Corticotropin-releasing Factor Research Articles

Oxytocin levels in response to CRH administration in hypopituitarism and hypothalamic damage: a randomized, crossover, placebo-controlled trial

Increasing evidence supports the presence of oxytocin deficiency (OXT-D) in patients with hypopituitarism and hypothalamic damage (HHD), that might be associated with neuropsychological deficits and sexual dysfunction, leading to worse quality of life (QoL). Therefore, identifying a provocative test to diagnose an OXT-D will be important. Corticotropin-releasing hormone (CRH) is a candidate for such a test as it increases oxytocin secretion in animal models. This study aimed to examine the effects of CRH on oxytocin release in HHD compared to healthy controls (HC) and to describe the psychopathology, sexual function and QoL and their associations with oxytocin. This is a single-blind, randomized, placebo-controlled, proof-of-concept study (NCT 04902235) with crossover assignment (CRH vs. placebo). Nineteen HHD patients (10 females) and 20 HC (11 females) completed two visits, receiving CRH or placebo in random order and completed validated questionnaires to assess psychopathology, sexual function and QoL. Samples were collected over 120 min to assess oxytocin. Linear mixed-effects regression model evaluated the change in oxytocin after CRH/placebo in HHD vs. HC. CRH administration did not impact oxytocin concentrations across groups over time (p = 0.97). HHD had greater psychopathology (most ps < 0.05), sexual dysfunction (p < 0.03) and worse QoL (p < 0.001) compared to HC, nevertheless, baseline oxytocin concentrations and area under the curve of oxytocin were not significantly associated with psychopathology, sexual function or QoL, neither in HHD or HC. In conclusion, CRH administration does not appear to be a suitable provocative test for diagnosing OXT-D in HHD. Identifying a reliable diagnostic test for OXT-D remains crucial. Alternative provocative tests or biomarkers should be explored.

The effects of corticotropin-releasing factor (CRF) and urocortins on the serotonin (hydroxytryptamine, 5HT) released from the raphe nuclei (RN).

Corticotropin-releasing factor (CRF) and urocortins (UCN1, UCN2 and UCN3) belong to the same CRF family of neuropeptides. They regulate the neuroendocrine, autonomic and behavioral responses to stress via two CRF receptors (CRF1 and CRF2). Stress, anxiety and depression affects the activity of the hypothalamic-pituitary-adrenal (HPA) axis and the serotoninergic neurotransmission, both being regulated by CRF and CRF-related peptides. However, the exact action of CRF and urocortins on the serotonin (5-hydroxytryptamine, 5HT) release was not fully elucidated yet. Therefore, the aim of the present study was to investigate the actions of CRF and urocortins on the 5HT released from the rat raphe nuclei (RN), the most important brain regions producing 5HT, and the participation of CRF receptors in these actions. In order to do so, male Wistar rats were used, their RN were isolated and dissected, and the RN slices were incubated with tritium-labelled 5HT, superfused and stimulated electrically. During superfusion, the RN slices were treated with CRF, UCN1, UCN2 or UCN3, and, when significant effect was observed, pretreated with selective CRF1 antagonist antalarmin or selective CRF2 antagonist astressin2B. The release of tritium-labelled 5HT from the RN was determined by liquid scintillation counting. CRF and UCN1 decreased significantly the tritium-labelled 5HT release from the RN, and these effects were reversed by antalarmin, but not by astressin2B. In addition, UCN3, but not UCN2, increased significantly the tritium-labelled 5HT release from the RN, and this effect was reduced by astressin2B, but not antalarmin. Our results indicate the existence of two apparently opposing CRF systems in the RN: activation of CRF1 by CRF and UCN1 may inhibit, whereas activation of CRF2 by UCN3 may stimulate the 5HT release. The dysbalance between CRF1 and CRF2 activation and, consequently, alteration of serotoninergic signalling may result in anxiety and depression, associated with hyperactivity of the HPA axis.

Hormonal mechanisms in the paraventricular nuclei associated with hyperalgesia in Parkinson's disease model rats.

Pain is a major non-motor symptom of Parkinson's disease (PD). The relationship between hyperalgesia and neuropeptides originating from paraventricular nucleus (PVN) in 6-hydroxydopamine (6-OHDA) rats has already been investigated for oxytocin (OXT), but not yet for arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH). The present study aimed to investigate the alterations in these neuropeptides following nociceptive stimulation in PD model rats and to examine the mechanisms of hyperalgesia. Dopaminergic nigrostriatal lesions were induced by injecting 6-OHDA into the medial forebrain bundle. Subcutaneous formalin injection into the vibrissa pad was performed in rats as a nociceptive stimulus in the orofacial region. Dopamine depletion's effect on nociception was assessed by counting the p-ERK-immunoreactive (-IR) cells in the trigeminal spinal subnucleus caudalis (Vc). The PD model rats induced by 6-OHDA injection (6-OHDA rats) showed a significantly higher number of p-ERK-IR cells in the Vc than the sham rats, confirming hyperalgesia in 6-OHDA rats. Then, we investigated the immunohistochemical responses to OXT, AVP, and CRH cells in the PVN and examined the changes in blood levels of these neuropeptides. As a result, formalin injection increased neuronal activity and blood levels of OXT and CRH in sham rats, but these were suppressed in the 6-OHDA rats. Contrarily, neuronal activity and blood level of AVP were unaffected by nociceptive stimuli and were significantly lower in 6-OHDA rats than in sham rats. Our findings suggest that OXT and CRH suppression is linked to hyperalgesia in PD, whereas AVP does not directly influence the observed hyperalgesia.

Effects of the combination of Epimedium brevicornum Maxim, Ligustrum lucidum Ait and Dexamethasone on asthmatic rats by endogenous glucocorticoid pathway.

The theory of traditional Chinese medicine (TCM) believes that kidney deficiency is the fundamental cause of chronic refractory asthma, accompanied by pathological changes such as airway remodeling and a reduction of endogenous glucocorticoid (GC) synthesis. The combination of Epimedium brevicornum Maxim (EB) and Ligustrum lucidum Ait (LL) is frequently used in TCM for kidney tonifying and the alleviation of asthma symptoms. This approach is based on Pei-Ben formula, a renowned treatment for asthma developed by the distinguished Shanghai Practitioner, Professor Huiguang Xu, over 30 years of clinical experience. Long-term use of exogenous GC in the treatment of asthma lead to the inhibition of endogenous GC synthesis and further hypothalamic-pituitary-adrenal (HPA) axis function. Our previous experiments confirmed that the combination of Epimedium brevicornum Maxim and Ligustrum lucidum Ait (EL) with dexamethasone (Dex) enhances kidney Yin and Yang, boosts endogenous GC levels, and improves airway remodeling and HPA axis function in asthmatic rats. However, the underlying mechanism remains unclear. This study aimed to investigate the regulatory effect of EL with Dex on endogenous GC pathway in asthmatic rats. We employed an ovalbumin (OVA)-induced asthma rat model and an OVA-induced asthma rat model with Metyrapone (Met, an inhibitor of endogenous GC synthesis) intervention to evaluate the effects of Dex, EL and their combination (EL+Dex) on asthma treatment. The assessment included the lung histopathology, GC receptors (GR) countent and GC-GR binding in bronchoalveolar lavage fluid (BALF), corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), corticosterone (CORT), cortisol (COR), interleukin 6 (IL-6), and immunoglobulin E (IgE) in serum, GC metabolites in urine, and hydroxysteroid dehydrogenase (HSD) 11B1, HSD11B2, cytochrome P450 family 11 subfamily B member 1 (CYP11B1) and steroidogenic factor 1 (SF1) in lung, liver, and adrenal gland. In the OVA-induced asthma model, we found that endogenous GC synthesis was suppressed in both the asthma group and the Dex group. The combination of EL and Dex could enhance HPA axis function, increase protein expression of key endogenous GC synthesis factors (HSD11B1, HSD11B2 in lung; CYP11B1, SF1 in adrenal; HSD11B2, CYP11B1 in liver), and improve the level of endogenous GC synthesis. In the OVA-induced asthma model with Met intervention, we observed a highly significant endogenous suppression in both the asthma+Met group and the Dex group. Additionally, the use of EL, either alone or in combination with Dex, demonstrated a significant effect in improving HPA axis function and enhancing the protein expression of key endogenous GC synthesis factors (HSD11B1, HSD11B2 in lung; HSD11B1, HSD11B2, CYP11B1, SF1 in adrenal; HSD11B1 in liver). In both asthma models, the combination of EL and Dex could relieve the pathological changes of airway inflammation and airway remodeling, and enhance GC-GR binding capacity. This study reveals that the combined use of EL and Dex could improve airway inflammation and airway remodeling in asthma through enhancing HPA axis function, endogenous GC synthesis, and GC-GR binding, offering a promising therapeutic strategy for asthma management.

Ashwagandha (Withania somnifera (L.) dunal) root extract containing withanolide a alleviates depression-like behavior in mice by enhancing the brain-derived neurotrophic factor pathway under unexpected chronic mild stress.

Ashwagandha (Withania somnifera (L.) Dunal) root or whole-plant extracts are used to treat anxiety, insomnia, and other nervous system disturbances. We evaluated the neuroprotective and antidepressant effects of ashwagandha root extract (ARE) on corticosterone-exposed HT-22cells and unpredictable chronic mild stress (UCMS)-challenged mice. The neuroprotective properties of ARE containing withanolide A were assessed in HT-22cells subjected to corticosterone-induced oxidative stress. Additionally, the effects of ARE on depression-like behavior, stress-related hormones, and inflammatory cytokine levels were evaluated in a mouse model of UCMS. In HT-22cells, ARE (100 and 200μg/mL) and its constituent, withanolide A (1.56 and 3.12μg/mL), mitigated corticosterone-induced increases in MAO activity, ROS, and MDA levels. Treatment also reversed corticosterone-induced reductions in BDNF, TrkB, p-AKT, p-ERK, and p-CREB and normalized Nrf2 and Keap1 levels, thereby elevating HO-1 expression. In UCMS mice, ARE improved behavioral outcomes, increased sucrose preference, and reduced immobility in the forced swimming test while enhancing activity in the open field test and elevated plus maze. ARE decreased the levels of stress hormones (corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone) and increased the levels of neurotransmitters (L-DOPA, 5-HTP, and serotonin). Histological analysis revealed that ARE reduced hippocampal cell loss. Additionally, ARE (60 and 100mg/kg) restored decreased levels of p-AKT, p-ERK, and p-CREB and lowered inflammation-related proteins (Cox2, iNOS, IL-6, IL-1β, TNF-α). These results indicate that ARE containing withanolide A exhibits notable neuroprotective and antidepressant properties.

A new pharmacological strategy against treatment-resistant depression

Major depressive disorder affects more than 50 million people in the world. However, 50% of patients don't respond to two or more drugs or psychotherapeutic treatments, named treatment-resistant depression (TRD). In this work, we propose a new augmentation treatment against TRD based on combining Fluoxetine (FLX) and the N-terminal fragment Galanin, GAL(1–15). In Wistar Kyoto (WKY) rats, akin to endogenous depression genetically, we evaluate GAL(1–15)’s impact on FLX-induced behaviours on tests measuring despair and anhedonia. We explored GALR2 involvement using the antagonist M871 and an in vivo model with siRNA 5-HT1A knockdown. Also, the 5-HT1AR was analyzed by autoradiography binding in several brain regions. We analyze the corticosterone levels and a dexamethasone-suppressed corticotropin-releasing hormone stimulation to study the HPA axis regulation. Our results shows that only the combination of FLX + GAL(1–15) induced antidepressant effects in the WKY animals in Behavioural tests related to despair. This combination also reduced corticosterone levels in the WKY animals and modulated the functional characteristics of the serotoninergic receptor 5-HT1A in the prefrontal cortex. These novel results suggest combining GAL(1–15) with FLX is a new potentiation strategy in TRD cases. It shows the innovative potential of the interactions between the galaninergic and serotonergic systems to find new strategies and drugs against resistant depression.

Enhanced engraftment of human haematopoietic stem cells via mechanical remodelling mediated by the corticotropin-releasing hormone

Enhanced engraftment of human haematopoietic stem cells via mechanical remodelling mediated by the corticotropin-releasing hormone

Inhibition of Glucocorticoid Synthesis by Metyrapone as an Approach to Study Their Contribution to Gastroprotection in Rats

According to the results of our research glucocorticoids produced in response to stress ulcerogenic stimuli are gastroprotective factors. The aim of this review article is to demonstrate, through the analysis of data obtained in our studies, that the inhibition of glucocorticoid synthesis by metyrapone can be an adequate and valuable approach for studying the contribution of glucocorticoids, produced during acute activation of the hypothalamic-pituitary-adrenocortical (HPA) axis, to gastroprotection in rats. When studying the contribution of glucocorticoids produced in response to moderate, normally non-ulcerogenic stressors or to the administration of the corticotropin-releasing factor (CRF) to gastroprotection, it was shown that the suppression of these hormones by metyrapone leads to: 1) the transformation of normally non-ulcerogenic stimuli into ulcerogenic ones; 2) the elimination of the gastroprotective effect of stress preconditioning; 3) the elimination of the gastroprotective effect of CRF. The effects of metyrapone were reproduced under conditions of suppressed glucocorticoid production using the selective CRF-1 receptor antagonist NBI 27914 and the blockade of glucocorticoid receptors with their antagonist RU38486. The data presented suggest that: a) glucocorticoids produced in response to moderate stress stimuli contribute to the protection of the gastric mucosa under these conditions and increase its resistance to subsequent ulcerogenic stimuli (i.e., they contribute to the gastroprotective effect of stress preconditioning); b) glucocorticoids produced in response to CRF administration participate in the realization of its gastroprotective action. The data obtained confirm that the activation of the HPA axis is a gastroprotective component of stress response, and stress-produced glucocorticoids are important gastroprotective factors.

Desmopressin is a safe and effective secretagogue to replace corticotropin-releasing hormone in petrosal sinus sampling.

Bilateral inferior petrosal sinus sampling (BIPSS) with corticotropin-releasing hormone (CRH) was the gold standard for distinguishing Cushing disease (CD) from ectopic ACTH secretion (EAS). CRH, however, is no longer available. To assess the reliability of BIPSS with desmopressin to differentiate CD from EAS. A retrospective study included patients who underwent BIPSS with desmopressin for ACTH-dependent hypercortisolism, with the whole diagnostic procedure in a single center. Fifty-eight patients with confirmed etiological diagnosis were included: 51 CD, 7 EAS. Forty-three CD patients (84.3%) had post-stimulation ratio ≥2 before stimulation and 6 of the other 8 (75%) had a ratio ≥3. All EAS patients were correctly diagnosed before and after stimulation. Sensitivity was 84.3% before stimulation and 92.2% combining pre- and post-stimulation results; specificity reached 100%. A ROC curve established optimal thresholds at 1.4 before stimulation and 1.7 after. Desmopressin is a good substitute for CRH, correcting diagnosis compared to baseline BIPSS in 12% of cases.

Regulation and function of the gill corticotropin-releasing factor system during osmoregulatory disturbances in Atlantic salmon.

While corticosteroids, including cortisol, have conserved osmoregulatory functions, the relative involvement of other stress-related hormones in osmoregulatory processes remains unclear. To address this gap, we initially characterized the gill corticotropin-releasing factor (CRF) system of Atlantic salmon (Salmo salar) and then determined: 1) how it is influenced by osmotic disturbances; 2) whether it is affected by cortisol; and 3) which physiological processes it regulates in the gills. Most CRF system components were expressed in the gills with CRF receptor 2 (crfr2a), CRF binding protein (crfbp1 and crfbp2), and urocortin 2 (ucn2a) being the most abundant. The development of seawater tolerance in migratory juveniles (i.e., smolts) was associated with a general transcriptional upregulation of CRF ligands, but transcript levels of crfr2a, crfbp2, crfb2, and ucn2a decreased by ∼50% following seawater transfer. Accordingly, transfer of seawater-acclimated fish into freshwater increased crfr2a and ucn2a levels. Cortisol treatment of cultured gill filaments had marked effects on the CRF system; however, these effects failed to fully replicate changes observed during in vivo experiments, suggesting direct contributions of the gill CRF system during osmotic disturbances. Indeed, activation of the CRF system in cultured filaments from freshwater-acclimated (but not seawater-acclimated) salmon had transcriptional effects on several physiological systems (e.g., endothelial permeability, angiogenesis, and immune regulation) which involved contributions by both CRF receptor subtypes. Overall, our results indicate that the gill CRF system is more active in hypoosmotic environments and directly contributes to the coordination of physiological responses following osmotic disturbances.

Targeting corticotropin-releasing hormone receptor type 1 (Crhr1) neurons: validating the specificity of a novel transgenic Crhr1-FlpO mouse

Corticotropin-releasing hormone (CRH) signaling through its cognate receptors, CRHR1 and CRHR2, contributes to diverse stress-related functions in the mammalian brain. Whereas CRHR2 is predominantly expressed in choroid plexus and blood vessels, CRHR1 is abundantly expressed in neurons in discrete brain regions, including the neocortex, hippocampus and nucleus accumbens. Activation of CRHR1 influences motivated behaviors, emotional states, and learning and memory. However, it is unknown whether alterations in CRHR1 signaling contribute to aberrant motivated behaviors observed, for example, in stressful contexts. These questions require tools to manipulate CRHR1 selectively. Here we describe and validate a novel Crhr1-FlpO mouse. Using bacterial artificial chromosome (BAC) transgenesis, we engineered a transgenic mouse that expresses FlpO recombinase in CRHR1-expressing cells. We used two independent methods to assess the specificity of FlpO to CRHR1-expressing cells. First, we injected Crhr1-FlpO mice with Flp-dependent viruses expressing fluorescent reporter molecules. Additionally, we crossed the Crhr1-FlpO mouse with a transgenic Flp-dependent reporter mouse. CRHR1 and reporter molecules were identified using immunocytochemistry and visualized via confocal microscopy in several brain regions in which CRHR1 expression and function is established. Expression of Flp-dependent viral constructs was highly specific to CRHR1-expressing cells in all regions examined (over 90% co-localization). In accord, robust and specific expression of the Flp-dependent transgenic reporter was observed in a reporter mouse, recapitulating endogenous CRHR1 expression. The Crhr1-FlpO mouse enables selective genetic access to CRHR1-expressing cells within the mouse brain. When combined with Cre-lox or site-specific recombinases, the mouse facilitates intersectional manipulations of CRHR1-expressing neurons.

Lateralization outcomes of bilateral inferior petrosal sinus sampling: desmopressin vs CRH.

Bilateral inferior petrosal sinus sampling (BIPSS) is the gold standard for localizing ACTH-dependent Cushing's syndrome (CS). While corticotropin-releasing hormone (CRH) was initially used for stimulation, desmopressin has become a common alternative. This research evaluates desmopressin's effectiveness in lateralizing Cushing's disease (CD) during BIPSS compared to CRH stimulation. The study included 33 individuals with ACTH-dependent CS who underwent BIPSS and had diagnoses confirmed by endoscopic endonasal transsphenoidal pituitary surgery (EETPS). Fourteen participants underwent BIPSS with CRH and 19 with desmopressin. A comparative analysis was conducted. BIPSS accurately lateralized 76% of cases, specifically, 71% with CRH and 79% with desmopressin (p = 0.2). For tumors < 6mm on MRI, overall accuracy was 82%, namely, 75% with CRH and 90% with desmopressin (p = 0.4). IPSS achieved 100% accuracy in the four cases with no lesion on preoperative MRI. This study demonstrates no significant difference in lateralization accuracy between desmopressin and CRH for IPSS. In challenging cases, especially those with microadenomas or non-lesional CD, desmopressin with IPSS aids in preoperative lateralization.

Early-life challenge enhances cortisol regulation in zebrafish larvae.

The hypothalamic-pituitary-adrenal (HPA) axis in mammals and the hypothalamic-pituitary-interrenal (HPI) axis in fish are open systems that adapt to the environment during development. Little is known about how this adaptation begins and regulates early stress responses. We used larval zebrafish to examine the impact of prolonged forced swimming at 5 days post-fertilization (dpf), termed early-life challenge (ELC), on cortisol responses, neuropeptide expression in the nucleus preopticus (NPO), and gene transcript levels. At 6 dpf, ELC-exposed larvae showed normal baseline cortisol but reduced reactivity to an initial stressor. Conversely, they showed increased reactivity to a second stressor within the 30-min refractory period, when cortisol responses are typically suppressed. ELC larvae had fewer corticotropin-releasing hormone (crh), arginine vasopressin (avp), and oxytocin (oxt)-positive cells in the NPO, with reduced crh and avp co-expression. Gene expression analysis revealed upregulation of genes related to cortisol metabolism (hsd11b2, cyp11c1), steroidogenesis (star), and stress modulation (crh, avp, oxt). These results suggest that early environmental challenge initiates adaptive plasticity in the HPI axis, tuning cortisol regulation to balance responsiveness and protection during repeated stress. Future studies should explore the broader physiological effects of prolonged forced swimming and its long-term impact on cortisol regulation and stress-related circuits.

Associations between hypothalamic-pituitary-adrenal (HPA) axis hormone levels, major depression features and antidepressant effects of ketamine.

Subanesthetic doses of (R,S)-ketamine (ketamine) have demonstrated rapid and robust antidepressant effects in individuals with depression. However, individual variability in response to ketamine exists, and current biomarkers of ketamine treatment response are not entirely understood. Preclinical evidence suggests a link between hypothalamic-pituitary-adrenal (HPA) axis activation, a determinant of the stress response system, and ketamine's efficacy in stressed mice exhibiting enhanced antidepressant responses. Here, we assessed the relationship between HPA axis, major depression features, and antidepressant response to ketamine in humans. We investigated 42 participants following medication washout with treatment-resistant depression who participated in a randomized, placebo-controlled, crossover trial receiving intravenous ketamine. Plasma levels of corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and cortisol were measured at baseline. Ketamine's antidepressant effects were assessed using the Montgomery-Asberg Depression Rating Scale. We found that baseline HPA axis hormone levels did not significantly moderate the antidepressant effects of ketamine. However, a negative association was observed between ACTH and CRF levels and the overall duration of depressive episodes, suggesting potential biomarker implications. Also, a negative correlation between baseline depressive scores and age of onset was observed, suggesting that the severity of depression might be greater if it develops at a younger age, indicating more enduring stress on the brain and body. Although we did not find a moderation effect of the plasma HPA axis hormones on the antidepressant effects of ketamine, moderation effects of the brain HPA axis hormones cannot be precluded and warrants further investigation. Importantly, our results implicate HPA axis components as potential biomarkers for the duration of depressive episodes.

Chronic stress induces behavioural changes through increased kynurenic acid by downregulation of kynurenine-3-monooxygenase with microglial decline.

Alterations in tryptophan-kynurenine (TRP-KYN) pathway are implicated in major depressive disorder (MDD). α7 nicotinic acetylcholine (α7nACh) receptor regulates the hypothalamic-pituitary-adrenal (HPA) axis. We have shown that deficiency of kynurenine 3-monooxygenase (KMO) induces depression-like behaviour via kynurenic acid (KYNA; α7nACh antagonist). In this study, we investigated the involvement of the TRP-KYN pathway in stress-induced behavioural changes and the regulation of the HPA axis. Mice were exposed to chronic unpredictable mild stress (CUMS) and subjected to behavioural tests. We measured TRP-KYN metabolites and the expression of their enzymes in the hippocampus. KMO heterozygous mice were used to investigate stress vulnerability. We also evaluated the effect of nicotine (s.c.) on CUMS-induced behavioural changes and an increase in serum corticosterone (CORT) concentration. CUMS decreased social interaction time but increased immobility time under tail suspension associated with increased serum corticosterone concentration. CUMS increased KYNA levels via KMO suppression with microglial decline in the hippocampus. Kmo+/- mice were vulnerable to stress: they exhibited social impairment and increased serum corticosterone concentration even after short-term CUMS. Nicotine attenuated CUMS-induced behavioural changes and increased serum corticosterone concentration by inhibiting the increase in corticotropin-releasing hormone. Methyllycaconitine (α7nACh antagonist) inhibited the attenuating effect of nicotine. CUMS-induced behavioural changes and the HPA axis dysregulation could be induced by the increased levels of KYNA via KMO suppression. KYNA plays an important role in the pathophysiology of MDD as an α7nACh antagonist. Therefore, α7nACh receptor is an attractive therapeutic target for MDD.

Peripherally Restricted Activation of Opioid Receptors Influences Anxiety-Related Behaviour and Alters Brain Gene Expression in a Sex-Specific Manner.

Although effects of stress-induced anxiety on the gastrointestinal tract and enteric nervous system (ENS) are well studied, how ENS dysfunction impacts behaviour is not well understood. We investigated whether ENS modulation alters anxiety-related behaviour in rats. We used loperamide, a potent μ-opioid receptor agonist that does not cross the blood-brain barrier, to manipulate ENS function and assess changes in behaviour, gut and brain gene expression, and microbiota profile. Sprague Dawley (male/female) rats were acutely dosed with loperamide (subcutaneous) or control solution, and their behavioural phenotype was examined using open field and elevated plus maze tests. Gene expression in the proximal colon, prefrontal cortex, hippocampus, and amygdala was assessed by RNA-seq and caecal microbiota composition determined by shotgun metagenome sequencing. In female rats, loperamide treatment decreased distance moved and frequency of supported rearing, indicating decreased exploratory behaviour and increased anxiety, which was associated with altered hippocampal gene expression. Loperamide altered proximal colon gene expression and microbiome composition in both male and female rats. Our results demonstrate the importance of the ENS for communication between gut and brain for normo-anxious states in female rats and implicate corticotropin-releasing hormone and gamma-aminobutyric acid gene signalling pathways in the hippocampus. This study also sheds light on sexually dimorphic communication between the gut and the brain. Microbiome and colonic gene expression changes likely reflect localised effects of loperamide related to gut dysmotility. These results suggest possible ENS pharmacological targets to alter gut to brain signalling for modulating mood.

Direct Modulation of CRH Nerve Terminal Function by Noradrenaline and Corticosterone.

Nerve terminals are the final point of regulation before neurosecretion. As such, neuromodulators acting on nerve terminals can exert significant influence on neural signaling. Hypothalamic corticotropin-releasing hormone (CRH) neurons send axonal projections to the median eminence where CRH is secreted to stimulate the hypothalamic-pituitary-adrenal (HPA) axis. Noradrenaline and corticosterone are two of the most important neuromodulators of HPA axis function; noradrenaline excites CRH neurons and corticosterone inhibits CRH neurons by negative feedback. Here, we used GCaMP6f Ca2+ imaging and measurement of nerve terminal CRH secretion using sniffer cells to determine whether these neuromodulators act directly on CRH nerve terminals in male mice. Contrary to expectations, noradrenaline inhibited action potential-dependent Ca2+ elevations in CRH nerve terminals and suppressed evoked CRH secretion. This inhibitory effect was blocked by α2-adrenoreceptor antagonism. Corticosterone also suppressed evoked CRH peptide secretion from nerve terminals, independent of action potential-dependent Ca2+ levels. This inhibition was prevented by the glucocorticoid receptor antagonist, RU486, and indicates that CRH nerve terminals may be a site of fast glucocorticoid negative feedback. Together these findings establish median eminence nerve terminals as a key site for regulation of the HPA axis.

Self-experience of a negative event alters responses to others in similar states through prefrontal cortex CRF mechanisms.

Our own experience of emotional events influences how we approach and react to others' emotions. Here we observe that mice exhibit divergent interindividual responses to others in stress (that is, preference or avoidance) only if they have previously experienced the same aversive event. These responses are estrus dependent in females and dominance dependent in males. Notably, silencing the expression of the corticotropin-releasing factor (CRF) within the medial prefrontal cortex (mPFC) attenuates the impact of stress self-experience on the reaction to others' stress. In vivo microendoscopic calcium imaging revealed that mPFC CRF neurons are activated more toward others' stress only following the same negative self-experience. Optogenetic manipulations confirmed that higher activation of mPFC CRF neurons is responsible for the switch from preference to avoidance of others in stress, but only following stress self-experience. These results provide a neurobiological substrate underlying how an individual's emotional experience influences their approach toward others in a negative emotional state.

Diverting glial glycolytic flux towards neurons is a memory-relevant role of Drosophila CRH-like signalling

An essential role of glial cells is to comply with the large and fluctuating energy needs of neurons. Metabolic adaptation is integral to the acute stress response, suggesting that glial cells could be major, yet overlooked, targets of stress hormones. Here we show that Dh44 neuropeptide, Drosophila homologue of mammalian corticotropin-releasing hormone (CRH), acts as an experience-dependent metabolic switch for glycolytic output in glia. Dh44 released by dopamine neurons limits glial fatty acid synthesis and build-up of lipid stores. Although basally active, this hormonal axis is acutely stimulated following learning of a danger-predictive cue. This results in transient suppression of glial anabolic use of pyruvate, sparing it for memory-relevant energy supply to neurons. Diverting pyruvate destination may dampen the need to upregulate glial glycolysis in response to increased neuronal demand. Although beneficial for the energy efficiency of memory formation, this mechanism reveals an ongoing competition between neuronal fuelling and glial anabolism.

Regulation of sleep amount by CRTC1 via transcription ofCrhin mice

The cAMP-response element binding protein (CREB) is required for regulation of daily sleep amount, whereas gain-of-function of CREB-regulated transcription coactivator 1 (CRTC1) causes severe insomnia in mice. However, the physiological functions of CRTCs and their downstream target genes in the regulation of sleep amount remain unclear. Here, we use adult brain chimeric (ABC)-expression/knockout platform for somatic genetics analysis of sleep in adult male mice. ABC-expression of constitutively active mutant CRTC1/2CAin the mouse brain neurons significantly reduces the amount of non-rapid eye movement sleep (NREMS) and/or REMS. Consistent with that SIK3 phosphorylates and inhibits CRTCs, ABC-expression of CRTC1/2/3CArescues the hypersomnia phenotype ofSleepy(Sik3Slp) mice. While ABC-Crtc2KOorCrtc3KOcauses no sleep phenotype, ABC-Crtc1KOor ABC-expression of dominant-negative CRTC (dnCRTC) results in modest reduction of NREMS amount accompanied with elevated NREMS delta power. Moreover, ABC-expression of CRTC1CAor dnCRTC in the excitatory neurons causes bidirectional changes of NREMS/REMS amount and/or NREMS delta power, whereas expression of CRTC1CAin the inhibitory neurons decreases REMS amount and increases NREMS delta power. The ability of CRTC1CAto regulate sleep requires its transactivation domain and CREB-binding domain and is dependent on CREB. Furthermore, we showed that inducible ABC-expression of corticotropin releasing hormone (Crh) and brain-derived neurotrophic factor (Bdnf)–two target genes of CRTCs–significantly reduces daily sleep amount. Notably, ABC-CrhKO, but notBdnfKO, rescues the insomnia phenotype of ABC-CRTC1CAmice. Taken together, these results indicate that CREB-CRTC1 complex regulates daily sleep amount by modulating the transcription ofCrhin the mouse brain neurons.Significance StatementCRTCs function as coactivators for CREB, a transcription factor required for the regulation of daily sleep amount in mice. Here, we found that CRTC1/2/3 function similarly to suppress NREMS and REMS in a CREB-dependent manner. Consistent with that CRTCs are substrates of SIK3 kinase, expression of constitutive active mutant CRTCsCArescue the hypersomnia phenotype ofSleepy(Sik3Slp) mice. Furthermore, we showed that CRTC1 reduces NREMS amount by upregulating the expression of neuropeptide CRH. These results elucidate the mechanism by which CRTCs regulates sleep amount and suggest a potential transcriptional mechanism in the stress-induced sleep/wake regulation.