Sex-Specific changes in stress circuitry of the nucleus tractus solitarius following food deprivation in two rat strains.

Food deprivation is used in many experimental models and is becoming increasingly prevalent in human diets. The impact of food deprivation on specific brain regions, including the nucleus of the tractus solitarius (NTS), a region that is involved in hunger and satiety sensing, remains to be determined. The NTS is a heterogeneous nucleus that includes corticotropin releasing factor receptor 1 (CRF1) neurons. CRF1 is implicated in both stress and appetite regulation, but the effects of food deprivation on CRF1 NTS neurons are unclear. We used immunofluorescence to examine the effects of 24-hour food deprivation on NTS activity in male and female Sprague-Dawley (SD) rats and CRF1-cre rats using cFos, an immediate early gene and neuronal marker of activation. NTS activity was increased in food deprived male but not female SD rats. In food deprived CRF1-cre rats, males had an increased proportion of active CRF1 + neurons with no change in females. In CRF1-cre rats, increased global NTS activity was observed in food deprived and refed males. Activation of CRF1 + neurons was also increased after deprivation but was reduced by refeeding. In females, food deprivation decreased global NTS activity that was then increased by refeeding, while CRF1 activity was unchanged. Collectively, these data suggest the NTS is differentially activated after food deprivation in a sex-specific manner, whereby males are more sensitive than females. These results provide insight into the role of brainstem stress circuitry in changes associated with conditions including intermittent fasting and eating disorders like anorexia.

The diverse role of corticotropin-releasing factor (CRF) and its CRF1 and CRF2 receptors under pathophysiological conditions: Insights into stress/anxiety, depression, and brain injury processes.

Corticotropin-releasing factor (CRF, corticoliberin) is a neuromodulatory peptide activating the hypothalamic-pituitary-adrenal (HPA) axis, widely distributed in the central nervous system (CNS) in mammals. In addition to its neuroendocrine effects, CRF is essential in regulating many functions under physiological and pathophysiological conditions through CRF1 and CRF2 receptors (CRF1R, CRF2R). This review aims to present selected examples of the diverse and sometimes opposite effects of CRF and its receptor ligands in various pathophysiological states, including stress/anxiety, depression, and processes associated with brain injury. It seems interesting to draw particular attention to the fact that CRF and its receptor ligands exert different effects depending on the brain structures or subregions, likely stemming from the varied distribution of CRFRs in these regions and interactions with other neurotransmitters. CRFR-mediated region-specific effects might also be related to brain site-specific ligand binding and the associated activated signaling pathways. Intriguingly, different types of CRF molecules can also influence the diverse actions of CRF in the CNS.

Chronic Nicotine Consumption and Withdrawal Regulate Melanocortin Receptor, CRF, and CRF Receptor mRNA Levels in the Rat Brain.

Alterations in the various neuropeptide systems in the mesocorticolimbic circuitry have been implicated in negative effects associated with drug withdrawal. The corticotropin-releasing factor (CRF) and α-melanocyte-stimulating hormone are two peptides that may be involved. This study investigated the regulatory effects of chronic nicotine exposure and withdrawal on the mRNA levels of melanocortin receptors (MC3R, MC4R), CRF, and CRF receptors (CRFR1 and CRFR2) expressed in the mesocorticolimbic system. Rats were given drinking water with nicotine or without nicotine (control group) for 12 weeks, after which they continued receiving nicotine (chronic exposure) or were withdrawn from nicotine for 24 or 48 h. The animals were decapitated following behavioral testing for withdrawal signs. Quantitative real-time PCR analysis demonstrated that nicotine exposure (with or without withdrawal) increased levels of CRF and CRFR1 mRNA in the amygdala, CRF mRNA in the medial prefrontal cortex, and CRFR1 mRNA in the septum. Nicotine withdrawal also enhanced MC3R and MC4R mRNA levels in different brain regions, while chronic nicotine exposure was associated with increased MC4R mRNA levels in the nucleus accumbens. These results suggest that chronic nicotine exposure and withdrawal regulate CRF and melanocortin signaling in the mesocorticolimbic system, possibly contributing to negative affective state and nicotine addiction.

Antagonists of the stress and opioid systems restore the functional connectivity of the prefrontal cortex during alcohol withdrawal through divergent mechanisms.

Chronic alcohol consumption leads to dependence and withdrawal symptoms upon cessation, contributing to persistent use. However, the brain network mechanisms by which the brain orchestrates alcohol withdrawal and how these networks are affected by pharmacological treatments remain elusive. Recent work revealed that alcohol withdrawal produces a widespread increase in coordinated brain activity and a decrease in modularity of the whole-brain functional network using single-cell whole-brain imaging of immediate early genes. This decreased modularity and functional hyperconnectivity are hypothesized to be novel biomarkers of alcohol withdrawal in alcohol dependence, which could potentially be used to evaluate the efficacy of new medications for alcohol use disorder. However, there is no evidence that current FDA-approved medications or experimental treatments known to reduce alcohol drinking in animal models can normalize the changes in whole-brain functional connectivity. In this report, we tested the effect of R121919, a CRF1 antagonist, and naltrexone, an FDA-approved treatment for alcohol use disorder, on whole-brain functional connectivity using the cellular marker FOS combined with graph theory and advanced network analyses. Results show that both R121919 and naltrexone restored the functional connectivity of the prefrontal cortex during alcohol withdrawal, but through divergent mechanisms. Specifically, R121919 increased FOS activation in the prefrontal cortex, partially restored modularity, and normalized connectivity, particularly in CRF1-rich regions, including the prefrontal, pallidum, and extended amygdala circuits. On the other hand, naltrexone decreased FOS activation throughout the brain, decreased modularity, and increased connectivity overall except for the Mu opioid receptor-rich regions, including the thalamus. These results identify the brain networks underlying the pharmacological effects of R121919 and naltrexone and demonstrate that these drugs restored different aspects of functional connectivity of the prefrontal cortex, pallidum, amygdala, and thalamus during alcohol withdrawal. Notably, these effects were particularly prominent in CRF1- and Mu opioid receptors-rich regions highlighting the potential of whole-brain functional connectivity using FOS as a tool for identifying neuronal network mechanisms underlying the pharmacological effects of existing and new medications for alcohol use disorder.

The Impact of Heroin Self-Administration and Environmental Enrichment on Ventral Tegmental CRF1 Receptor Expression.

BACKGROUND: There is a strong link between chronic stress and vulnerability to drug abuse and addiction. Corticotropin releasing factor (CRF) is central to the stress response that contributes to continuation and relapse to heroin abuse. Chronic heroin exposure can exacerbate CRF production, leading to dysregulation of the midbrain CRF-dopamine-glutamate interaction. METHODS: Here we investigated the role of midbrain CRF1 receptors in heroin self-administration and assessed neuroplasticity in CRF1 receptor expression in key opioid addiction brain regions. RESULTS: Infusions of antalarmin (a CRF1 receptor antagonist) into the ventral tegmental area (VTA) dose dependently reduced heroin self-administration in rats but had no impact on food reinforcement or locomotor activity in rats. Using RNAscope in situ hybridization, we found that heroin, but not saline, self-administration upregulated CRF1 receptor mRNA in the VTA, particularly on dopamine neurons. AMPA GluR1 and dopamine reuptake transporter mRNA in VTA neurons were not affected by heroin. The western-blot assay showed that CRF1 receptors were upregulated in the VTA and nucleus accumbens. No significant changes in CRF1 protein expression were detected in the prefrontal cortex, insula, dorsal hippocampus, and substantia nigra. In addition, we found that 15 days of environmental enrichment implemented after heroin self-administration does not reverse upregulation of VTA CRF1 receptor mRNA but it downregulates dopamine transporter mRNA. CONCLUSIONS: Overall, these data suggest that heroin self-administration requires stimulation of VTA CRF1 receptors and upregulates their expression in brain regions involved in reinforcement. Such long-lasting neuroadaptations may contribute to continuation of drug use and relapse due to stress exposure and are not easily reversed by EE exposure.

Corticotropin releasing factor-1 receptor antagonism associated with favorable outcomes of male reproductive health biochemical parameters.

Background: Disruption in androgen profiles and testicular adrenal rest tumors in males with congenital adrenal hyperplasia (CAH) can negatively affect sexual activity and fertility. Adrenal hyperandrogenism suppresses gonadotropin secretion and testicular adrenal rest tumors (TARTS), despite being noncancerous lesions, cause obstructive azoospermia and impaired testosterone (T) production. Circulating T in men with uncontrolled CAH is often predominantly adrenal in origin, which is reflected in high androstenedione/testosterone ratios (A4/T). Therefore, decreased luteinizing hormone (LH) levels and an increased A4/T are markers of impaired fertility in these individuals. Methods: Oral tildacerfont 200 to 1000 mg once daily (QD) (n=10) or 100 to 200 mg twice daily (n=9 and 7) for 2 weeks (Study 201), and 400 mg QD (n=11) for 12 weeks (Study 202). Outcomes measured changes from baseline in A4, T, A4/T, and LH. Results: Mean T levels increased in Study 201 from 375.5 ng/dL to 390.5 ng/dL at week 2 (n=9), 485.4 ng/dL at week 4 (n=4) and 420.7 ng/dL at week 6 (n=4). In Study 202, T levels fluctuated in the normal range from 448.4 ng/dL at baseline to 412.0 ng/dL at week 12. Mean LH levels increased in Study 201 from 0.68 IU/L to 1.59 IU/L at week 2 (n=10), 1.62 IU/L at week 4 (n=5) and 0.85 IU/L at week 6 (n=4). In Study 202, mean LH levels increased from 0.44 IU/L at baseline to 0.87 IU/L at week 12. Mean A4/T decreased across both studies. In Study 201, the mean A4/T changed from a baseline of 1.28 to 0.59 at week 2 (n=9), 0.87 at week 4 (n=4), and 1.03 at week 6 (n=4). In Study 202, the A4/T decreased from baseline of 2.44 to 0.68 at week 12. Four men were hypogonadal at baseline; all experienced improved A4/T and 3/4 (75%) reached levels <1. Conclusion: Tildacerfont treatment demonstrated clinically meaningful reductions in A4 levels, and A4/T with concomitant increased LH levels indicating increased testicular T production. The data suggests improvement in hypothalamic-pituitary-gonadal axis function, but more data is required to confirm favorable male reproductive health outcomes.

Sex Differences in the Impact of Electronic Nicotine Vapor on Corticotropin-Releasing Factor Receptor 1 Neurons in the Mouse Ventral Tegmental Area.

Nicotine engages dopamine neurons in the ventral tegmental area (VTA) to encode reward and drive the development of nicotine addiction, however how nicotine alters a stress associated VTA population remains unclear. Here, we used male and female CRF1-GFP mice and nicotine vapor exposure to examine the effects of nicotine in VTA corticotropin-releasing factor receptor 1 (CRF1) neurons. We use immunohistochemistry and electrophysiology to examine neuronal activity, excitability, and inhibitory signaling. We found that VTA CRF1 neurons are mainly dopaminergic and project to the nucleus accumbens (NAc; VTA-NAcCRF1 neurons). VTA-NAcCRF1 neurons show greater phasic inhibition in naive females and greater focal nicotine-induced increases in firing in naive males. Following acute nicotine vapor exposure, phasic inhibition was not altered, but focal nicotine-induced tonic inhibition was enhanced in females and diminished in males. Acute nicotine vapor exposure did not affect firing in VTA-NAcCRF1 neurons, but females showed lower baseline firing and higher focal nicotine-induced firing. Activity (cFos) was increased in the CRF1 dopaminergic VTA population in both sexes, but with greater increases in females. Following chronic nicotine vapor exposure, both sexes displayed reduced basal phasic inhibition and the sex difference in tonic inhibition following acute vapor exposure was no longer observed. Additionally, activity of the CRF1 dopaminergic VTA population was no longer elevated in either sex. These findings reveal sex-dependent and exposure-dependent changes in mesolimbic VTA-NAc CRF1 neuronal activity, inhibitory signaling, and nicotine sensitivity following nicotine vapor exposure. These changes potentially contribute to nicotine-dependent behaviors and the intersection between stress, anxiety, and addiction.SIGNIFICANCE STATEMENT Nicotine is known to engage reward systems in the brain historically centering the neurotransmitter dopamine however, how nicotine impacts other neurons in the reward pathway is less clear. The current study investigates the impact of acute and chronic electronic nicotine vapor exposure in a genetically-defined cell population containing the stress receptor corticotropin-releasing factor 1 (CRF1) that is located in the reward circuitry. This study employs functional measures of neuronal activity and identifies important sex differences in nicotine's effects across time and exposure.

Corticotropin-releasing factor receptor agonists decrease interstitial cells of Cajal in murine colon.

BACKGROUND: Peripheral corticotropin-releasing factor (CRF) has been reported to affect gastrointestinal motility through corticotropin-releasing factor receptor located in enteric nervous system (ENS), but less is known about of the relationship between peripheral CRF and interstitial cells of Cajal (ICC). METHODS: Mice were intraperitoneally injected with CRF receptor agonists to determine their effects on colonic ICC. Chronic heterotypic stress (CHeS) was applied to mice to determine endogenous CRF-CRF receptor signaling on colonic ICC. RESULTS: We found that stressin1, a selective CRF receptor 1 (CRF1 ) agonist, significantly increased the expression of CRF1 but had no effect on the expression of CRF2 in the smooth muscles of murine colon. The protein expression of c-Kit, Anoctamin-1 (ANO1), and stem cell factor (SCF) in the colonic smooth muscles was significantly decreased in stressin1-treated mice. Accordingly, 2-(4-Chloro-2-methylphenoxy)-N'-(2-methoxybenzylidene) acetohydrazide (Ani 9), a selective ANO1 blocker, had a less significant inhibitory effect on CMMC in stressin1-treated mice compared to the saline-treated ones. Similarly, we also found that ICC and ANO1 were reduced in the colonic smooth muscles of mice by treatment with sauvagine (ip), a CRF2 agonist. However, different with stressin1, sauvagine decreased the expression of CRF2 besides increasing CRF1 expression in the colonic smooth muscles. Similar results of CRF1 and c-Kit expressions were also obtained from the colon of CHeS-treated mice. CONCLUSION: All these results suggest that CRF may be involved in the abnormality of colonic motility through peripheral CRF1 to decrease the number and function of ICC, which provides a potential target for treating stress-induced gastrointestinal motility disorder.

Identification of potential antagonists of CRF1R for possible treatment of stress and anxiety neuro-disorders using structure-based virtual screening and molecular dynamics simulation.

G protein-coupled-receptors (GPCRs) are the largest family of cell surface receptors with tremendous therapeutic potential. They mediate signal transduction activities via G protein-dependent signaling pathways, G protein-independent signaling pathways, and other complicated regulatory processes. The corticotropin-releasing factor receptor type 1 (CRF1R) is a member of class B GPCRs that is predominantly found in the central nervous system, where it plays a key role in stress-related neuro-disorders. To date, no drug targeting this receptor has been approved, partly due to inadequate understanding of the activation mechanism of class B GPCRs. Previously, using MD simulation, we demonstrated that the CRF1R complexed with a small-molecule antagonist CP-376395 maintains a conformation of its transmembrane domain (TMD). Here, using the most abundant structures derived from those simulations, we carried out a structure-based virtual screening of ZINC15 "Druglike" library containing approximately 17 million compounds. The docking complexes of the CRF1R with the top 30 hits were submitted to MD simulation to examine the stability of ligand binding mode. Furthermore, MM-GBSA binding energy calculations were performed on all the complexes to rank them with improving accuracy. Hit 1 (ZINC000046079839) and hit 20 (ZINC000032907937) span the allosteric site of the CRF1R, persistently forming interactions with transmembrane helices 3 and 6. These interactions are likely to keep the receptor in an inactive state since both transmembrane helices play a critical role in the activation of the receptor.

Chronic stress-induced synaptic changes to corticotropin-releasing factor-signaling in the bed nucleus of the stria terminalis.

The sexually dimorphic bed nucleus of the stria terminalis (BNST) is comprised of several distinct regions, some of which act as a hub for stress-induced changes in neural circuitry and behavior. In rodents, the anterodorsal BNST is especially affected by chronic exposure to stress, which results in alterations to the corticotropin-releasing factor (CRF)-signaling pathway, including CRF receptors and upstream regulators. Stress increases cellular excitability in BNST CRF+ neurons by potentiating miniature excitatory postsynaptic current (mEPSC) amplitude, altering the resting membrane potential, and diminishing M-currents (a voltage-gated K+ current that stabilizes membrane potential). Rodent anterodorsal and anterolateral BNST neurons are also critical regulators of behavior, including avoidance of aversive contexts and fear learning (especially that of sustained threats). These rodent behaviors are historically associated with anxiety. Furthermore, BNST is implicated in stress-related mood disorders, including anxiety and Post-Traumatic Stress Disorders in humans, and may be linked to sex differences found in mood disorders.

Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior.

Corticotropin-releasing factor type-1 (CRF1) receptors are critical to stress responses because they allow neurons to respond to CRF released in response to stress. Our understanding of the role of CRF1-expressing neurons in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF1+ cells in rats. Here, we describe the generation and validation of a transgenic CRF1-Cre-tdTomato rat. We report that Crhr1 and Cre mRNA expression are highly colocalized in both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in tdTomato+ neurons are similar to those previously reported in GFP+ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be targeted to CeA CRF1+ cells via virally delivered Cre-dependent transgenes, that transfected Cre/tdTomato+ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like and nocifensive behaviors. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the brain, and that the expression pattern of Cre-tdTomato cells is similar to the known expression pattern of CRF1 cells. Given the accuracy of expression in the CRF1-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF1+ neurons can now be performed in assays that require the use of rats as the model organism.

Molecular analysis of nematode-responsive defence genes CRF1, WRKY45, and PR7 in Solanum lycopersicum tissues during the infection of plant-parasitic nematode species of the genus Meloidogyne.

Several pathogens, including nematodes, have severe effects on plant development and growth, and immense populations of parasitic nematodes may cause plant death and crop loss. Obligate plant-parasitic nematodes and root-knot nematodes belonging to the genus Meloidogyne are significant parasites in crops. During nematode infection, damage-associated molecular patterns play a role in the activation of plant defence responses to pathogens. Several genes are involved in Meloidogyne parasitism. However, the expression of nematode-responsive genes CRF1, WRKY45, and PR7 during infection with different parasitic nematode species is not well understood. Therefore, this study aimed to reveal plant responses to differential gene expression of nematode-responsive genes in tomato plants, and their relationship to nematode reproduction and comparative phylogeny. Molecular methods for gene expression, greenhouse work for nematode reproduction, and phylogenetic analysis were used to determine nematode-plant interactions. The results revealed that differential gene expression of CRF1, WRKY45, and PR7 depended on the nematode species. The relative CRF1 gene expression reached its highest level at 3 dpi, following nematode infection. In conclusion, plant defense responses disturbed the expression of nematode-responsive genes, and the differential expression of nematode-responsive genes was affected by nematode species and nematode parasitism.

Yeast Crf1p: An activator in need is an activator indeed.

Ribosome biogenesis is an energetically costly process, and tight regulation is required for stoichiometric balance between components. This requires coordination of RNA polymerases I, II, and III. Lack of nutrients or the presence of stress leads to downregulation of ribosome biogenesis, a process for which mechanistic target of rapamycin complex I (mTORC1) is key. mTORC1 activity is communicated by means of specific transcription factors, and in yeast, which is a primary model system in which transcriptional coordination has been delineated, transcription factors involved in regulation of ribosomal protein genes include Fhl1p and its cofactors, Ifh1p and Crf1p. Ifh1p is an activator, whereas Crf1p has been implicated in maintaining the repressed state upon mTORC1 inhibition. Computational analyses of evolutionary relationships have indicated that Ifh1p and Crf1p descend from a common ancestor. Here, we discuss recent evidence, which suggests that Crf1p also functions as an activator. We propose a model that consolidates available experimental evidence, which posits that Crf1p functions as an alternate activator to prevent the stronger activator Ifh1p from re-binding gene promoters upon mTORC1 inhibition. The correlation between retention of Crf1p in related yeast strains and duplication of ribosomal protein genes suggests that this backup activation may be important to ensure gene expression when Ifh1p is limiting. With ribosome biogenesis as a hallmark of cell growth, failure to control assembly of ribosomal components leads to several human pathologies. A comprehensive understanding of mechanisms underlying this process is therefore of the essence.

The CRF1 receptor mediates social behavior deficits induced by opiate withdrawal.

Poor sociability and aggressive behavior are key clinical features of opioid use disorders. The corticotropin-releasing factor (CRF) system may mediate behavioral effects of substances of abuse but its implication in substance-induced social behavior deficits and outward-directed hostility remains largely unknown. CRF signaling is mediated by two receptor types, termed CRF1 and CRF2 . The present study aimed at understanding the role for the CRF1 receptor in social and aggressive behavior induced by withdrawal from repeated opiate administration. Thus, wild-type (CRF1 +/+), CRF1 receptor heterozygous (CRF1 +/-), and null mutant (CRF1 -/-) female and male mice were treated with saline or escalating doses of morphine (20-100 mg/kg, i.p.) during six consecutive days and tested in the three-chamber task for sociability (i.e., preference for an unfamiliar same-sex conspecific vs. an object) 7 days after the last administration. Moreover, aggressive biting behavior toward the unfamiliar conspecific was assessed during the three-chamber test. Opiate withdrawal disrupted sociability in CRF1 +/+ and CRF1 +/-, but not in CRF1 -/-, female mice, without affecting aggressive biting behavior in any genotype. In contrast, opiate withdrawal did not affect sociability but increased aggressive biting behavior in male mice, independently of CRF1 receptor-deficiency. Nevertheless, in opiate-withdrawn CRF1 +/+, but not CRF1 +/- and CRF1 -/-, male mice, sociability directly correlated with aggressive biting behavior, suggesting a role for the CRF1 receptor in hostility-linked social approach. These findings demonstrate the implication of the CRF1 receptor in social behavior deficits associated with repeated opiate administration and withdrawal, revealing a new potential target for the treatment of opioid use disorders.

Activation of CRF1 receptors expressed in brainstem autonomic nuclei stimulates colonic enteric neurons and secreto-motor function in male rats.

BACKGROUND: Hypothalamic corticotropin-releasing factor (CRF) receptor 1 (CRF1 ) plays a role in acute stress-related stimulation of colonic motor function. Less is known on CRF1 signaling in the brainstem. METHODS: We investigate CRF1 expression in the brainstem and the colonic response to 4th ventricle (4V) injection of CRF and urocortin (Ucn) 2 (3 µg/rat) in chronically cannulated male rats. KEY RESULTS: Transcripts of CRF1 wild-type 1a and splice variants 1c, 1e, 1f, 1o along with three novel variants 1a-2 (desK-110 in exon 5), 1p (-exon 7), and 1q (exon 5 extension) were identified in the pons and medulla. The area postrema, nucleus tractus solitarius, dorsal motor nucleus of the vagus, locus coeruleus, and Barrington's nucleus isolated by laser capture microdissection expressed 1a, 1a-2, and 1p but not 1q. Compared to 4V vehicle, 4V CRF induced fecal pellet output (FPO) and diarrhea that were blocked by the CRF antagonist, astressin-B. CRF2 agonist, Ucn2 had no effect on basal or CRF-induced FPO. CRF actions were correlated with the induction of c-Fos immunoreactivity in myenteric neurons of the proximal and distal colon (pC, dC) and submucosal neurons of dC. c-Fos immunoreactivity occurred in 39% and 37% of myenteric cholinergic and 7% and 58% of nitrergic neurons in the pC and dC, respectively. CONCLUSIONS & INFERENCES: CRF1a and its splice variants are expressed in brainstem nuclei, and activation of CRF1 signaling at the level of the brainstem stimulates colonic secretory-motor function through activation of colonic enteric neurons.

Ventromedial prefrontal cortex CRF1 receptors modulate the tachycardic activity of baroreflex.

Ventral medial prefrontal cortex (vMPFC) glutamatergic neurotransmission has a facilitatory role on cardiac baroreflex activity which is mediated by NMDA receptors activation. Corticotrophin releasing factor receptors type1 and 2 (CRF1 and CRF2), present in the vMPFC, are colocalized in neurons containing glutamate vesicles, suggesting that such receptors may be involved in glutamate release in this cortical area. Therefore, our hypothesis is that the CRF1 and CRF2 receptors can modulate the baroreflex bradycardic and tachycardic responses. In order to prove this assumption, male Wistar rats had bilateral stainless steel guide cannula implanted into the vMPFC, and baroreflex was activated by intravenous infusion of phenylephrine or sodium nitroprusside through a vein catheter. A second catheter was implanted into the femoral artery for cardiovascular measurements. The CRF1 receptor antagonist administration in either infralimbic cortex (IL) or prelimbic cortex (PL), vMPFC regions, was unable to change the bradycardic responses but increased the slope of the baroreflex tachycardic activity. Microinjection of the CRF2 receptor antagonist into the IL and PL did not alter ether bradycardic nor tachycardic baroreflex responses. The administration of the non-selective CRF receptors agonist, urocortin in these areas, did not modify bradycardic responses but decreased tachycardia slope of the baroreflex. CRF1 receptor antagonist administration prior to non-selective CRF agonist in vMPFC prevented the tachycardic responses reduction. However, CRF2 receptor antagonism could not prevent the effect of CRF receptors agonist. These results suggest that IL and PL CRF1 but not CRF2 receptors have an inhibitory role on the baroreflex tachycardic activity. Furthermore, they have no influence on baroreflex bradycardic activity.

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.

Involvement of the ventral, but not dorsal, hippocampus in anxiety-like behaviors in mice exposed to the elevated plus maze: participation of CRF1 receptor and PKA pathway.

BACKGROUND: The hippocampus is a limbic structure involved in anxiety-like behaviors. We aimed to evaluate the role of the dorsal (DH) and ventral (VH) hippocampus in anxiety-like behaviors in the elevated plus maze (EPM). METHODS: We inhibited these brain regions using cobalt chloride (CoCl2: 1.0 nmol) microinjections. We also investigated the involvement of corticotropin-releasing factor (CRF) action and protein kinase A (PKA) pathway using intra-DH and intra-VH microinjections of the CRF1 receptor antagonist CP376395 (0, 3.0, or 6.0 nmol) and the PKA inhibitor H-89 (0, 2.5, or 5.0 nmol). RESULTS: The results indicated that intra-VH CoCl2 microinjection increased the percentage of time spent and entries in the open arms. The mice also exhibited fewer stretch attend postures in the protected area and increased percentage of open arm entries. Further, intra-VH injection of 3.0 nmol CP376395 increased time spent in the open arms. Intra-DH injection of 6.0 nmol CP376395 increased the frequency of unprotected head dipping, whereas intra-VH injection of 6 nmol CP376395 increased the frequency of protected head dipping. Intra-VH, but not intra-DH, microinjection of 2.5 nmol H-89 increased the percentages of open arm entries and time spent in the open arms. Microinjection of 2.5 and 5.0 nmol H-89 reduced the frequency of protected head dipping behavior. CONCLUSIONS: This study demonstrated that VH modulates anxiety-like behaviors in EPM. Moreover, CRF and the cAMP/PKA pathway seem to modulate these effects.

Discovery of a stable tripeptide targeting the N-domain of CRF1 receptor.

The corticotropin-releasing factor (CRF) and its CRF1 receptor (CRF1R) play a central role in the maintenance of homeostasis. Malfunctioning of the CRF/CRF1R unit is associated with several disorders, such as anxiety and depression. Non-peptide CRF1R-selective antagonists have been shown to exert anxiolytic and antidepressant effects on experimental animals. However, none of them is in clinical use today because of several side effects, thus demonstrating the need for the development of other more suitable CRF1R antagonists. In an effort to develop novel CRF1R antagonists we designed, synthesized and chemically characterized two tripeptide analogues of CRF, namely (R)-LMI and (S)-LMI, having their Leu either in R (or D) or in S (or L) configuration, respectively. Their design was based on the crystal structure of the N-extracellular domain (N-domain) of CRF1R/CRF complex, using a relevant array of computational methods. Experimental evaluation of the stability of synthetic peptides in human plasma has revealed that (R)-LMI is proteolytically more stable than (S)-LMI. Based on this finding, (R)-LMI was selected for pharmacological characterization. We have found that (R)-LMI is a CRF antagonist, inhibiting (1) the CRF-stimulated accumulation of cAMP in HEK 293 cells expressing the CRF1R, (2) the production of interleukins by adipocytes and (3) the proliferation rate of RAW 264.7 cells. (R)-LMI likely blocked agonist actions by interacting with the N-domain of CRF1R as suggested by data using a constitutively active chimera of CRF1R. We propose that (R)-LMI can be used as an optimal lead compound in the rational design of novel CRF antagonists.

Agonist bias and agonist-dependent antagonism at corticotrophin releasing factor receptors.

The corticotropin-releasing factor (CRF) receptors represent potential drug targets for the treatment of anxiety, stress, and other disorders. However, it is not known if endogenous CRF receptor agonists display biased signaling, how effective CRF receptor antagonists are at blocking different agonists and signaling pathways or how receptor activity-modifying proteins (RAMPs) effect these processes. This study aimed to address this by investigating agonist and antagonist action at CRF1 and CRF2 receptors. We used CRF1 and CRF2 receptor transfected Cos7 cells to assess the ability of CRF and urocortin (UCN) peptides to activate cAMP, inositol monophosphate (IP1 ), and extracellular signal-regulated kinase 1/2 signaling and determined the ability of antagonists to block agonist-stimulated cAMP and IP1 accumulation. The ability of RAMPs to interact with CRF receptors was also examined. At the CRF1 receptor, CRF and UCN1 activated signaling in the same manner. However, at the CRF2 receptor, UCN1 and UCN2 displayed similar signaling profiles, whereas CRF and UCN3 displayed bias away from IP1 accumulation over cAMP. The antagonist potency was dependent on the receptor, agonist, and signaling pathway. CRF1 and CRF2 receptors had no effect on RAMP1 or RAMP2 surface expression. The presence of biased agonism and agonist-dependent antagonism at the CRF receptors offers new avenues for developing drugs tailored to activate a specific signaling pathway or block a specific agonist. Our findings suggest that the already complex CRF receptor pharmacology may be underappreciated and requires further investigation.