only known cognate receptor for UCN2/3. Here, we ascertained the role of CRF2 in glucose clearance, insulin sensitivity, and other parameters associated with metabolic syndrome in a mouse model of nutritional stress.MethodsWild-type (WT) and Crhr2−/− (null) mice of both sexes were fed either normal chow diet or HFD. After 8 weeks, blood glucose levels in response to glucose and insulin challenge were determined. Change in body and fat mass, plasma insulin, and lipid profile were assessed. Histological evaluation of liver sections was performed.ResultsHere, we show that genotype (Crhr2), sex, and diet were all independent variables in the regulation of blood glucose levels, body and fat mass gain/redistribution, and insulin resistance. Surprisingly, CRF2-deficient mice (Crhr2−/−) male mice showed similarly impaired glucose clearance on HFD and chow. HFD-fed female Crhr2−/− mice redistributed their fat depots that were distinct from wild-type females and male mice on either diet. Blood cholesterol and low-density lipoprotein (LDL) levels were elevated significantly in male Crhr2−/− mice; female Crhr2−/− mice were protected. Male, but not female Crhr2−/− mice developed peripheral insulin resistance. HFD, but not chow-fed wild-type male mice developed hepatic macrovesicular steatosis. In contrast, livers of Crhr2−/− male mice showed microvesicular steatosis on either diet, whereas livers of female mice on this 8-week HFD regimen did not develop steatosis.ConclusionsCRF2 receptor dysregulation is a sexually dimorphic risk factor in development of pre-diabetic and metabolic symptoms.

DNA methylation levels are associated with CRF1 receptor antagonist treatment outcome in women with post-traumatic stress disorder

BackgroundWe have previously evaluated the efficacy of the CRF1 receptor antagonist GSK561679 in female PTSD patients. While GSK561679 was not superior to placebo overall, it was associated with a significantly stronger symptom reduction in a subset of patients with probable CRF system hyperactivity, i.e., patients with child abuse and CRHR1 SNP rs110402 GG carriers. Here, we test whether blood-based DNA methylation levels within CRHR1 and other PTSD-relevant genes would be associated with treatment outcome, either overall or in the high CRF activity subgroup.ResultsTherefore, we measured CRHR1 genotypes as well as baseline and post-treatment DNA methylation from the peripheral blood in the same cohort of PTSD-diagnosed women treated with GSK561679 (N = 43) or placebo (N = 45). In the same patients, we assessed DNA methylation at the PTSD-relevant genes NR3C1 and FKBP5, shown to predict or associate with PTSD treatment outcome after psychotherapy. We observed significant differences in CRHR1 methylation after GSK561679 treatment in the subgroup of patients with high CRF activity. Furthermore, NR3C1 baseline methylation significantly interacted with child abuse to predict PTSD symptom change following GSK561679 treatment.ConclusionsOur results support a possible role of CRHR1 methylation levels as an epigenetic marker to track response to CRF1 antagonist treatment in biologically relevant subgroups. Moreover, pre-treatment NR3C1 methylation levels may serve as a potential marker to predict PTSD treatment outcome, independent of the type of therapy. However, to establish clinical relevance of these markers, our findings require replication and validation in larger studies.Trial registrationNCT01018992. Registered 6 November 2009.

Sex differences in morphine-induced trafficking of mu-opioid and corticotropin-releasing factor receptors in locus coeruleus neurons

The locus coeruleus (LC)-norepinephrine (NE) system is a key nucleus in which endogenous opioid and stress systems intersect to regulate the stress response. LC neurons of male rats become sensitized to stress following chronic morphine administration. Whether sex dictates this pattern of opioid-induced plasticity has not been demonstrated. Delineating the neurobiological adaptations produced by chronic opioids will enhance our understanding of stress vulnerability in opioid-dependent individuals, and may reveal how stress negatively impacts addiction recovery. In the present study, the effect of chronic morphine on the subcellular distribution of mu-opioid (MOR) and CRF receptors (CRFR) was investigated in the LC of male and female rats using immunoelectron microscopy. Results showed that placebo-treated females exhibited higher MOR and CRFR cytoplasmic distribution ratio when compared to placebo-treated males. Chronic morphine exposure induced a shift in the distribution of MOR immunogold-silver particles from the plasma membrane to the cytoplasm selectively in male LC neurons. Interestingly, chronic morphine exposure induced CRFR recruitment to the plasma membrane of both male and female LC neurons. These findings provide a potential mechanism by which chronic opioid administration increases stress vulnerability in males and females via an increase in surface availability of CRFR in LC neurons. However, our results also support the notion that cellular adaptations to chronic opioids differ across the sexes as redistribution of MOR following morphine exposure was only observed in male LC neurons.

Altered colonic sensory and barrier functions by CRF: roles of TLR4 and IL-1

Visceral allodynia and increased colonic permeability are considered to be crucial pathophysiology of irritable bowel syndrome (IBS). Corticotropin-releasing factor (CRF) and immune-mediated mechanisms have been proposed to contribute to these changes in IBS, but the precise roles have not been determined. We explored these issues in rats in vivo. The threshold of visceromotor response, i.e., abdominal muscle contractions induced by colonic balloon distention was electrophysiologically measured. Colonic permeability was estimated by quantifying the absorbed Evans blue in colonic tissue. Intraperitoneal injection of CRF increased the permeability, which was blocked by astressin, a non-selective CRF receptor antagonist, but astressin2-B, a selective CRF receptor subtype 2 (CRF2) antagonist did not modify it. Urocortin 2, a selective CRF2 agonist inhibited the increased permeability by CRF. Eritoran, a toll-like receptor 4 (TLR4) antagonist or anakinra, an interleukin-1 receptor antagonist blocked the visceral allodynia and the increased gut permeability induced by CRF. Subcutaneous injection of lipopolysaccharide (immune stress) or repeated water avoidance stress (WAS, psychological stress), 1 h daily for 3 days induced visceral allodynia and increased gut permeability (animal IBS models), which were also blocked by astressin, eritoran or anakinra. In conclusion, stress-induced visceral allodynia and increased colonic permeability were mediated via peripheral CRF receptors. CRF induced these visceral changes via TLR4 and cytokine system, which were CRF1 dependent, and activation of CRF2 inhibited these CRF1-triggered responses. CRF may modulate immune system to alter visceral changes, which are considered to be pivotal pathophysiology of IBS.

Changes in striatal dopamine release and locomotor activity following acute withdrawal from chronic nicotine are mediated by CRF1, but not CRF2, receptors

The aim of the present study was to investigate the participation of corticotropin-releasing factor (CRF) receptors (CRF1 and CRF2) in the alterations of the dorsal and ventral striatal dopamine release and the vertical and horizontal locomotor activity observed in rats following chronic nicotine treatment and consequent acute withdrawal. In this purpose, male Wistar rats were exposed to repeated intraperitoneal (ip) injection with nicotine or saline solution for 7 days. On the 8th day or the 9th day the rats were injected intracerebroventricularly (icv) with selective CRF1 antagonist antalarmin or selective CRF2 antagonist astressin2B or saline solution. Thirty minutes after the icv injection the changes of the horizontal and vertical locomotor activity were recorded in an in vivo conducta system. Immediately after the behavioral recordings the changes of the dorsal and ventral striatal dopamine release were determined in an in vitro superfusion system. On the 8th day, the horizontal and vertical locomotor activities and the dorsal and ventral striatal dopamine releases increased significantly in nicotine-treated rats, compared to the saline-treated ones. On the 9th day, the horizontal locomotor activity and the dorsal striatal dopamine release increased significantly, whereas the vertical locomotor activity and the ventral striatal dopamine release decreased significantly in nicotine-treated rats, compared to the saline-treated ones. All the changes observed were attenuated significantly by antalarmin, but not astressin2B. The present study demonstrates that the changes of striatal dopamine release and locomotor activity observed following chronic nicotine treatment and consequent acute withdrawal are mediated by CRF1, but not CRF2, receptor.

Mesopontine cholinergic inputs to midbrain dopamine neurons drive stress-induced depressive-like behaviors

Stressful life events are primary environmental factors that markedly contribute to depression by triggering brain cellular maladaptations. Dysregulation of ventral tegmental area (VTA) dopamine neurons has been causally linked to the appearance of social withdrawal and anhedonia, two classical manifestations of depression. However, the relevant inputs that shape these dopamine signals remain largely unknown. We demonstrate that chronic social defeat (CSD) stress, a preclinical paradigm of depression, causes marked hyperactivity of laterodorsal tegmentum (LDTg) excitatory neurons that project to the VTA. Selective chemogenetic-mediated inhibition of cholinergic LDTg neurons prevent CSD-induced VTA DA neurons dysregulation and depressive-like behaviors. Pro-depressant outcomes are replicated by pairing activation of LDTg cholinergic terminals in the VTA with a moderate stress. Prevention of CSD outcomes are recapitulated by blocking corticotropin-releasing factor receptor 1 within the LDTg. These data uncover a neuro-circuitry of depressive-like disorders and demonstrate that stress, via a neuroendocrine signal, profoundly dysregulates the LDTg.

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.

Cannabidiol regulates the expression of hypothalamus-pituitary-adrenal axis-related genes in response to acute restraint stress.

Research interest has grown around the potential therapeutic use of cannabidiol in mood-related disorders, due to its anxiolytic and antidepressant-like effects. These have been partially attributed to its action as an allosteric modulator of 5-HTR1A. However, the exact mechanism supporting cannabidiol properties remains unclear. To assess the effects of cannabidiol on different targets of the hypothalamus-pituitary-adrenal axis under baseline and stress conditions. We administered cannabidiol (5 mg/kg, 15 mg/kg or 30 mg/kg, intraperitoneally) or vehicle to male C57BL/6J mice 90 min before single restraint stress exposure (20 min). Using real-time polymerase chain reaction analysis, we measured alterations in the relative gene expression of corticotropin-releasing factor in the paraventricular nucleus, pro-opiomelanocortin in the arcuate nucleus of the hypothalamus, glucocorticoid receptor in the hippocampus, and serotonin 5-HTR1A receptor in the hippocampus and amygdala. Under baseline conditions, cannabidiol did not modify any element of the hypothalamus-pituitary-adrenal axis. In contrast, all doses induced alterations in 5-HTR1A in the amygdala and hippocampus. Interestingly, cannabidiol at low (5 mg/kg) and intermediate doses (15 mg/kg) successfully blocked the effects induced by acute stress on corticotropin-releasing factor, pro-opiomelanocortin and glucocorticoid receptor gene expression. Also, restraint stress induced the opposite effects in 5-HTR1A gene expression in the hippocampus and amygdala, an effect not seen in mice treated with cannabidiol at low doses. Taken together, these data suggest the ability of cannabidiol to regulate acute stress hypothalamus-pituitary-adrenal axis activation might be explained, at least in part, by its action on 5-HTR1A receptors.