Corticotropin-releasing Factor Receptor Research Articles

Selective antagonism of CRF1 receptor by a substituted pyrimidine.

The corticotrophin-releasing factor (CRF) and its type 1 receptor (CRF1R) regulate the hypothalamic-pituitary-adrenal axis, as well as other systems, thus playing a crucial role in the maintenance of homeostasis. Non-peptide CRF1R-selective antagonists exert therapeutic effects on experimental animals with abnormal regulation of their homeostatic mechanisms. However, none of them is as yet in clinical use. In an effort to develop novel small non-peptide CRF1R-selective antagonists, we have synthesized a series of substituted pyrimidines described in a previous study. These small molecules bind to CRF1R, with analog 3 having the highest affinity. Characteristic structural features of analog 3 are a N,N-bis(methoxyethyl)amino group at position 6 and a methyl in the alkythiol group at position 5. Based on the binding profile of analog 3, we selected it in the present study for further pharmacological characterization. The results of this study suggest that analog 3 is a potent CRF1R-selective antagonist, blocking the ability of sauvagine, a CRF-related peptide, to stimulate cAMP accumulation in HEK 293 cells via activation of CRF1R, but not via CRF2R. Moreover, analog 3 blocked sauvagine to stimulate the proliferation of macrophages, further supporting its antagonistic properties. We have also constructed molecular models of CRF1R to examine the interactions of this receptor with analog 3 and antalarmin, a prototype CRF1R-selective non-peptide antagonist, which lacks the characteristic structural features of analog 3. Our data facilitate the design of novel non-peptide CRF1R antagonists for clinical use.

Alterations in effort-related decision-making induced by stimulation of dopamine D1, D2, D3, and corticotropin-releasing factor receptors in nucleus accumbens subregions.

Nucleus accumbens (NAc) dopamine (DA) plays an integral role in overcoming effort costs, as blockade of D1 and D2 receptors reduces the choice of larger, more-costly rewards. Similarly, the stress neuropeptide corticotropin-releasing factor (CRF) modulates DA transmission and mediates stress-induced alterations in effort-related choice. The current study explored how excessive stimulation of different DA receptors within the NAc core and shell alters effort-related decision-making and compared these effects to those induced by CRF stimulation. Male Long Evans rats were well-trained on an effort-discounting task wherein they choose between a low-effort/low-reward and a high-effort/high-reward lever where the effort requirement increased over blocks (2-20 presses). Dopamine D1 (SKF 81297, 0.2-2μg), D2/3 (quinpirole, 1-10μg), or D3 (PD 128,907, 1.5-3μg) receptor agonists, or CRF (0.5μg), were infused into the NAc core or shell prior to testing. Stimulation of D2/3 receptors with quinpirole in the NAc core or shell markedly reduced the choice of high-effort option and increase choice latencies, without altering preference for larger vs smaller rewards. Stimulation of D1 or D3 receptors did not alter choice, although SKF 81297 infusions into the shell reduced response vigor. In comparison, core infusions of CRF flattened the discounting curve, reducing effortful choice when costs were low and increasing it when costs were high. Excessive stimulation of NAc D2 receptors has detrimental effects on effort-related decision-making. Furthermore, CRF stimulation induces dissociable effects on decision-making compared with those induced the effects of stimulation of different DA receptors.

Stress Engages Novel Beta‐Adrenergic Receptor and CRF1 Receptor Mediated Glutamatergic Signaling in the Ventral Bed Nucleus of the Stria Terminalis

Pathological stress reactivity is a critical factor in many mental health disorders. In order to better treat pathological stress‐related mental health disorders, a better understanding of the neurocircuitry governing homeostatic stress responses is needed. The bed nucleus of the stria terminalis (BNST) is a heterogenous limbic structure that plays a crucial role in the central nervous system's response to stress. During normal stress responding, norepinephrine signaling in the BNST is increased and modulates glutamatergic transmission. Prior experiments showed that stimulating beta1‐adrenergic receptors (β1‐ARs) enhances glutamatergic transmission in the dorsal lateral BNST (dBNST) through the modulation of corticotropin releasing factor (CRF) signaling. However, whether this mechanism is conserved in other subregions of the BNST has not been well‐studied. Therefore, this study sought to determine the mechanism by which β‐AR stimulation might modulate glutamatergic transmission in the ventral BNST (vBNST). Using whole‐cell patch clamp electrophysiology, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from vBNST neurons in brain slices from adult male C57BL/6J mice. Mice were either exposed to one‐hour restraint stress or left as stress‐naïve prior to obtaining the slices. At baseline, sEPSC frequency was significantly higher in stressed than naïve mice (p<0.05). In both naïve and stressed mice, 10‐min bath application of the non‐selective β‐AR agonist, isoproterenol (3 μM), significantly increased vBNST sEPSC frequency (p<0.001). In both conditions, the isoproterenol effect was blocked by the selective β1‐AR antagonist betaxolol (10 μM), indicating that isoproterenol is acting through a β1‐AR mediated mechanism. The CRF1 receptor antagonist, NBI 27914 (1 μM), blocked the isoproterenol effect, indicating similar β1‐AR/CRF1 receptor circuit interactions in both the dorsal and ventral BNST. Surprisingly, although 10‐min application of the β2‐AR agonist clenbuterol (10 μM) did not change sEPSC frequency in naïve mice (p>0.05), clenbuterol did significantly increase sEPSC frequency in stressed mice (p<0.05). NBI 27914 blocked the clenbuterol effect in stressed mice, indicating a role for CRF1 receptors in this β2‐AR circuit. Additionally, using RNAscope in‐situ hybridization, expression of β2‐AR mRNA was upregulated following stress, specifically in CRF expressing neurons. These results suggest that stress induces de novo β2‐AR expression in vBNST CRF neurons that act to stimulate glutamate release within this brain region. These results uncover novel stress neurocircuitry in the vBNST and further investigation of this circuit may lead to possible therapeutic targets for treatment of chronic stress.Support or Funding InformationThis work funded by NIH grant AA22937.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methamphetamine and cocaine administration increases CRF‐R2 and decreases S100‐beta astrocytic immunoreactivity in the VTA

Dopamine neurons in the ventral tegmental area (VTA) play a crucial role in the reinforcing properties of abused drugs including methamphetamine and cocaine. However, there is evidence suggesting the involvement of non‐dopaminergic transmitters, including glutamate and corticotropin‐releasing factor (CRF), on psychostimulant effects in the VTA. Astrocytes can express a large variety of receptors for neurotransmitters such as glutamate which could indirectly influence neurotransmission in the VTA. Additionally, corticotropin‐releasing factor (CRF) receptors have been reported on astrocytes in the cerebellum, although no one has yet identified them on astrocytes in the VTA. CRF in the VTA plays a role in stress‐induced relapse and drug‐seeking behavior, but the localization of its effects are not completely understood. Although tissue from mouse VTA shows a high level of CRF1 receptor transcript and lower levels of CRF2 receptor transcript, both receptors play a functional role in regulating VTA synaptic activity.Here, we aimed to use immunofluorescence to identify the effect of methamphetamine and cocaine administration on astrocytic glutamate and CRF receptors. We treated adult male mice with i.p. injections of methamphetamine (3 mg/kg), cocaine (10 mg/kg), or saline (vehicle) for 12 days. Brains were then removed and sectioned (40 μm thickness) in the coronal plane. The tissue was then processed for immunofluorescence using tyrosine hydroxylase as a dopaminergic cell marker and S100‐beta as a marker for astrocytes. We examined the level of glutamate and glutamate/aspartate transporters (GLT‐1 and GLAST) and CRF receptors. Images were captured using fluorescent microscopy and processed using ImageJ software. The results showed a ~30% decrease in GLT‐1 and GLAST immunofluorescence in brains of mice treated with cocaine or methamphetamine compared to saline. In addition, we observed increased labelling of CRF2 receptors in drug‐treated groups, a decrease of S100beta, and an increase of co‐staining of these markers. Our results suggest that administration of either methamphetamine or cocaine increases immunoreactivity for CRF2 receptors in the VTA, an effect that is most pronounced in S100‐ beta positive cells.Support or Funding InformationThis work was supported by National Institute on Aging grant R01 AG052606.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Drinking patterns and alterations in several brain protein levels after binge drinking during adolescence followed by intermittent stress and ethanol drinking during adulthood in male and female C57BL/6J mice

Alcohol abuse is a serious problem in our society with stress likely increasing the risk for escalation to harmful drinking. We are continuing to explore the effect of prior binge drinking on subsequent drinking behaviors and the response to intermittent predator odor stress (PS) exposure (a model of traumatic stress). We assessed responses in mice that were adolescent at the time of the binge drinking sessions, as we are aware that the adolescent brain is particularly vulnerable to ethanol. Male and female C57BL/6J adolescent mice had either 7 binge ethanol sessions (binge) or drank water (control). Following an abstinence period, all mice (now adults) were provided free access to 10% ethanol (10E) and water via lickometers for 4 weeks, with 30 min exposure to PS during the middle 2 weeks (2X/week). For mice exposed to the binge paradigm during adolescence, drinking levels changed across time after repeated PS. In males, ethanol intake increased over baseline by 34% in the binge group vs unchanged in the control group. In females, ethanol intake was increased by 57% (binge) and 38% (control) over baseline. At 24 hr after the final drinking session, brains were harvested and frozen for Western Blot analyses versus separate naïve age‐matched mice. Prefrontal cortex (PFC) and hippocampus (HIPP) were assayed for levels of the GABAA receptor (GABAAR) alpha2 and alpha 4 subunits, P450scc (a steroidogenesis enzyme), and CRF R1 (corticotropin releasing factor receptor) with values compared to naïve mice. We found divergent responses across proteins by sex and brain area. In male PFC, there reduced levels for GABAAR alpha 4 subunit levels (to 0.86 ± 0.04% for control and 0.85 ± 0.04% for binge vs naïve) and a decrease in P450scc levels of 084 ± 0.04% (control) and 0.89 ± 0.04% (binge) whereas the male HIPP also showed a trend towards reduced levels of the GABAAR alpha 4 subunit (0.90 ± 0.04% and 0.85 ± 0.03% for control and binge, respectively) but an increase in CRF‐R1 levels to 1.26 ± 0.05% for control and 1.28 ± 0.04% for binge. In contrast, the female PFC had decreased levels of the GABAAR alpha 2 subunit (0.77 ± 0.12% for control and 0.74 ± 0.07% for binge) with a similar decrease in alpha 4 subunit levels as in male PFC (0.84 ± 0.11% for control and 0.78 ± 0.09% for binge). Also similar to male, female HIPP had increased levels for CRF‐R1 levels of 1.14 ± 0.05% for control and 1.15 ± 0.05% for binge. And while P450scc levels were decreased (0.84 ± 0.04% for both groups) in female HIPP, a similar change was only observed in male PFC. The similar changes in the binge and control groups vs naïve animals suggest that the repeated PS and ethanol drinking history during adulthood influenced neuroadaptations in brain proteins more than the adolescent binge drinking experience. Taken together, these data continue to support the suggestion that a history of ethanol drinking and stress may interact to increase the risk for harm from subsequent drinking with important sex differences in drinking behaviors and brain protein changes.Support or Funding InformationSupported by BX001070 and BX002966 (DF) from the Department of Veteran AffairsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Research on the Effects of the Chronic Treatment With Different Doses of Urocortin 2 in Heart Failure Rats.

Corticotropin-releasing factor (CRF) receptor type 2 (CRF2) exists in both cardiomyocytes and neurocytes. The purpose of this research was to explore if chronic treatment with urocortin 2 (UCN2), a CRF2 receptor agonist, at different doses can improve prognosis and regulate the expression of CRF2 receptor and calcium handling proteins without any adverse effects on behavior in heart failure. Heart failure was established in Sprague-Dawley rats and was confirmed by echocardiography. Heart failure rats were injected intraperitoneally with UCN2 (5, 10, or 20 µg·kg−1·d−1) for 30 days. Survival rate, cardiac function, expressions of cardiac CRF2 receptor, RyR2, SERCA2, and hypothalamic and hippocampal c-FOS, CRF receptor type 1 (CRF1) and CRF2 receptor were determined. Behavior was evaluated by Morris Water-Maze and Open-Field tests. Results showed that chronic peripheral UCN2 treatment improved survival rate in a dose–response manner and increased cardiac function and expression of CRF2 receptor and SERCA2 in heart failure, especially at the high dosage. Moreover, cellular-fos (c-FOS), CRF1 receptor, and CRF2 receptor expressions of both hypothalamic and hippocampal tissues were significantly increased in high dosage group. Furthermore, the behavior tests suggested that chronic UCN2 treatment did not exacerbate stress/anxiety-like behavior in HF. In conclusion, chronic peripheral treatment with UCN2 increases survival in a dose–response manner in heart failure rats without inducing stress/anxiety-like behavior.

The blockade of corticotropin-releasing factor 1 receptor attenuates anxiety-related symptoms and hypothalamus-pituitary-adrenal axis reactivity in mice with mild traumatic brain injury.

Recent studies have shown that mild traumatic brain injury (mTBI) is associated with higher risk for anxiety-related disorders. Dysregulation in the hypothalamus-pituitary-adrenal (HPA) axis following mTBI has been proposed to be involved in the development of neurobehavioral abnormalities; however, the underlying mechanisms are largely unknown. The aim of this study was to determine whether the corticotropin-releasing-factor-1 (CRF-1) receptor is involved in the regulation of anxiety-related symptoms in a mouse model of mTBI. Animals with or without mTBI received intracerebroventricular injections of a CRF-1 receptor agonist (CRF; 0.01 nmol/mouse) or antagonist (antalarmin; 1 µg/mouse) for 5 days, and then the animals were subjected to anxiety tests (light-dark box and zero maze). The levels of adrenocorticotropic hormone and corticosterone, the most important markers of HPA axis, were also measured after behavioral tests. Our results indicated that mTBI-induced anxiety-related symptoms in mice through increased levels of adrenocorticotropic hormone and corticosterone, showing HPA axis hyperactivity. Interestingly, activation of CRF receptor by a subthreshold dose of CRF resulted in significant increases in anxiety-like behaviors and HPA axis response to stress, whereas blockade of CRF receptors by a subthreshold dose of antalarmin decreased anxiety-related symptoms and HPA axis response to stress in mTBI-induced mice. Collectively, these findings suggest that the CRF-1 receptor plays an important role in the regulation of anxiety-related behaviors following mTBI induction in mice and support the hypothesis that blockade of the CRF-1 receptor may be a promising therapeutic target for anxiety-related disorders in patients with TBI.

MP11-13 CENTRALLY ADMINISTERED BOMBESIN INDUCES FREQUENT URINATION VIA BRAIN CORTICOTROPIN-RELEASING FACTOR RECEPTOR TYPE 1 IN RATS

INTRODUCTION AND OBJECTIVES:Psychological stress exacerbates bladder dysfunction such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC). We previously reported that bombesin (BB), a stress-related neuropeptide, centrally induces facilitation of the rat micturition reflex. Brain BB is reported to activate hypothalamo-pituitary-adrenal axis and sympathetic nerves, as representative responses to stressful conditions, via brain corticotropin-releasing factor (CRF), another stress-related neuropeptide. Therefore, we examined whether brain CRF is involved in the BB-induced frequent urination in rats.METHODS:(1) In urethane anesthetized (0.8 g/kg, ip) male Sprague-Dawley (SD) rats (300-400 g), a catheter was inserted into the bladder to perform cystometry (CMG, 12 ml/h saline infusion). Astressin (a non-selective CRF receptor antagonist, 1 or 3 nmol/rat) was intracerebroventricularly (icv) pretreated 60 min before BB administration (0.03 nmol/rat, icv). CMG was started 60 min b...

Comparison of Colonic Baseline Ion Secretion and Responses to Stress‐related Peptides between Male and Female Mice

Background and AimsSex plays an important role in the symptoms of irritable bowel syndrome (IBS) and responses to treatment. Constipation‐predominant IBS (IBS‐C) is more prevalent in women, whereas diarrhea‐predominant IBS (IBS‐D) is more common in men. Stress has been shown to increase intestinal ion secretion and may exacerbate symptoms in IBS‐D. Most of the previous preclinical studies investigating the effects of stress on gut function were performed in male animals. The aim of the present study was to determine if there was a sex difference in baseline ion secretion in the mouse colon and/or its responses to stress‐related peptides, including corticotrophin releasing hormone (CRF), urocortin 1 (Ucn1), Ucn2, and Ucn3.MethodsAdult C57BL/6J mice (47 males and 20 females) were used in the present study. Mucosa/submucosa preparations from the proximal colon were mounted in Ussing flux chambers for measurement of short‐circuit current (Isc) as an indicator of mucosal ion secretion.ResultsMale mice were found to exhibit a significantly (p < 0.05) higher baseline Isc (70.90 ± 4.84 μA/cm2, n = 47) in the proximal colon than females (52.92 ± 4.40 μA/cm2, n = 20). In addition, male mice exhibited a greater increase in colonic Isc (ΔIsc) than did female mice in response to 1 μM of CRF (10.26 ± 1.31 μA/cm2 vs. 4.59 ± 1.21 μA/cm2, n = 5/group, p < 0.05), 1 μM of Ucn1 (16.65 ± 2.23 μA/cm2 vs. 6.80 ± 1.16 μA/cm2, n = 5/group, p < 0.01), 1 μM of Ucn2 (21.76 ± 4.58 μA/cm2 vs. 6.72 ± 0.57 μA/cm2, n = 5/group, p < 0.05), or 1 μM of Ucn3 (41.41 ± 8.91 μA/cm2 vs. 7.86 ± 1.47 μA/cm2, n = 5, p < 0.01). Colonic Isc response to the selective CRF1 receptor agonist, stressin I (1 μM) was similar in male and female mice (10.19 ± 2.19 μA/cm2 vs. 7.08 ± 5.16 μA/cm2, n = 4–5/group, p > 0.05). In male mice, Ucn3 caused a significantly higher ΔIsc than stressin I, CRF, Ucn1, or Ucn2. Contrastingly, female mice displayed a similar and weaker ΔIsc response to these peptides.ConclusionsMarked sex differences were observed in colonic baseline ion secretion and responses to stress‐related peptides. Male mice demonstrated a significantly higher baseline ion secretion in the proximal colon when compared with their female counter parts. In addition, CRF, Ucn1, Unc2, and Ucn3 evoked a greater increase in colonic ion secretion in male mice than female mice. Ucn3 is more efficacious than the other stress‐related peptides to evoke ion secretion in the male mouse colon, suggesting a predominant role of the CRF2 receptors in stimulation colonic ion secretion in males. Female mice, however, exhibited low level responses to these stress‐related peptides, and no significant differences were seen between these peptides‐evoked responses. The results suggest that the greater colonic baseline ion secretion and responses to stress‐related peptides in male mice may contribute to the high prevalence of IBS‐D in males.Support or Funding InformationNIH R15 DK097460‐01A1 (SL) and UWL graduate research service and educational leadership grant (AK). Howard Hua is a summer research fellow from Wheeler High School, Marietta, GA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dehydroepiandrosterone sulfate improves visceral sensation and gut barrier in a rat model of irritable bowel syndrome

Stress-induced altered visceral sensation and impaired gut barrier play an important role in the pathophysiology of irritable bowel syndrome (IBS). These responses were demonstrated to be peripheral corticotropin-releasing factor (CRF) dependent and also mediated via proinflammatory cytokine in animal IBS model. Dehydroepiandrosterone sulfate (DHEA-S) is known to have anti-inflammatory properties by suppressing proinflammatory cytokine release. We hypothesized that DHEA-S improves stress-induced visceral changes and is beneficial for IBS treatment. We explored the effects of DHEA-S on lipopolysaccharide (LPS)- or repeated water avoidance stress (WAS)-induced visceral allodynia and increased colonic permeability (rat IBS models). The threshold of visceromotor response, i.e. abdominal muscle contractions induced by colonic balloon distention was electrophysiologically measured. Colonic permeability was estimated in vivo by quantifying the absorbed Evans blue in colonic tissue. DHEA-S abolished visceral allodynia and colonic hyperpermeability induced by LPS in a dose-dependent manner. It also blocked repeated WAS- or peripheral injection of CRF-induced visceral changes. These effects by DHEA-S in LPS model were reversed by bicuculline, a γ-aminobutyric acid (GABA)A receptor antagonist, NG-nitro-L-arginine methyl ester, a nitric oxide (NO) synthesis inhibitor, naloxone, an opioid receptor antagonist, or sulpiride, a dopamine D2 receptor antagonist. However, domperidone, a peripheral dopamine D2 receptor antagonist did not modify the effects. Peripheral injection of astressin2-B, a selective CRF receptor subtype 2 (CRF2) antagonist also reversed these effects. In conclusion, DHEA-S blocked stress-induced visceral changes via GABAA, NO, opioid, central dopamine D2 and peripheral CRF2 signaling. DHEA-S may be useful for IBS treating.

Pretreatment with a CRF antagonist amplifies feeding inhibition induced by fourth ventricular cocaine- and amphetamine-regulated transcript peptide

BackgroundPre-treatment with the corticotropin-releasing factor antagonist α-helical CRF9-41 prevents inhibition of gastric emptying by cocaine-and amphetamine-regulated transcript peptide at a dorsal hindbrain level, but its inhibition of sucrose intake is not affected. This is suggestive of separable underlying mechanisms of action in the caudal brainstem for cocaine-and amphetamine-regulated transcript peptide with regard to food intake and gastrointestinal functions. Here we further examine cocaine-and amphetamine-regulated transcript peptide—corticotropin-releasing factor receptor interactions in caudal brainstem controls of solid food intake. Injections of combinations of vehicle, cocaine-and amphetamine-regulated transcript peptide (0.5 μg or 1 μg) or α-helical CRF9-41 were given into the fourth cerebral ventricle of rats. Nocturnal solid food intake was recorded over 22 h.ResultsPre-treatment with α-helical CRF9-41 into the fourth ventricle significantly increased the responsivity to cocaine-and amphetamine-regulated transcript peptide on hypophagia. In a separate control experiment, α-helical CRF9-41 pre-treatment blocked CRF-induced food intake inhibition indicative of its antagonistic effectiveness.ConclusionsWe conclude that an endogenous Corticotropin-releasing factor agonist may modulate suppression of food intake caused by cocaine-and amphetamine-regulated transcript peptide at a dorsal hindbrain level in the absence of stress. A potential caudal brainstem mechanism whereby cocaine-and amphetamine-regulated transcript peptide effects on food intake is attenuated via corticotropin-releasing factor receptor activity causing tonic inhibition, is suggested.

Anorexigenic effects of mesotocin in chicks are genetic background-dependent and are associated with changes in the paraventricular nucleus and lateral hypothalamus

Mesotocin (MT) decreases food intake and induces hyperthermia in chicks although hypothalamic mechanisms are unknown. The purpose of this study was thus to investigate effects of receptor antagonists and MT on feeding behavior and hypothalamic physiology. Intracerebroventricular injection of 2.5 nmol into broiler chicks was associated with decreased food intake for 180 min and water intake from 60 to 180 min. Cloacal temperatures were elevated in chicks injected with 0.156 and 0.625 nmol at 30 and 60 min, and up to 180 min in those injected with 2.5 nmol. MT also increased temperatures and decreased food and water intake in chicks from lines selected for low (LWS) or high (HWS) body weight with a higher dose threshold but longer food intake response in HWS chicks. An oxytocin receptor antagonist prevented MT-mediated changes in food intake but not water intake or temperature. Yohimbine, an α2-adrenergic receptor antagonist, did not affect food intake, temperature, or MT-mediated effects. MT increased c-Fos immunoreactivity in the paraventricular nucleus (PVN) and lateral hypothalamus (LH). Hypothalamic agouti-related peptide, corticotropin-releasing factor receptor sub-type 1, and melanocortin receptor 3 mRNAs increased in response to MT. There was increased MT mRNA in the LH and L-aromatic amino acid decarboxylase mRNA in the PVN of MT-injected chicks. In conclusion, MT induced anorexia and hyperthermia and reduced water intake. MT was associated with activation of the PVN and LH and differences in the mRNA abundance of some appetite-associated factors, thus implicating these nuclei and several signaling pathways in the effects observed.

Isoflavones Alter Hypothalamic–Pituitary–Adrenal Axis Response Following Photoperiod Alteration

Photoperiod and diet are factors known to modulate the hypothalamic–pituitary–adrenal (HPA) axis. Specifically, shifts in photoperiod have been previously linked with affective and anxiety disorders. Furthermore, isoflavones have been shown to mediate behavioral outcome in response to the environment of the animal. Here, we investigated the effect of photoperiod alteration on the HPA axis and how the addition of isoflavones might modulate the response to stress. Male C57BL/6J mice were maintained on either a 12:12 or a 16:8 light–dark (LD) cycle for 10 days, and fed a diet of either standard rodent chow or an isoflavone free (IF) chow beginning 3 weeks prior to light alteration. Consistent with previous work, mice in the shorter active period (16:8 LD cycle) showed increased basal corticosterone (CORT) secretion. In the absence of isoflavones, this response was attenuated. Increases in mineralcorticoid (MR) and glucocorticoid (GR) receptor mRNA expression were seen in the pituitary following photoperiod alteration. However, animals fed the standard isoflavone rich chow showed increases in the ratio of MR:GR mRNA in the anterior bed nucleus of the stria terminalis following photoperiod alteration. Decreases in corticotrophin-releasing factor receptor 1 (CRFR1) mRNA expression were seen in animals fed the IF chow in the amygdala, prefrontal cortex and ventral hippocampus. These data suggest that alterations in CORT secretion following photoperiod alteration may be mediated through differences in CRFR1 gene expression or changes in MR:GR mRNA ratios. These findings provide insight into the potential mechanisms by which the HPA axis adapts to photoperiod and diet.

Interactions between RAMP2 and CRF receptors: The effect of receptor subtypes, splice variants and cell context

Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1α is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1β. It has been shown previously that co-expression of CRFR1β with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptor-like receptor (CLR). This study investigated the ability of CRFR1α, CRFR1β and CRFR2β to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [ΔCTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1α and CRFR1β enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([ΔCTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2β co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction. Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2β (histidine) in this interface region may impair CRFR2β:RAMP2 interaction by electrostatic repulsion.

Psychophysiological treatment outcomes: Corticotropin-releasing factor type 1 receptor antagonist increases inhibition of fear-potentiated startle in PTSD patients.

After exposure to a traumatic event, a subset of people develop post-traumatic stress disorder (PTSD). One of the key deficits in PTSD is regulation of fear, and impaired inhibition of fear-potentiated startle (FPS) has been identified as a potential physiological biomarker specific to PTSD. As part of a larger clinical trial, this study investigated the effects of a CRF receptor 1 antagonist, GSK561679, on inhibition of fear-potentiated startle during a conditional discrimination fear-conditioning paradigm, termed AX+/BX-. Prior research using this paradigm has demonstrated deficits in inhibition of conditioned fear in several PTSD populations. The randomized, double-blind, placebo-controlled clinical trial compared fear inhibition between female PTSD participants taking 350mg/day GSK561679 (n=47 pre- and 29 post-treatment) and patients taking a placebo pill (n=52 pre- and 30 post-treatment) daily for 6weeks. There was no significant difference between the two groups in their acquisition of fear or discrimination between threat and safety cues, and no pre-post-treatment effect on these measures. However, there was a significant effect of treatment on inhibition of FPS during the AB trials in the AX+/BX- transfer test (p<0.05). While all PTSD participants showed typical impairments in fear inhibition prior to treatment, GSK561679 enhanced fear inhibition post-treatment, independent of clinical effects. The current study suggests that CRF receptor 1 antagonism may have specific effects within neural circuitry mediating fear inhibition responses, but not overall symptom presentation, in PTSD.

Hypothalamic mechanism of corticotropin-releasing factor’s anorexigenic effect in Japanese quail (Coturnix japonica)

Central administration of corticotropin-releasing factor (CRF), a 41-amino acid peptide, is associated with anorexigenic effects across various species, with particularly potent reductions in food intake in rodents and chickens (Gallus gallus domesticus), a species for which the most is known. The purpose of the current study was to determine the hypothalamic mechanism of CRF-induced anorexigenic effects in 7 day-old Japanese quail (Coturnix japonica), a less-intensely-selected gallinaceous relative to the chicken that can provide more evolutionary perspective. After intracerebroventricular (ICV) injection of 2, 22, or 222 pmol of CRF, a dose-dependent decrease in food intake was observed that lasted for 3 and 24 h for the 22 and 222 pmol doses, respectively. The 2 pmol dose had no effect on food or water intake. The numbers of c-Fos immunoreactive cells were increased in the paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) at 1 h post-injection in quail injected with 22 pmol of CRF. The hypothalamic mRNA abundance of proopiomelanocortin, melanocortin receptor subtype 4, CRF, and CRF receptor sub-type 2 was increased at 1 h in quail treated with 22 pmol of CRF. Behavior analyses demonstrated that CRF injection reduced feeding pecks and jumps and increased the time spent standing. In conclusion, results demonstrate that the anorexigenic effects of CRF in Japanese quail are likely influenced by the interaction between CRF and melanocortin systems and that injection of CRF results in species-specific behavioral changes.

Plasma Corticotropin-Releasing Factor Receptors and B7-2⁺ Extracellular Vesicles in Blood Correlate with Irritable Bowel Syndrome Disease Severity.

Extracellular vesicles (EVs) are composed of bilayer membranes that are released by different cell types and are present in bodily fluids, such as blood, urine, and bile. EVs are thought to play a key role in intracellular communication. Based on their size and density, EVs are classified into small, medium, or large EVs. Cargo composition in EVs reflects physiological changes in health and disease. Patients with irritable bowel syndrome (IBS) exhibit visceral hypersensitivity and mood disorders. Stressful episodes often precede disease symptoms in IBS patients. Stress-induced symptoms include, but are not limited to, abdominal pain and mood swings. Perceived stress responses are mediated by two known G protein-coupled receptors (GPCRs), corticotropin-releasing factor receptor 1 and 2 (CRFRs). CRFRs belong to the Class B secretin receptor family of GPCRs. Here, we show that CRFRs were present in human and murine plasma, and in EVs purified from mouse serum. CRFRs were present in plasma from IBS patients and healthy controls. EVs secreted from immune cells influence both adaptive and innate immune responses via exchange of EVs between different immune cell types. B7-2 (CD86), a plasma membrane antigen-presenting protein, is present on EVs secreted from dendritic, B-, and mast cells, whereas CD9 is present on EVs secreted from dendritic and intestinal epithelial cells. We found that plasma CRFR levels positively correlated with B7-2+ EVs (R = 0.8597, p < 0.0001), but no association was seen with CD9+ EVs. Plasma CRFRs expression negatively correlated with IBS severity scores. Our data suggests that plasma EVs from immune cells carry CRFRs as cargos and influence cell-cell communication in health and disease.

Exploring Pairwise Chemical Crosslinking To Study Peptide-Receptor Interactions.

Pairwise crosslinking is a powerful technique to characterize interactions between G protein coupled receptors and their ligands in the live cell. In this work, the "thiol trapping" method, which exploits the proximity-enhanced reaction between haloacetamides and cysteine, is examined to identify intermolecular pairs of vicinal positions. By incorporating cysteine into the corticotropin-releasing factor receptor and either α-chloro- or α-bromoacetamide groups into its ligands, it is shown that thiol trapping provides highly reproducible signals and a low background, and represents a valid alternative to classical "disulfide trapping". The method is advantageous if reducing agents are required during sample analysis. Moreover, it can provide partially distinct spatial constraints, thus giving access to a wider dataset for molecular modeling. Finally, by applying recombinant mini-Gs, GTPγS, and Gαs-depleted HEK293 cells to modulate Gs coupling, it is shown that yields of crosslinking increase in the presence of elevated levels of Gs.

Mom doesn't care: When increased brain CRF system activity leads to maternal neglect in rodents.

Mothers are the primary caregivers in mammals, ensuring their offspring's survival. This strongly depends on the adequate expression of maternal behavior, which is the result of a concerted action of "pro-maternal" versus "anti-maternal" neuromodulators such as the oxytocin and corticotropin-releasing factor (CRF) systems, respectively. When essential peripartum adaptations fail, the CRF system has negative physiological, emotional and behavioral consequences for both mother and offspring often resulting in maternal neglect. Here, we provide an elaborate and unprecedented review on the implications of the CRF system in the maternal brain. Studies in rodents have advanced our understanding of the specific roles of brain regions such as the limbic bed nucleus of the stria terminalis, medial preoptic area and lateral septum even in a CRF receptor subtype-specific manner. Furthermore, we discuss potential interactions of the CRF system with other neurotransmitters like oxytocin and noradrenaline, and present valuable translational aspects of the recent research.

Prefrontal Corticotropin-Releasing Factor (CRF) Neurons Act Locally to Modulate Frontostriatal Cognition and Circuit Function.

The PFC and extended frontostriatal circuitry support higher cognitive processes that guide goal-directed behavior. PFC-dependent cognitive dysfunction is a core feature of multiple psychiatric disorders. Unfortunately, a major limiting factor in the development of treatments for PFC cognitive dysfunction is our limited understanding of the neural mechanisms underlying PFC-dependent cognition. We recently demonstrated that activation of corticotropin-releasing factor (CRF) receptors in the caudal dorsomedial PFC (dmPFC) impairs higher cognitive function, as measured in a working memory task. Currently, there remains much unknown about CRF-dependent regulation of cognition, including the source of CRF for cognition-modulating receptors and the output pathways modulated by these receptors. To address these issues, the current studies used a viral vector-based approach to chemogenetically activate or inhibit PFC CRF neurons in working memory-tested male rats. Chemogenetic activation of caudal, but not rostral, dmPFC CRF neurons potently impaired working memory, whereas inhibition of these neurons improved working memory. Importantly, the cognition-impairing actions of PFC CRF neurons were dependent on local CRF receptors coupled to protein kinase A. Additional electrophysiological recordings demonstrated that chemogenetic activation of caudal dmPFC CRF neurons elicits a robust degradation of task-related coding properties of dmPFC pyramidal neurons and, to a lesser extent, medium spiny neurons in the dorsomedial striatum. Collectively, these results demonstrate that local CRF release within the caudal dmPFC impairs frontostriatal cognitive and circuit function and suggest that CRF may represent a potential target for treating frontostriatal cognitive dysfunction.SIGNIFICANCE STATEMENT The dorsomedial PFC and its striatal targets play a critical role in higher cognitive function. PFC-dependent cognitive dysfunction is associated with many psychiatric disorders. Although it has long-been known that corticotropin-releasing factor (CRF) neurons are prominent within the PFC, their role in cognition has remained unclear. Using a novel chemogenetic viral vector system, the present studies demonstrate that PFC CRF neurons impair working memory via activation of local PKA-coupled CRF receptors, an action associated with robust degradation in task-related frontostriatal neuronal coding. Conversely, suppression of constitutive PFC CRF activity improved working memory. Collectively, these studies provide novel insight into the neurobiology of cognition and suggest that CRF may represent a novel target for the treatment of cognitive dysfunction.