Influence of copper source and dietary inclusion level on growth performance of weaned pigs and expression of trace element related genes in the small intestine.

Copper is routinely supplemented to weanling pig diets at concentrations above nutritional requirements to enhance growth performance. We hypothesised that this effect depends on the source of Cu and its dietary concentration. We tested this in weaned pigs (26 d of age) over a 35-d period using a 2 × 3 factorial arrangement with two Cu-sources (CuSO4 and Cu2O, monovalent copper oxide, CoRouge®) and three supplementary dietary Cu-levels (15, 80 and 160 mg Cu/kg) as respective factors. Increasing Cu level linearly increased (P < 0.001) final BW and daily gain. These effects tended (P = 0.09) to be greater with Cu2O than CuSO4. Feed conversion ratio decreased linearly (P < 0.001) with increasing dietary Cu content, independent of Cu source. Plasma Cu, Zn and Fe levels were unaffected, whereas liver Cu content increased quadratically (P < 0.001) with increasing dietary Cu content, with a larger increase (P < 0.001) with CuSO4 than Cu2O. Bile Cu content increased quadratically (P = 0.025) with increasing Cu content, irrespective of Cu source. RT-qPCR analysis revealed that increasing Cu content quadratically (P = 0.009) increased duodenal but not ileal metallothionein 1A (MT1A) mRNA, with greater effect (P = 0.010) of CuSO4. Regardless of the Cu source, increasing Cu dose linearly increased (P = 0.006) duodenal DMT1/SLC11A2 mRNA but decreased ZIP4/SLC39A4 mRNA in duodenum (P < 0.001) and ileum (P < 0.005). ZnT10/SLC30A10 mRNA was significantly (P = 0.021) and numerically (P = 0.061) greater with Cu2O compared to CuSO4, in duodenum and ileum, respectively. Copper content quadratically modulated duodenal but not ileal transferrin receptor (P = 0.029) and ferric reductase CYBRD1 mRNA (P = 0.022). In hypothalamus, high Cu dose (P = 0.024) and Cu2O as source (P = 0.028) reduced corticotropin-releasing hormone (CRH) mRNA. Low versus high CuSO4 increased corticotropin-releasing hormone receptor (CRHR2) mRNA, while low Cu2O had the opposite effect (P = 0.009). In conclusion, incremental Cu intake enhanced growth performance, with a tendency for a greater effect of Cu2O. The lower increase in duodenal MT1A mRNA and liver Cu content indicates that less Cu from Cu2O was absorbed by gut and sequestered in liver. Thus, high Cu absorption is not essential for its growth-promoting effect and dietary Cu may affect intestinal Fe and Zn absorption via the active, transcellular route. The effects on hypothalamic CRH and CRHR2 expression indicate a role for the hypothalamus in mediating the effects of Cu on growth performance.

Larger hypothalamic subfield volumes in patients with chronic insomnia disorder and relationships to levels of corticotropin-releasing hormone.

The hypothalamus is a well-established core structure in the sleep-wake cycle. While previous studies have not consistently found whole hypothalamus volume changes in chronic insomnia disorder (CID), differences may exist at the smaller substructural level of the hypothalamic nuclei. The study aimed to investigate the differences in total and subfield hypothalamic volumes, between CID patients and healthy controls (HCs) in vivo, through an advanced deep learning-based automated segmentation tool. A total of 150 patients with CID and 155 demographically matched HCs underwent T1-weighted structural magnetic resonance scanning. We utilized FreeSurfer v7.2 for automated segmentation of the hypothalamus and its five nuclei. Additionally, correlation and causal mediation analyses were performed to investigate the association between hypothalamic volume changes, insomnia symptom severity, and hypothalamus-pituitary-adrenal (HPA) axis-related blood biomarkers. CID patients exhibited larger volumes in the right anterior inferior, left anterior superior, and left posterior subunits of the hypothalamus compared to HCs. Moreover, we observed a positive association between blood corticotropin-releasing hormone (CRH) levels and insomnia severity, with anterior inferior hypothalamus (a-iHyp) hypertrophy mediating this relationship. In conclusion, we found significant volume increases in several hypothalamic subfield regions in CID patients, highlighting the central role of the HPA axis in the pathophysiology of insomnia.

[Effect of Zuogui Jiangtang Jieyu Formula on intestinal flora and short-chain fatty acid metabolism in rats with diabetes mellitus complicated with depression].

This study aimed to investigate the regulatory effects of Zuogui Jiangtang Jieyu Formula(ZJJ) on the intestinal flora, short chain fatty acids(SCFAs), and neuroinflammation in rats with diabetes mellitus complicated depression(DD). The DD model was established in rats and model rats were randomly divided into a model group, a positive drug(metformin + fluoxetine) group, a ZJJ low-dose group, and a ZJJ high-dose group, with eight rats in each group. Another eight rats were assigned to the blank group. Subsequently, depressive-like behavior test was conducted on the rats, and cerebrospinal fluid samples were collected to measure pro-inflammatory cytokines [interleukin-1ß(IL-1ß), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α)]. Blood serum samples were collected to measure proteins related to the hypothalamic-pituitary-adrenal axis(HPA axis), including corticotropin-releasing hormone(CRH), adrenocorticotropic hormone(ACTH), and cortisol(CORT), as well as glucose metabolism. Gut contents were collected from each group for 16S rRNA sequencing analysis of intestinal flora and SCFAs sequencing. The results indicated that ZJJ not only improved glucose metabolism in DD rats(P<0.01) but also alleviated depressive-like behavior(P<0.05) and HPA axis hyperactivity(P<0.05 or P<0.01). Besides, it also improved the neuroinflammatory response in the brain, as evidenced by a significant reduction in pro-inflammatory cytokines in cerebrospinal fluid(P<0.05 or P<0.01). Additionally, ZJJ improved the intestinal flora, causing the intestinal flora in DD rats to resemble that of the blank group, characterized by an increased Firmicutes abundance. ZJJ significantly increased the levels of SCFAs(acetic acid, butyric acid, valeric acid, and isovaleric acid)(P<0.01). Therefore, it is deduced that ZJJ can effectively ameliorate intestinal flora dysbiosis, regulate SCFAs, and thereby improve both glucose metabolism disturbances and depressive-like behavior in DD.

Hypothalamic CRF neurons facilitate brain reward function.

Aversive stimuli activate corticotropin-releasing factor (CRF)-expressing neurons in the paraventricular nucleus of hypothalamus (PVNCRF neurons) and other brain stress systems to facilitate avoidance behaviors. Appetitive stimuli also engage the brain stress systems, but their contributions to reward-related behaviors are less well understood. Here, we show that mice work vigorously to optically activate PVNCRF neurons in an operant chamber, indicating a reinforcing nature of these neurons. The reinforcing property of these neurons is not mediated by activation of the hypothalamic-pituitary-adrenal (HPA) axis. We found that PVNCRF neurons send direct projections to the ventral tegmental area (VTA), and selective activation of these projections induced robust self-stimulation behaviors, without activation of the HPA axis. Similar to the PVNCRF cell bodies, self-stimulation of PVNCRF-VTA projection was dramatically attenuated by systemic pretreatment of CRF receptor 1 or dopamine D1 receptor (D1R) antagonist and augmented by corticosterone synthesis inhibitor metyrapone, but not altered by dopamine D2 receptor (D2R) antagonist. Furthermore, we found that activation of PVNCRF-VTA projections increased c-Fos expression in the VTA dopamine neurons and rapidly triggered dopamine release in the nucleus accumbens (NAc), and microinfusion of D1R or D2R antagonist into the NAc decreased the self-stimulation of these projections. Together, our findings reveal an unappreciated role of PVNCRF neurons and their VTA projections in driving reward-related behaviors, independent of their core neuroendocrine functions. As activation of PVNCRF neurons is the final common path for many stress systems, our study suggests a novel mechanism underlying the positive reinforcing effect of stressful stimuli.

Moxibustion ameliorates visceral hypersensitivity by regulating hypothalamus-spinal cord-colon axis in rats with irritable bowel syndrome with diarrhea. / 基于下丘脑-脊髓-ç»“è‚ è½´æŽ¢è®¨è‰¾ç¸æ”¹å–„è ¹æ³»åž‹è‚ æ˜“æ¿€ç»¼åˆå¾å¤§é¼ å† è„é«˜æ•æ„Ÿçš„ä½œç”¨æœºåˆ¶.

OBJECTIVES: To observe the effect of moxibustion intervention on the hypothalamus-spinal cord-colon axis of rats with irritable bowel syndrome with diarrhea (IBS-D) and explore the mechanism of moxibustion in improving visceral hypersensitivity in rats with IBS-D. METHODS: A total of 36 SD rats were randomly divided into normal, model, and moxibustion groups, with 12 rats in each group. The IBS-D model was established by maternal separation + acetic acid stimulation + chronic restraint. Rats of the moxibustion group received bilateral moxibustion on "Tianshu" (ST25) and "Shangjuxu" (ST37) for 15 min, once a day for 7 consecutive days. The body weight, loose stool rate, and minimum threshold volume of abdominal withdrawal reflex (AWR) were measured before and after moxibustion intervention, respectively. The histopathological changes in the colon tissue were observed after HE staining. The number of colonic mucosal mast cells (MCs) was measured by toluidine blue staining. The activation of MCs was determined by tryptase positive expression level and examined by immunohistochemical staining. The content, protein and mRNA expression levels and positive expression levels of corticotropin releasing factor (CRF), substance P (SP), and calcitonin gene-related peptide (CGRP) in the hypothalamus, spinal cord and colon tissues were measured by ELISA, Western blot, real-time fluorescent quantitative PCR and immunofluorescence staining, respectively. RESULTS: Compared with the normal group, the loose stool rate was increased (P<0.01)；the body weight and minimum threshold volume of AWR were decreased (P<0.01)；the inflammatory infiltration of colon tissues was obvious；the number of MCs and positive expression level of tryptase in the colon tissue were increased (P<0.01)；the contents, positive expression le-vels, protein and mRNA expression levels of CRF, SP and CGRP in the hypothalamus, spinal cord and colon tissues were increased (P<0.01, P<0.05) in the model group. After the intervention, compared with the model group, all these indicators showed opposite trends (P<0.01, P<0.05) in the moxibustion group. CONCLUSIONS: Moxibustion can improve visceral hypersensitivity in rats with IBS-D, and its mechanism may be related to regulating the hypothalamic-spinal-colon axis to reduce the release of CRF, SP and CGRP, and thus to inhibite MC in colon tissue.

Neonatal di-(2-ethylhexyl)phthalate exposure induces permanent alterations in secretory CRH neuron characteristics in the hypothalamus paraventricular region of adult male rats.

Corticotrophin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) play a critical role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis. Early-life exposure to di-(2-ethylhexyl) phthalate (DEHP) has been associated with an increased risk of developing psychiatric disorders in adulthood. The present work was designed to explore the impact of neonatal exposure to DEHP on adult PVN CRH neuronal activity. DEHP or vehicle was given to male rat pups from PND16 to PND22. Then, anxiety-like behaviors, serum corticosterone and testosterone, immunohistochemistry, western blotting, fluorescence in situ hybridization and acute ex vivo slice electrophysiological recordings were used to evaluate the influence of DEHP on adult PVN secretory CRH neurons. Neonatal DEHP-exposed rats exhibited enhanced anxiety-like behaviors in adults, with an increase in CORT. Secretory CRH neurons showed higher spontaneous firing activity but could be inhibited by GABAAR blockers. CRH neurons displayed fewer firing spikes, prolonged first-spike latency, depolarizing shifts in GABA reversal potential and strengthened GABAergic inputs, as indicated by increases in the frequency and amplitude of sIPSCs. Enhancement of GABAergic transmission was accompanied by upregulated expression of GAD67 and downregulated expression of GABABR1, KCC2 and GAT1. These findings suggest that neonatal exposure to DEHP permanently altered the characteristics of secretory CRH neurons in the PVN, which may contribute to the development of psychiatric disorders later in life.

Neuroscience: Neurons that use a stress hormone increase eating.

A neuropeptide called corticotropin-releasing hormone (CRH) is known for stress signaling in the brain. A study now shows that a small population of CRH-expressing neurons situated in the lateral hypothalamus area are involved in sensing olfactory food cues and promoting food consumption in mice.

Hypothalamic CRH neurons represent physiological memory of positive and negative experience.

Recalling a salient experience provokes specific behaviors and changes in the physiology or internal state. Relatively little is known about how physiological memories are encoded. We examined the neural substrates of physiological memory by probing CRHPVN neurons of mice, which control the endocrine response to stress. Here we show these cells exhibit contextual memory following exposure to a stimulus with negative or positive valence. Specifically, a negative stimulus invokes a two-factor learning rule that favors an increase in the activity of weak cells during recall. In contrast, the contextual memory of positive valence relies on a one-factor rule to decrease activity of CRHPVN neurons. Finally, the aversive memory in CRHPVN neurons outlasts the behavioral response. These observations provide information about how specific physiological memories of aversive and appetitive experience are represented and demonstrate that behavioral readouts may not accurately reflect physiological changes invoked by the memory of salient experiences.

Growth differentiation factor-15 stimulates the synthesis of corticotropin-releasing factor in hypothalamic 4B cells.

Growth differentiation factor-15 (GDF15) is a stress-activated cytokine that regulates cell growth and inflammatory and stress responses. We previously reported the role and regulation of GDF15 in pituitary corticotrophs. Dexamethasone increases Gdf15 gene expression levels and production. GDF15 suppresses adrenocorticotropic hormone synthesis in pituitary corticotrophs and subsequently mediates the negative feedback effect of glucocorticoids. Here, we analyzed corticotropin-releasing factor (Crf) promoter activity in hypothalamic 4B cells transfected with promoter-driven luciferase reporter constructs. The effects of time and GDF15 concentration on Crf mRNA levels were analyzed using quantitative real-time polymerase chain reaction. Glial cell-derived neurotrophic factor family receptor α-like (GFRAL) protein is expressed in 4B cells. GDF15 increased Crf promoter activity and Crf mRNA levels in 4B cells. The protein kinase A and C pathways also contributed to the GDF15-induced increase in Crf gene expression. GDF15 stimulates GFRAL, subsequently increasing the phosphorylation of AKT, an extracellular signal-related kinase, and the cAMP response element-binding protein. Therefore, GDF15-dependent pathways may be involved in regulating Crf expression under stressful conditions in hypothalamic cells.

Repeated toluene and cyclohexane inhalation produces differential effects on HPA and HPT axes in adolescent male rats.

Misused volatile solvents typically contain toluene (TOL) as the main psychoactive ingredient. Cyclohexane (CHX) can also be present and is considered a safer alternative. Solvent misuse often occurs at early stages of life, leading to permanent neurobehavioral impairment and growth retardation. However, a comprehensive examination of the effects of TOL and CHX on stress regulation and energy balance is lacking. Here, we compared the effect of a binge-pattern exposure to TOL or CHX (4,000 or 8,000 ppm) on body weight, food intake, the hypothalamus-pituitary-adrenal (HPA) and hypothalamus-pituitary-thyroid (HPT) axes in male adolescent Wistar rats. At 8,000 ppm, TOL decreased body weight gain without affecting food intake. In addition, TOL and CHX altered the HPA and HPT axes' function in a solvent- and concentration-dependent manner. The highest TOL concentration produced HPA axis hyperactivation in animals not subjected to stress, which was evidenced by increased corticotropin-releasing-factor (CRF) release from the median eminence (ME), elevated adrenocorticotropin hormone (ACTH) and corticosterone serum levels, and decreased CRF mRNA levels in the hypothalamic paraventricular nucleus (PVN). TOL (8,000 ppm) also increased triiodothyronine (T3) serum levels, decreased pro-thyrotropin-releasing-hormone (pro-TRH) mRNA transcription in the PVN, pro-TRH content in the ME, and serum thyroid stimulating hormone (TSH) levels. CHX did not affect the HPA axis. We propose that the increased HPT axis activity induced by TOL can be related to the impaired body weight gain associated with inhalant misuse. These findings may contribute to a better understanding of the effects of the misused solvents TOL and CHX.

Dual action of serotonin on local excitatory and inhibitory neural circuits regulating the corticotropin-releasing factor neurons in the paraventricular nucleus of the hypothalamus.

Serotonergic neurons originating from the raphe nuclei have been proposed to regulate corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVH). Since glutamate- and γ-aminobutyric acid (GABA)-containing neurons, constituting the hypothalamic local circuits, innervate PVH CRF neurons, we examined whether they mediate the actions of serotonin (5-hydroxytryptamine [5-HT]) on CRF neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in PVH CRF neurons, under whole cell patch-clamp, using the CRF-modified yellow fluorescent protein (Venus) ΔNeo mouse. Serotonin elicited an increase in the frequency of sEPSCs in 77% of the cells and a decrease in the frequency of sIPSCs in 71% of the cells, tested in normal medium. Neither the amplitude nor decay time of sEPSC and sIPSC was affected, thus the site(s) of action of serotonin may be presynaptic. In the presence of tetrodotoxin (TTX), serotonin had no significant effects on either parameter of sEPSC or sIPSC, indicating that the effects of serotonin are action potential-dependent, and that the presynaptic interneurons are largely intact within the slice; distant neurons may exist, though, since some 20%-30% of neurons did not respond to serotonin without TTX. We next examined through what receptor subtype(s) serotonin exerts its effects on presynaptic interneurons. DOI (5-HT2A/2C agonist) mimicked the action of serotonin on the sIPSCs, and the serotonin-induced decrease in sIPSC frequency was inhibited by a selective 5-HT2C antagonist RS102221. 8-OH-DPAT (5-HT1A/7 agonist) mimicked the action of serotonin on the sEPSCs, and the serotonin-induced increase in sEPSC frequency was inhibited by a selective 5-HT7 antagonist SB269970. Thus, serotonin showed a dual action on PVH CRF neurons, by upregulating glutamatergic- and downregulating GABAergic interneurons; the former may partly be mediated by 5-HT7 receptors, whereas the latter by 5-HT2C receptors. The CRF-Venus ΔNeo mouse was useful for the electrophysiological examination.

Impact and Role of Hypothalamic Corticotropin Releasing Hormone Neurons in Withdrawal from Chronic Alcohol Consumption in Female and Male Mice.

Worldwide, alcohol use and abuse are a leading risk of mortality, causing 5.3% of all deaths (World Health Organization, 2022). The endocrine stress system, initiated by the peripheral release of corticotropin releasing hormone (CRH) from primarily glutamatergic neurons in the paraventricular nucleus of the hypothalamus (PVN), is profoundly linked with alcohol use, abuse, and relapse (Blaine and Sinha, 2017). These PVN CRH-releasing (PVNCRH) neurons are essential for peripheral and central stress responses (Rasiah et al., 2023), but little is known about how alcohol affects these neurons. Here, we show that two-bottle choice alcohol consumption blunts the endocrine-mediated corticosterone response to stress during acute withdrawal in female mice. Conversely, using slice electrophysiology, we demonstrate that acute withdrawal engenders a hyperexcitable phenotype of PVNCRH neurons in females that is accompanied by increased glutamatergic transmission in both male and female mice. GABAergic synaptic transmission was unaffected by alcohol history. We then tested whether chemogenetic inhibition of PVNCRH neurons would restore stress response in female mice with a history of alcohol drinking in the looming disk test, which mimics an approaching predator threat. Accordingly, inhibition of PVNCRH neurons reduced active escape in hM4Di alcohol history mice only. This study indicates that stress-responsive PVNCRH neurons in females are particularly affected by a history of alcohol consumption. Interestingly, women have indicated an increase in heavy alcohol use to cope with stress (Rodriguez et al., 2020), perhaps pointing to a potential underlying mechanism in alcohol-mediated changes to PVNCRH neurons that alter stress response.SIGNIFICANCE STATEMENT Paraventricular nucleus of the hypothalamus neurons that release corticotropin releasing hormone (PVNCRH) are vital for stress response. These neurons have been understudied in relation to alcohol and withdrawal despite profound relations between stress, alcohol use disorders (AUD), and relapse. In this study, we use a variety of techniques to show that acute withdrawal from a history of alcohol impacts peripheral stress response, PVNCRH neurons, and behavior. Specifically, PVNCRH are in a hyperactive state during withdrawal, which drives an increase in active stress coping behaviors in female mice only. Understanding how alcohol use and withdrawal affects stress responding PVNCRH neurons may contribute to finding new potential targets for the treatment of alcohol use disorder.

CRH-R2 signalling modulates feeding and circadian gene expression in hypothalamic mHypoA-2/30 neurons.

The hypothalamic type 2 corticotropin releasing hormone receptor (CRH-R2) plays critical roles in homeostatic regulation, particularly in fine tuning stress recovery. During acute stress, the CRH-R2 ligands CRH and urocortins promote adaptive responses and feeding inhibition. However, in rodent models of chronic stress, over-exposure of hypothalamic CRH-R2 to its cognate agonists is associated with urocortin 2 (Ucn2) resistance; attenuated cAMP-response element binding protein (CREB) phosphorylation and increased food intake. The molecular mechanisms involved in these altered CRH-R2 signalling responses are not well described. In the present study, we used the adult mouse hypothalamus-derived cell line mHypoA-2/30 to investigate CRH-R2 signalling characteristics focusing on gene expression of molecules involved in feeding and circadian regulation given the role of clock genes in metabolic control. We identified functional CRH-R2 receptors expressed in mHypoA-2/30 cells that differentially regulate CREB and AMP-activated protein kinase (AMPK) phosphorylation and downstream expression of the appetite-regulatory genes proopiomelanocortin (Pomc) and neuropeptide Y (Npy) in accordance with an anorexigenic effect. We studied for the first time the effects of Ucn2 on clock genes in native and in a circadian bioluminescence reporter expressing mHypoA-2/30 cells, detecting enhancing effects of Ucn2 on mRNA levels and rhythm amplitude of the circadian regulator Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), which could facilitate anorexic responses in the activity circadian phase. These data uncover novel aspects of CRH-R2 hypothalamic signalling that might be important in regulation of circadian feeding during stress responses.

Ghrelin Action in the PVH of Male Mice: Accessibility, Neuronal Targets, and CRH Neurons Activation.

The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.

Vasopressin Expressed in Hypothalamic CRF Neurons Causes Impaired Water Diuresis in Secondary Adrenal Insufficiency.

Patients with secondary adrenal insufficiency can present with impaired free water excretion and hyponatremia, which is due to the enhanced secretion of vasopressin (AVP) despite increased total body water. AVP is produced in magnocellular neurons in the paraventricular nucleus of the hypothalamus (PVH) and supraoptic nucleus and in parvocellular corticotropin-releasing factor (CRF) neurons in the PVH. This study aimed to elucidate whether magnocellular AVP neurons or parvocellular CRF neurons coexpressing AVP are responsible for the pathogenesis of hyponatremia in secondary adrenal insufficiency. The number of CRF neurons expressing copeptin, an AVP gene product, was significantly higher in adrenalectomized AVP-floxed mice (AVPfl/fl) than in sham-operated controls. Adrenalectomized AVPfl/fl mice supplemented with aldosterone showed impaired water diuresis under ad libitum access to water or after acute water loading. They became hyponatremic after acute water loading, and it was revealed under such conditions that aquaporin-2 (AQP2) protein levels were increased in the kidney. Furthermore, translocation of AQP2 to the apical membrane was markedly enhanced in renal collecting duct epithelial cells. Remarkably, all these abnormalities observed in the mouse model for secondary adrenal insufficiency were ameliorated in CRF-AVP-/- mice that lacked AVP in CRF neurons. Our study demonstrates that CRF neurons in the PVH are responsible for the pathogenesis of impaired water excretion in secondary adrenal insufficiency.

Olfactory modulation of stress-response neural circuits.

Stress responses, which are crucial for survival, are evolutionally conserved throughout the animal kingdom. The most common endocrine axis among stress responses is that triggered by corticotropin-releasing hormone neurons (CRHNs) in the hypothalamus. Signals of various stressors are detected by different sensory systems and relayed through individual neural circuits that converge on hypothalamic CRHNs to initiate common stress hormone responses. To investigate the neurocircuitry mechanisms underlying stress hormone responses induced by a variety of stressors, researchers have recently developed new approaches employing retrograde transsynaptic viral tracers, providing a wealth of information about various types of neural circuits that control the activity of CRHNs in response to stress stimuli. Here, we review earlier and more recent findings on the stress neurocircuits that converge on CRHNs, focusing particularly on olfactory systems that excite or suppress the activities of CRHNs and lead to the initiation of stress responses. Because smells are arguably the most important signals that enable animals to properly cope with environmental changes and survive, unveiling the regulatory mechanisms by which smells control stress responses would provide broad insight into how stress-related environmental cues are perceived in the animal brain.

Electroacupuncture in the treatment of IBS in rats: investigation of the mechanisms of CRH+ neurons in the paraventricular nucleus.

Electroacupuncture (EA) is well documented to treat irritable bowel syndrome (IBS). However, the mechanism of the central nervous system related to IBS and acupuncture stimulation is still not well known. In this study, a rat model of IBS was established by cold-restraint comprehensive stresses for 15 days, and it was found that the levels of corticotropin-releasing hormone (CRH), corticosterone (CORT), and adrenocorticotropic hormone (ACTH) in the peripheral serum were increased; the visceral sensitivity was enhanced; and the intestinal motility was accelerated, specifically, there was an enhancement in the discharge frequency of neurons in the paraventricular nucleus (PVN). EA treatment for 3 days, 20 min/day, alleviated the increase in the levels of CRH, CORT, and ACTH in the peripheral serum of rats, reduced the visceral sensitivity of IBS rats, and inhibited colon movement and discharge frequency of the neurons in the PVN. In addition, EA could reduce the excitability of CRH neurons and the expression of corticotropin-releasing hormone receptor 1 (CRHR1) and corticotropin-releasing hormone receptor 2 (CRHR2) in PVN. At the same time, the expression of CRH, CRHR1, and CRHR2 in the peripheral colon was decreased. Taken together, EA appears to regulate intestinal functional activity through the central CRH nervous system, revealing the central regulation mechanism of EA in IBS rats, and providing a scientific research basis for the correlation among the meridians, viscera, and brain.NEW & NOTEWORTHY The purpose of this research was to determine the central regulatory mechanism of electroacupuncture (EA) in rats with irritable bowel syndrome (IBS). Our results showed that combined with the serum changes in corticotropin-releasing hormone (CRH), corticosterone (CORT), and adrenocorticotropic hormone (ACTH), the improvement of IBS by EA was related to them. Furthermore, EA could regulate intestinal functional activity through the central CRH+ nervous system.

Electroacupuncture Ameliorates Hypothalamic‒Pituitary‒Adrenal Axis Dysfunction Induced by Surgical Trauma in Mice Through the Hypothalamic Oxytocin System.

Electroacupuncture (EA) can effectively reduce surgical stress reactions and promote postoperative recovery, but the mechanisms remain unclear. The present study aims to examine the effects of EA on the hyperactivity of the hypothalamic‒pituitary‒adrenal (HPA) axis and investigate its potential mechanisms. Male C57BL/6 mice were subjected to partial hepatectomy (HT). The results showed that HT increased the concentrations of corticotrophin-releasing hormone (CRH), corticosterone (CORT), and adrenocorticotropic hormone (ACTH) in the peripheral blood and upregulated the expression of CRH and glucocorticoid receptors (GR) proteins in the hypothalamus. EA treatment significantly inhibited the hyperactivity of the HPA axis by decreasing the concentration of CRH, CORT, and ACTH in peripheral blood and downregulating the expression of CRH and GR in the hypothalamus. Moreover, EA treatment reversed the HT-induced downregulation of oxytocin (OXT) and oxytocin receptor (OXTR) in the hypothalamus. Furthermore, intracerebroventricular injection of the OXTR antagonist atosiban blocked the effects of EA. Thus, our findings implied that EA mitigated surgical stress-induced HPA axis dysfunction by activating the OXT/OXTR signaling pathway.

Disruption of tonic endocannabinoid signalling triggers cellular, behavioural and neuroendocrine responses consistent with a stress response.

BACKGROUND AND PURPOSE: Endocannabinoid (eCB) signalling gates many aspects of the stress response, including the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is controlled by corticotropin releasing hormone (CRH) producing neurons in the paraventricular nucleus of the hypothalamus (PVN). Disruption of eCB signalling increases drive to the HPA axis, but the mechanisms subserving this process are poorly understood. EXPERIMENTAL APPROACH: Using an array of cellular, endocrine and behavioural readouts associated with activation of CRH neurons in the PVN, we evaluated the contributions of tonic eCB signalling to the generation of a stress response. KEY RESULTS: The CB1 receptor antagonist/inverse agonist AM251, neutral antagonist NESS243 and NAPE PLD inhibitor LEI401 all uniformly increased Fos in the PVN, unmasked stress-linked behaviours, such as grooming, and increased circulating CORT, recapitulating the effects of stress. Similar effects were also seen after direct administration of AM251 into the PVN, while optogenetic inhibition of PVN CRH neurons ameliorated stress-like behavioural changes produced by disruption of eCB signalling. CONCLUSIONS AND IMPLICATIONS: These data indicate that under resting conditions, constitutive eCB signalling restricts activation of the HPA axis through local regulation of CRH neurons in the PVN.

Recruitment of Corticotropin-Releasing Hormone (CRH) Neurons in Categorically Distinct Stress Reactions in the Mouse Brain.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular hypothalamic nucleus (PVH) are in the position to integrate stress-related information and initiate adaptive neuroendocrine-, autonomic-, metabolic- and behavioral responses. In addition to hypophyseotropic cells, CRH is widely expressed in the CNS, however its involvement in the organization of the stress response is not fully understood. In these experiments, we took advantage of recently available Crh-IRES-Cre;Ai9 mouse line to study the recruitment of hypothalamic and extrahypothalamic CRH neurons in categorically distinct, acute stress reactions. A total of 95 brain regions in the adult male mouse brain have been identified as containing putative CRH neurons with significant expression of tdTomato marker gene. With comparison of CRH mRNA and tdTomato distribution, we found match and mismatch areas. Reporter mice were then exposed to restraint, ether, high salt, lipopolysaccharide and predator odor stress and neuronal activation was revealed by FOS immunocytochemistry. In addition to a core stress system, stressor-specific areas have been revealed to display activity marker FOS. Finally, activation of CRH neurons was detected by colocalization of FOS in tdTomato expressing cells. All stressors resulted in profound activation of CRH neurons in the hypothalamic paraventricular nucleus; however, a differential activation of pattern was observed in CRH neurons in extrahypothalamic regions. This comprehensive description of stress-related CRH neurons in the mouse brain provides a starting point for a systematic functional analysis of the brain stress system and its relation to stress-induced psychopathologies.