Corticotropin releasing factor alters the functional diversity of accumbal cholinergic interneurons.

Cholinergic interneurons (ChIs) provide the main source of acetylcholine in the striatum and have emerged as a critical modulator of behavioral flexibility, motivation, and associative learning. In the dorsal striatum (DS), ChIs display heterogeneous firing patterns. Here, we investigated the spontaneous firing patterns of ChIs in the nucleus accumbens (NAc) shell, a region of the ventral striatum. We identified four distinct ChI firing signatures: regular single-spiking, irregular single-spiking, rhythmic bursting, and a mixed-mode pattern composed of bursting activity and regular single spiking. ChIs from females had lower firing rates compared with males and had both a higher proportion of mixed-mode firing patterns and a lower proportion of regular single-spiking neurons compared with males. We further observed that across the estrous cycle, the diestrus phase was characterized by higher proportions of irregular ChI firing patterns compared with other phases. Using pooled data from males and females, we examined how the stress-associated neuropeptide corticotropin releasing factor (CRF) impacts these firing patterns. ChI firing patterns showed differential sensitivity to CRF. This translated into differential ChI sensitivity to CRF across the estrous cycle. Furthermore, CRF shifted the proportion of ChI firing patterns toward more regular spiking activity over bursting patterns. Finally, we found that repeated stressor exposure altered ChI firing patterns and sensitivity to CRF in the NAc core, but not the NAc shell. These findings highlight the heterogeneous nature of ChI firing patterns, which may have implications for accumbal-dependent motivated behaviors.NEW & NOTEWORTHY Cholinergic interneurons (ChIs) within the dorsal and ventral striatum can exert a major influence on network output and motivated behaviors. However, the firing patterns and neuromodulation of ChIs within the ventral striatum, specifically the nucleus accumbens (NAc) shell, are understudied. Here, we report that NAc shell ChIs have heterogeneous ChI firing patterns that are labile and can be modulated by the stress-linked neuropeptide corticotropin releasing factor (CRF) and by the estrous cycle.

Peripubertal antagonism of corticotropin-releasing factor receptor 1 results in sustained, sex-specific changes in behavioral plasticity and the transcriptomic profile of the amygdala.

Peripuberty is a significant period of neurodevelopment with long-lasting effects on the brain and behavior. Blocking type 1 corticotropin-releasing factor receptors (CRFR1) in neonatal and peripubertal rats attenuates detrimental effects of early-life stress on neural plasticity, behavior, and stress hormone action, long after exposure to the drug has ended. CRFR1 antagonism can also impact neural and behavioral development in the absence of stressful stimuli, suggesting sustained alterations under baseline conditions. To investigate this further, we administered a CRFR1 antagonist (CRFR1a), R121919, to young adolescent male and female rats across 4 days. Following each treatment, rats were tested for locomotion, social behavior, mechanical allodynia, or PPI of the acoustic startle reflex. Acute CRFR1 blockade immediately reduced PPI in peripubertal males, but not females. In adulthood, each assay was repeated without CRFR1a exposure to test for long-term effects of the adolescent treatment, with males continuing to experience deficits in PPI, while females displayed altered locomotion, PPI, and social behavior. The amygdala was collected to measure long-term effects on gene expression in pathways related to neural plasticity and neurodevelopmental disorders. Relative expression of cannabinoid type 1 receptors (CB1R), which mediate sensorimotor and HPA function, was also measured. In the adult amygdala, peripubertal CRFR1a induced alterations in pathways related to neural plasticity and stress in males and lower expression of CB1R protein in females. Understanding how acute exposure to neuropharmacological agents can have sustained impacts on brain and behavior, in the absence of further exposures, has important clinical implications for adolescent psychiatric treatment protocols.

Cells and circuits for amygdala neuroplasticity in the transition to chronic pain.

Maladaptive plasticity is linked to the chronification of diseases such as pain, but the transition from acute to chronic pain is not well understood mechanistically. Neuroplasticity in the central nucleus of the amygdala (CeA) has emerged as a mechanism for sensory and emotional-affective aspects of injury-induced pain, although evidence comes from studies conducted almost exclusively in acute pain conditions and agnostic to cell type specificity. Here, we report time-dependent changes in genetically distinct and projection-specific CeA neurons in neuropathic pain. Hyperexcitability of CRF projection neurons and synaptic plasticity of parabrachial (PB) input at the acute stage shifted to hyperexcitability without synaptic plasticity in non-CRF neurons at the chronic phase. Accordingly, chemogenetic inhibition of the PB→CeA pathway mitigated pain-related behaviors in acute, but not chronic, neuropathic pain. Cell-type-specific temporal changes in neuroplasticity provide neurobiological evidence for the clinical observation that chronic pain is not simply the prolonged persistence of acute pain.

CRF release from a unique subpopulation of accumbal neurons constrains action-outcome acquisition in reward learning.

BACKGROUND: The nucleus accumbens (NAc) mediates reward learning and motivation. Despite an abundance of neuropeptides, peptidergic neurotransmission from the NAc has not been integrated into current models of reward learning. The existence of a sparse population of neurons containing corticotropin releasing factor (CRF) has been previously documented. Here we provide a comprehensive analysis of their identity and functional role in shaping reward learning. METHODS: To do this, we took a multidisciplinary approach that included florescent in situ hybridization (Nmice ≥ 3), tract tracing (Nmice = 5), ex vivo electrophysiology (Ncells ≥ 30), in vivo calcium imaging with fiber photometry (Nmice ≥ 4) and use of viral strategies in transgenic lines to selectively delete CRF peptide from NAc neurons (Nmice ≥ 4). Behaviors used were instrumental learning, sucrose preference and spontaneous exploration in an open field. RESULTS: Here we show that the vast majority of NAc CRF-containing (NAcCRF) neurons are spiny projection neurons (SPNs) comprised of dopamine D1-, D2- or D1/D2-containing SPNs that primarily project and connect to the ventral pallidum and to a lesser extent the ventral midbrain. As a population, they display mature and immature SPN firing properties. We demonstrate that NAcCRF neurons track reward outcomes during operant reward learning and that CRF release from these neurons acts to constrain initial acquisition of action-outcome learning, and at the same time facilitates flexibility in the face of changing contingencies. CONCLUSION: We conclude that CRF release from this sparse population of SPNs is critical for reward learning under normal conditions.

The effects of corticotropin-releasing factor on motor learning.

Corticotropin-releasing factor (CRF) is mainly secreted from the hypothalamic paraventricular nuclei and plays a crucial role in stress-related responses. Recent studies have reported that CRF is a neuromodulator in the central nervous system. In the cerebellum, CRF is essential for the induction of long-term depression (LTD) at the parallel fiber-Purkinje cell synapses. Given that LTD is thought to be one of the fundamental mechanisms of motor learning, CRF may affect motor learning. However, the role of CRF in motor learning in vivo remains unclear. In this study, we aimed to examine the role of CRF in motor learning. This was achieved through a series of behavioral experiments involving the in vivo administration of CRF and its antagonists. Rats injected with CRF directly into the cerebellum exhibited superior performance on the rotarod test, especially during initial training phases, compared to control subjects. Conversely, rats receiving a CRF receptor antagonist demonstrated reduced endurance on the rotating rod compared to controls. Notably, CRF mRNA expression levels in the cerebellum did not show significant variance between the CRF-injected and control groups. These findings imply a critical role of endogenous CRF in cerebellar motor learning and suggest that exogenous CRF can augment this process. (199 words).

Effect of sex on chronic stress induced alterations in hindbrain catecholaminergic system and autonomic dysfunction resulting in gastrointestinal dysmotility.

It has been reported that the clinical symptoms of functional dyspepsia (FD) exacerbate upon stress while the gender-related factors have been incompletely understood. This study aims to investigate the role of sex in chronic heterotypic stress (CHS)-induced autonomic and gastric motor dysfunction. For CHS, the rats were exposed to the combination of different stressors for 7 consecutive days. Subsequently, electrocardiography was recorded in anesthetized rats to evaluate heart rate variability (HRV) for the determination of autonomic outflow and sympathovagal balance. Solid gastric emptying (GE) was measured in control and CHS-loaded male and female rats. The immunoreactivities of catecholaminergic cell marker tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), corticotropin releasing factor (CRF), and estrogen receptor (ER-α/ß) were evaluated in medullary and pontine brainstem sections by immunohistochemistry. Compared with the controls, CHS significantly delayed GE in males but not in females. There was no significant sex-related difference in parasympathetic indicator HF under either control or CHS conditions. Sympathetic indicator LF was significantly higher in control females compared to the males. The higher sympathetic output in females was found to be attenuated upon CHS; in contrast, the elevated sympathetic output was detected in CHS-loaded males. No sex- or stress-related effect was observed on ChAT immunoreactivity in the dorsal motor nucleus of N.vagus (DMV). In males, greater number of TH-ir cells was observed in the caudal locus coeruleus (LC), while they were more densely detected in the rostral LC of females. Regardless of sex, CHS elevated immunoreactivity of TH throughout the LC. Under basal conditions, greater number of TH-ir cells was detected in the rostral ventrolateral medulla (RVLM) of females. In contrast, CHS remarkably increased the number of TH-ir cells in the RVLM of males which was found to be decreased in females. There was no sex-related alteration in TH immunoreactivity in the nucleus tractus solitarius (NTS) of control rats, while CHS affected both sexes in a similar manner. Compared with females, CRF immunoreactivity was prominently observed in control males, while both of which were stimulated by CHS. ER-α/ß was found to be co-expressed with TH in the NTS and LC which exhibit no alteration related to either sex or stress status. These results indicate a sexual dimorphism in the catecholaminergic and the CRF system in brainstem which might be involved in the CHS-induced autonomic and visceral dysfunction occurred in males.

Sex-specific alterations in visual properties induced by single prolonged stress model.

Patients with post-traumatic stress disorder (PTSD) exhibit sex differences in symptomology, with women more likely to report higher rates of intrusive and avoidance symptoms than men, underscoring the need for sex-informed approaches to research and treatment. Our study delved into the sex-specific aspects of stress-induced visual impairments using the single prolonged stress (SPS) model, a partially validated rodent model for PTSD. Male SPS mice exhibit heightened optimal spatial frequency (SF) of primary visual cortex (V1) neurons, while female counterparts exhibit decreased optimal temporal frequency (TF) of V1 neurons. This phenomenon persisted until the 29th day after SPS modeling, and it may be the physiological basis for the observed increase in visual acuity in male SPS mice in visual water task. Furthermore, our study found that corticotropin-releasing factor receptor 1 regulated optimal TF and optimal SF of V1 in mice, but did not exhibit sex differences. These findings indicated that severe stress induces sex-specific effects on visual function.

Cholinergic interneurons in the nucleus accumbens are a site of cellular convergence for corticotropin-releasing factor and estrogen regulation in male and female mice.

Cholinergic interneurons (ChIs) act as master regulators of striatal output, finely tuning neurotransmission to control motivated behaviours. ChIs are a cellular target of many peptide and hormonal neuromodulators, including corticotropin-releasing factor, opioids, insulin and leptin, which can influence an animal's behaviour by signalling stress, pleasure, pain and nutritional status. However, little is known about how sex hormones via estrogen receptors influence the function of these other neuromodulators. Here, we performed in situ hybridisation on mouse striatal tissue to characterise the effect of sex and sex hormones on choline acetyltransferase (Chat), estrogen receptor alpha (Esr1) and corticotropin-releasing factor type 1 receptor (Crhr1) expression. Although we did not detect sex differences in ChAT protein levels in the dorsal striatum or nucleus accumbens, we found that female mice have more Chat mRNA-expressing neurons than males in both the dorsal striatum and nucleus accumbens. At the population level, we observed a sexually dimorphic distribution of Esr1- and Crhr1-expressing ChIs in the ventral striatum that was negatively correlated in intact females, which was abolished by ovariectomy and not present in males. Only in the NAc did we find a significant population of ChIs that co-express Crhr1 and Esr1 in females and to a lesser extent in males. At the cellular level, Crhr1 and Esr1 transcript levels were negatively correlated only during the estrus phase in females, indicating that changes in sex hormone levels can modulate the interaction between Crhr1 and Esr1 mRNA levels.

Effects of Repetitive Mild Traumatic Brain Injury on Corticotropin-Releasing Factor Modulation of Lateral Habenula Excitability and Motivated Behavior.

Mild traumatic brain injury (mTBI) is a significant health burden due to mTBI-related chronic debilitating cognitive and psychiatric morbidities. Recent evidence from our laboratory suggests a possible dysregulation within reward/motivational circuit function at the level of a subcortical structure, the lateral habenula (LHb), where we demonstrated a causal role for hyperactive LHb in mTBI-induced motivational deficits in self-care grooming behavior in young adult male mice when exposed to mTBI during late adolescence (at ∼8 weeks old). In this study, we extended this observation by further characterizing neurobehavioral effects of this repetitive closed head injury model of mTBI in both young adult male and female mice on LHb excitability, corticotropin releasing factor (CRF) modulation of LHb activity, and behavioral responses of motivation to self-care behavior and approach versus avoidance behavior in the presence of a social- or threat-related stimulus. We show that mTBI increases LHb spontaneous tonic activity in female mice similar to what we previously observed in male mice, as well as promoting LHb neuronal hyperexcitability and hyperpolarization-induced LHb bursting in both male and female mice. Interestingly, mTBI only increases LHb intrinsic excitability in male mice coincident with higher levels of the hyperpolarization-activated cation currents (HCN/Ih) and reduces levels of the M-type potassium currents while potentiating M-currents without altering intrinsic excitability in LHb neurons of female mice. Because persistent dysregulation of brain CRF systems is suggested to contribute to chronic psychiatric morbidities and that LHb neurons are highly responsive to CRF, we tested whether the LHb CRF subsystem becomes engaged following mTBI. We found that in vitro inhibition of CRF receptor type 1 (CRFR1) within the LHb reverses mTBI-induced enhancement of LHb tonic activity and hyperexcitability in both sexes, suggesting that an augmented intra-LHb CRF-CRFR1-mediated signaling contributes to the overall LHb hyperactivity following mTBI. Behaviorally, mTBI diminishes motivation for self-care grooming in female mice as in male mice. mTBI also alters defensive behaviors in the looming shadow task by shifting the innate defensive behaviors toward more passive action locking rather than escape behaviors in response to an aerial threat in both male and female mice, as well as prolonging the latency to escape responses in female mice. While this model of mTBI reduces social preference in male mice, it induces higher social novelty seeking during the novel social encounters in both male and female mice. Overall, our study provides further translational validity for the use of this pre-clinical model of mTBI for investigation of mTBI-related reward circuit dysfunction and mood/motivation-related behavioral deficits in both sexes while uncovering a few sexually dimorphic neurobehavioral effects of this model that may differentially affect young males and females when exposed to this type of mTBI during late adolescence.

Glucose attenuates the long-term adverse neurodevelopment effect of neonate pain stimulus via CRF/GR in rats.

BACKGROUND: Neonates undergo numerous painful procedures throughout their hospitalization. Repeated procedural pain may cause adverse long-term effects. Glucose as a non-pharmacological analgesia, is used for neonate pain management. In this study, potential mechanism of attenuate pain induced by glucose in neurodevelopment effect of neonate pain stimulus was investigated. METHODS: Neonatal rats to perform a repetitive injury model and glucose intervention model in the postnatal day 0-7(P0-7). Pain thresholds were measured by von Frey test weekly. The puberty behavioral outcome, tissue loss and protein expression in hippocampus were analyzed. RESULTS: Oral administration of glucose after repeated pain stimulation can maintain the hippocampal structure in, and reduce the expressions of corticotropin releasing factor (CFR) and glucocorticoid receptor (GR), therefore, resulted in long-term threshold of pain and cognitive improvement. CONCLUSION: Exposure to neonatal repeated procedural pain causes persistent mechanical hypersensitivity and the dysfunction of spatial memory retention at puberty. In addition, glucose can relieve these adverse effects, possibly via decreasing CRF/GR levels to change the hypothalamus-pituitary-adrenal (HPA) axis.

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.

Effects of Repetitive Mild Traumatic Brain Injury on Corticotropin-Releasing Factor Modulation of Lateral Habenula Excitability and Motivated Behavior.

Mild traumatic brain injury (mTBI) is a significant health burden due to mTBI-related chronic debilitating cognitive and psychiatric morbidities. Recent evidence from our laboratory suggests a possible dysregulation within reward/motivational circuit function at the level of a subcortical structure, the lateral habenula (LHb), where we demonstrated a causal role for hyperactive LHb in mTBI-induced motivational deficits in self-care grooming behavior in young adult male mice when exposed to mTBI injury during late adolescence (at ~8 weeks old). Here we extended this observation by further characterizing neurobehavioral effects of this repetitive closed head injury model of mTBI in both young adult male and female mice on LHb excitability, corticotropin releasing factor (CRF) modulation of LHb activity, and behavioral responses of motivation to self-care behavior, and approach versus avoidance behavior in the presence of a social- or threat-related stimulus. We show that mTBI increases LHb spontaneous tonic activity in female mice similar to what we previously observed in male mice as well as promoting LHb neuronal hyperexcitability and hyperpolarization-induced LHb bursting in both male and female mice. Interestingly, mTBI only increases LHb intrinsic excitability in male mice coincident with higher levels of the hyperpolarization-activated cation currents (HCN/Ih) and reduces levels of the M-type potassium currents while potentiating M-currents without altering intrinsic excitability in LHb neurons of female mice. Since persistent dysregulation of brain CRF systems is suggested to contribute to chronic psychiatric morbidities and that LHb neurons are highly responsive to CRF, we then tested whether LHb CRF subsystem becomes engaged following mTBI. We found that in vitro inhibition of CRF receptor type 1 (CRFR1) within the LHb normalizes mTBI-induced enhancement of LHb tonic activity and hyperexcitability in both sexes, suggesting that an augmented intra-LHb CRF-CRFR1-mediated signaling contributes to the overall LHb hyperactivity following mTBI. Behaviorally, mTBI diminishes motivation for self-care grooming in female mice as in male mice. mTBI also alters defensive behaviors in the looming shadow task by shifting the innate defensive behaviors towards more passive action-locking rather than escape behaviors in response to an aerial threat in both male and female mice as well as prolonging the latency to escape responses in female mice. While, this model of mTBI reduces social preference in male mice, it induces higher social novelty seeking during the novel social encounters in both male and female mice. Overall, our study provides further translational validity for the use of this preclinical model of mTBI for investigation of mTBI-related reward circuit dysfunction and mood/motivation-related behavioral deficits in both sexes while uncovering a few sexually dimorphic neurobehavioral effects of this model that may differentially affect young males and females when exposed to this type of mTBI injury during late adolescence.

Mechanisms of Peitu Yimu acupuncture in repairing intestinal mucosal barrier by regulating CRF/CRFR1 pathway in diarrhea-predominant irritable bowel syndrome rats. / 培土抑木针法调节CRF/CRFR1é€šè·¯ä¿®å¤è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾å¤§é¼ è‚ é»è†œå±éšœçš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To investigate the effect of Peitu Yimu(strengthening spleen and soothing liver) acupuncture on intestinal mucosal barrier function and corticotropin-releasing factor (CRF)/CRF receptor 1 (CRFR1) pathway in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), so as to explore its underlying mechanism in alleviating IBS-D. METHODS: Forty female SD rats were randomly divided into blank, model, electroacupuncture (EA), and agonist groups, with 10 rats in each group. Except for the blank group, rats in the other groups were given folium sennae infusion by gavage combined with chronic unpredictable mild stress to establish IBS-D model. Rats in the EA group received acupuncture at "Tianshu"(ST25) and EA at "Zusanli"(ST36) and "Taichong"(LR3) (2 Hz/15 Hz) on one side for 20 min, with the side chosen alternately every other day, for 14 days after modeling. Rats in the agonist group received acupuncture 30 min after intravenous injection of CRFR1 agonist urocortin, with the same manipulation method and time as the EA group. Before and after intervention, visceral pain threshold and stool Bristol scores were measured. Elevated plus maze test and open field test were used to detect anxiety and depression like behavior of rats. ELISA was used to detect the contents of CRF and CRFR1 in rats serum. Immunohistochemistry was used to detect the positive expressions of CRF, CRFR1, zonula occludens protein 1(ZO-1), occlusal protein(Occludin), and closure protein 1 (Claudin-1) in colon tissue. RESULTS: Compared with the blank group, the visceral pain threshold, open arm time percentage (OT%), total distance of movement in the open field test, and positive expression of ZO-1, Occludin, and Claudin-1 in colon were decreased (P<0.01, P<0.05), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were increased (P<0.01) in the model group. After intervention and compared with the model group, the visceral pain threshold, OT%, total distance of movement in the open field test, and positive expressions of ZO-1, Occludin, and Claudin-1 in colon were increased (P<0.05, P<0.01), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were decreased (P<0.01) in the EA group；the Bristol stool scores, serum CRF content, and CRF positive expression in colon were significantly decreased in the agonist group (P<0.01). CONCLUSIONS: Peitu Yimu acupuncture can significantly improve visceral hypersensitivity and anxiety-depression state in IBS-D rats. Its mechanism may be related to the inhibition of CRF/CRFR1 pathway and restoration of intestinal tight junction protein expressions.

Chronic pain enhances excitability of corticotropin-releasing factor-expressing neurons in the oval part of the bed nucleus of the stria terminalis.

We previously reported that enhanced corticotropin-releasing factor (CRF) signaling in the bed nucleus of the stria terminalis (BNST) caused the aversive responses during acute pain and suppressed the brain reward system during chronic pain. However, it remains to be examined whether chronic pain alters the excitability of CRF neurons in the BNST. In this study we investigated the chronic pain-induced changes in excitability of CRF-expressing neurons in the oval part of the BNST (ovBNSTCRF neurons) by whole-cell patch-clamp electrophysiology. CRF-Cre; Ai14 mice were used to visualize CRF neurons by tdTomato. Electrophysiological recordings from brain slices prepared from a mouse model of neuropathic pain revealed that rheobase and firing threshold were significantly decreased in the chronic pain group compared with the sham-operated control group. Firing rate of the chronic pain group was higher than that of the control group. These data indicate that chronic pain elevated neuronal excitability of ovBNSTCRF neurons.

Teneurin C-terminal associated peptide (TCAP)-1 attenuates the development and expression of naloxone-precipitated morphine withdrawal in male Swiss Webster mice.

RATIONALE: Corticotropin-releasing factor (CRF), the apical stress-inducing hormone, exacerbates stress and addictive behaviors. TCAP-1 is a peptide that directly inhibits both CRF-mediated stress and addiction-related behaviors; however, the direct action of TCAP-1 on morphine withdrawal-associated behaviors has not previously been examined. OBJECTIVE: To determine whether TCAP-1 administration attenuates behavioral and physiological consequences of morphine withdrawal in mice. METHODS: Mice were administered via subcutaneous route TCAP-1 either before or after initial morphine exposure, after which jumping behavior was quantified to assess the effects of TCAP-1 on naloxone-precipitated morphine withdrawal. As a comparison, mice were treated with nonpeptide CRF1 receptor antagonist CP-154,526. In one experiment, plasma corticosterone (CORT) was also measured as a physiological stress indicator. RESULTS: Pretreatment with TCAP-1 (10-250 nmol/kg) before morphine treatment significantly inhibited the development of naloxone-precipitated withdrawal. TCAP-1 (250-500 nmol/kg) treatment administered after morphine treatment attenuated the behavioral expression of naloxone-precipitated withdrawal. TCAP-1 (250 nmol/kg) treatment during morphine treatment was more effective than the optimal dosing of CP-154,526 (20 mg/kg) at suppressing the behavioral expression of naloxone-precipitated withdrawal, despite similar reduction of withdrawal-induced plasma CORT level increases. CONCLUSIONS: These findings establish TCAP-1 as a potential therapeutic candidate for the prevention and treatment of morphine withdrawal.

CRF binding protein activity in the hypothalamic paraventricular nucleus is essential for stress adaptations and normal maternal behaviour in lactating rats.

To ensure the unrestricted expression of maternal behaviour peripartum, activity of the corticotropin-releasing factor (CRF) system needs to be minimised. CRF binding protein (CRF-BP) might be crucial for this adaptation, as its primary function is to sequester freely available CRF and urocortin1, thereby dampening CRF receptor (CRF-R) signalling. So far, the role of CRF-BP in the maternal brain has barely been studied, and a potential role in curtailing activation of the stress axis is unknown. We studied gene expression for CRF-BP and both CRF-R within the paraventricular nucleus (PVN) of the hypothalamus. In lactating rats, Crh-bp expression in the parvocellular PVN was significantly higher and Crh-r1 expression in the PVN significantly lower compared to virgin rats. Acute CRF-BP inhibition in the PVN with infusion of CRF(6-33) increased basal plasma corticosterone concentrations under unstressed conditions in dams. Furthermore, while acute intra-PVN infusion of CRF increased corticosterone secretion in virgin rats, it was ineffective in vehicle (VEH)-pre-treated lactating rats, probably due to a buffering effect of CRF-BP. Indeed, pre-treatment with CRF(6-33) reinstated a corticosterone response to CRF in lactating rats, highlighting the critical role of CRF-BP in maintaining attenuated stress reactivity in lactation. To our knowledge, this is the first study linking hypothalamic CRF-BP activity to hypothalamic-pituitary-adrenal axis regulation in lactation. In terms of behaviour, acute CRF-BP inhibition in the PVN under non-stress conditions reduced blanket nursing 60 min and licking/grooming 90 min after infusion compared to VEH-treated rats, while increasing maternal aggression towards an intruder. Lastly, chronic intra-PVN inhibition of CRF-BP strongly reduced maternal aggression, with modest effects on maternal motivation and care. Taken together, intact activity of the CRF-BP in the PVN during the postpartum period is essential for the dampened responsiveness of the stress axis, as well as for the full expression of appropriate maternal behaviour.

Inhibiting CRF Projections from the Central Amygdala to Lateral Hypothalamus and Amygdala Deletion of CRF Alters Binge-Like Ethanol Drinking in a Sex-Dependent Manner.

Background: Binge alcohol drinking is a dangerous pattern of consumption that can contribute to the development of more severe alcohol use disorders (AUDs). Importantly, the rate and severity of AUDs has historically differed between men and women, suggesting that there may be sex differences in the central mechanisms that modulate alcohol (ethanol) consumption. Corticotropin releasing factor (CRF) is a centrally expressed neuropeptide that has been implicated in the modulation of binge-like ethanol intake, and emerging data highlight sex differences in central CRF systems. Methods: In the present report we characterized CRF+ neurocircuitry arising from the central nucleus of the amygdala (CeA) and innervating the lateral hypothalamus (LH) in the modulation of binge-like ethanol intake in male and female mice. Results: Using chemogenetic tools we found that silencing the CRF+ CeA to LH circuit significantly blunted binge-like ethanol intake in male, but not female, mice. Consistently, genetic deletion of CRF from neurons of the CeA blunted ethanol intake exclusively in male mice. Furthermore, pharmacological blockade of the CRF type-1 receptor (CRF1R) in the LH significantly reduced binge-like ethanol intake in male mice only, while CRF2R activation in the LH failed to alter ethanol intake in either sex. Finally, a history of binge-like ethanol drinking blunted CRF mRNA in the CeA regardless of sex. Conclusions: These observations provide novel evidence that CRF+ CeA to LH neurocircuitry modulates binge-like ethanol intake in male, but not female mice, which may provide insight into the mechanisms that guide known sex differences in binge-like ethanol intake.

Alterations in hippocampal cholinergic dynamics following CRF infusions into the medial septum of male and female rats.

Corticoptropin releasing factor (CRF) is implicated in stress-related physiological and behavioral changes. The septohippocampal pathway regulates hippocampal-dependent mnemonic processes, which are affected in stress-related disorders, and given the abundance of CRF receptors in the medial septum (MS), this pathway is influenced by CRF. Moreover, there are sex differences in the MS sensitivity to CRF and its impact on hippocampal function. However, the mechanisms underlying these associations remain elusive. In the present study, we utilized an in vivo biosensor-based electrochemistry approach to examine the impact of MS CRF infusions on hippocampal cholinergic signaling dynamics in male and female rats. Our results show increased amplitudes of depolarization-evoked phasic cholinergic signals in the hippocampus following MS infusion of CRF at the 3 ng dose as compared to the infusion involving artificial cerebrospinal fluid (aCSF). Moreover, a trend for a sex × infusion interaction indicated larger cholinergic transients in females. On the contrary, intraseptal infusion of a physiologically high dose (100 ng) of CRF produced a subsequent reduction in phasic cholinergic transients in both males and females. The assessment of tonic cholinergic activity over 30 min post-infusion revealed no changes at the 3 ng CRF dose in either sex, but a significant infusion × sex interaction indicated a reduction in females at the 100 ng dose of CRF as compared to the aCSF. Taken together, our results show differential, dose-dependent modulatory effects of MS CRF on the dynamics of phasic and tonic modes of cholinergic signaling in the hippocampus of male and female rats. These cholinergic signaling modes are critical for memory encoding and maintaining arousal states, and may underlie sex differences in cognitive vulnerability to stress and stress-related psychiatric disorders.

Activation of CRF/CRFR1 Signaling in the Central Nucleus of the Amygdala Contributes to Chronic Stress-Induced Exacerbation of Neuropathic Pain by Enhancing GluN2B-NMDA Receptor-Mediated Synaptic Plasticity in Adult Male Rats.

Exacerbation of pain by chronic stress and comorbidity of pain with stress-related disorders such as depression and post-traumatic stress disorder, represent significant clinical challenges. Previously we have documented that chronic forced swim (FS) stress exacerbates neuropathic pain in spared nerve injury (SNI) rats, associated with an up-regulation of GluN2B-containing N-methyl-D-aspartate receptors (GluN2B-NMDARs) in the central nucleus of the amygdala (CeA). However, the molecular mechanisms underlying chronic FS stress (CFSS)-mediated exacerbation of pain sensitivity in SNI rats still remain unclear. In this study, we demonstrated that exposure of CFSS to rats activated the corticotropin-releasing factor (CRF)/CRF receptor type 1 (CRFR1) signaling in the CeA, which was shown to be necessary for CFSS-induced depressive-like symptoms in stressed rats, and as well, for CFSS-induced exacerbation of pain hypersensitivity in SNI rats exposed to chronic FS stress. Furthermore, we discovered that activation of CRF/CRFR1 signaling in the CeA upregulated the phosphorylation of GluN2B-NMDARs at tyrosine 1472 (pGluN2BY1472) in the synaptosomal fraction of CeA, which is highly correlated to the enhancement of synaptic GluN2B-NMDARs expression that has been observed in the CeA in CFSS-treated SNI rats. In addition, we revealed that activation of CRF/CRFR1 signaling in the CeA facilitated the CFSS-induced reinforcement of long-term potentiation as well as the enhancement of NMDAR-mediated excitatory postsynaptic currents in the basolateral amygdala (BLA)-CeA pathway in SNI rats. These findings suggest that activation of CRF/CRFR1 signaling in the CeA contributes to chronic stress-induced exacerbation of neuropathic pain by enhancing GluN2B-NMDAR-mediated synaptic plasticity in rats subjected to nerve injury. PERSPECTIVE: Our present study provides a novel mechanism for elucidating stress-induced hyperalgesia and highlights that the CRF/CRFR1 signaling and the GluN2B-NMDAR-mediated synaptic plasticity in the CeA may be important as potential therapeutic targets for chronic stress-induced pain exacerbation in human neuropathic pain. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Investigating intestinal mast cell dynamics during acute heat stress in growing pigs.

Objectives were to examine the temporal pattern of intestinal mast cell dynamics and the effects of a mast cell stabilizer (ketotifen [Ket]) during acute heat stress (HS) in growing pigs. Crossbred barrows (n = 42; 32.3 ±â€…1.9 kg body weight [BW]) were randomly assigned to 1 of 7 environmental-therapeutic treatments: (1) thermoneutral (TN) control (TNCon; n = 6), (2) 2 h HS control (2 h HSCon; n = 6), (3) 2 h HS + Ket (2 h HSKet; n = 6); (4) 6 h HSCon (n = 6), (5) 6 h HSKet (n = 6), (6) 12 h HSCon (n = 6), or (7) 12 h HSKet (n = 6). Following 5 d of acclimation to individual pens, pigs were enrolled in two experimental periods (P). During P1 (3 d), pigs were housed in TN conditions (21.5 ±â€…0.8 °C) for the collection of baseline measurements. During P2, TNCon pigs remained in TN conditions for 12 h, while HS pigs were exposed to constant HS (38.1 ±â€…0.2 °C) for either 2, 6, or 12 h. Pigs were euthanized at the end of P2, and blood and tissue samples were collected. Regardless of time or therapeutic treatment, pigs exposed to HS had increased rectal temperature, skin temperature, and respiration rate compared to their TNCon counterparts (1.9 °C, 6.9° C, and 119 breaths/min; P < 0.01). As expected, feed intake and BW gain markedly decreased in HS pigs relative to their TNCon counterparts (P < 0.01). Irrespective of therapeutic treatment, circulating corticotropin-releasing factor decreased from 2 to 12 h of HS relative to TNCon pigs (P < 0.01). Blood cortisol increased at 2 h of HS (2-fold; P = 0.04) and returned to baseline by 6 h. Plasma histamine (a proxy of mast cell activation) remained similar across thermal treatments and was not affected by Ket administration (P > 0.54). Independent of Ket or time, HS increased mast cell numbers in the jejunum (94%; P < 0.01); however, no effects of HS on mast cell numbers were detected in the ileum or colon. Jejunum and ileum myeloperoxidase area remained similar among treatments (P > 0.58) but it tended to increase (12%; P = 0.08) in the colon in HSCon relative to TNCon pigs. Circulating lymphocytes and basophils decreased in HSKet relative to TN and HSCon pigs (P ≤ 0.06). Blood monocytes and eosinophils were reduced in HS pigs relative to their TNCon counterparts (P < 0.01). In summary, HS increased jejunum mast cell numbers and altered leukocyte dynamics and proinflammatory biomarkers. However, Ket administration had no effects on mast cell dynamics measured herein.

Heat stress (HS) affects various physiological, metabolic, and endocrine parameters, ostensibly due to reduced intestinal barrier integrity and the ensuing immune response. Evidence indicates that generalized "stress" may be a critical component of HS-induced leaky gut, a mechanism likely mediated by mast cells. Mast cell activation has been extensively associated with various stress-related intestinal inflammatory conditions; however, its contribution to intestinal barrier dysfunction during HS remains unclear. Thus, this study was designed to evaluate mast cell dynamics during an acute HS challenge and to assess the effects a mast cell stabilizer on biomarkers of intestinal inflammation. Herein, HS induced a rapid increase in circulating cortisol, increased jejunum mast cell numbers, and altered metabolism, leukocyte dynamics, and proinflammatory biomarkers. Contrary to our hypothesis, HS did not alter circulating histamine (a biomarker of mast cell activation), and mast cell stabilization did not affect mast cell numbers nor altered histamine concentrations. Altogether, our observations support a connection between HS and intestinal mast cell infiltration that may contribute to the pathophysiology of intestinal dysfunction during a heat load.