Cocaine- And Amphetamine-regulated Transcript Research Articles

Orphan peptide and G protein-coupled receptor signalling in alcohol use disorder.

Neuropeptides and G protein-coupled receptors (GPCRs) have long been, and continue to be, one of the most popular target classes for drug discovery in CNS disorders, including alcohol use disorder (AUD). Yet, orphaned neuropeptide systems and receptors (oGPCR), which have no known cognate receptor or ligand, remain understudied in drug discovery and development. Orphan neuropeptides and oGPCRs are abundantly expressed within the brain and represent an unprecedented opportunity to address brain function and may hold potential as novel treatments for disease. Here, we describe the current literature regarding orphaned neuropeptides and oGPCRs implicated in AUD. Specifically, in this review, we focus on the orphaned neuropeptide cocaine- and amphetamine-regulated transcript (CART), and several oGPCRs that have been directly implicated in AUD (GPR6, GPR26, GPR88, GPR139, GPR158) and discuss their potential and pitfalls as novel treatments, and progress in identifying their cognate receptors or ligands.

Cotadutide (GLP-1/Glucagon dual receptor agonist) modulates hypothalamic orexigenic and anorexigenic neuropeptides in obese mice

The hypothalamic neuropeptides linked to appetite and satiety were investigated in obese mice treated with cotadutide (a dual receptor agonist of glucagon-like peptide 1 (GLP-1R)/Glucagon (GCGR)). Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Each group was further divided, adding cotadutide treatment and forming groups C, CC, HF, and HFC for four additional weeks. The hypothalamic arcuate neurons were labeled by immunofluorescence, and protein expressions (Western blotting) for neuropeptide Y (NPY), proopiomelanocortin (POMC), agouti-related protein (AgRP), and cocaine- and amphetamine-regulated transcript (CART). Cotadutide enhanced POMC and CART neuropeptides and depressed NPY and AGRP neuropeptides. In addition, gene expressions (RT-qPCR) determined that Lepr (leptin receptor) and Calcr (calcitonin receptor) were diminished in HF compared to C but enhanced in CC compared to C and HFC compared to HF. Besides, Socs3 (suppressor of cytokine signaling 3) was decreased in HFC compared to HF, while Sst (somatostatin) was higher in HFC compared to HF; Tac1 (tachykinin 1) and Mc4r (melanocortin-4-receptor) were lower in HF compared to C but increased in HFC compared to HF. Also, Glp1r and Gcgr were higher in HFC compared to HF. In conclusion, the findings are compelling, demonstrating the effects of cotadutide on hypothalamic neuropeptides and hormone receptors of obese mice. Cotadutide modulates energy balance through the gut-brain axis and its associated signaling pathways. The study provides insights into the mechanisms underlying cotadutide's anti-obesity effects and its possible implications for obesity treatment.

Are decreased cocaine- and amphetamine regulated transcript and Agouti- related peptide levels associated Eating behavior in medication-free children with attention deficit and hyperactivity disorder?

This study aimed to investigate plasma levels of cocaine- and amphetamine-regulated transcript (CART), agouti-related protein (AgRP), cholecystokinin (CCK) and peptide YY (PYY) and their relationship with eating behaviors among children with attention deficit hyperactivity disorder (ADHD) and healthy controls. A total of 94 medication-free children with ADHD and 82 controls aged 8–14 years were included in this study. The Plasma levels of CART, AgRP, CCK and PYY were measured using enzyme-linked immunosorbent assay kits. The Children's Eating Behavior Questionnaire (CEBQ) was used to assess eating behaviors in children. CART and AgRP levels were found to be significantly lower in the ADHD group than in the control group, while CCK levels were found to be significantly higher in the ADHD group than in the control group. However, there was no significant difference in PYY levels between the groups. Compared to controls, those with ADHD demonstrated significantly higher scores on the CEBQ subscales of food responsiveness, emotional overeating, desire to drink, enjoyment of food, and food fussiness, and significantly lower scores on the slowness of eating subscale. CART was significantly correlated with emotional overeating and enjoyment of food scores, while AgRP was significantly correlated with emotional undereating scores. Covariance analysis was performed by controlling potential confounders such as body mass index, age and sex, and the results were found to be unchanged. It was concluded that CART, AgRP, and CCK may play a potential role in the pathogenesis of ADHD.

Bisphenol A Effects on Neurons’ Neurochemical Character in the Urinary Bladder Intramural Ganglia of Domestic Pigs

Bisphenol A (BPA), a substance globally used to produce plastics, is part of many everyday items, including bottles, food containers, electronic elements, and others. It may penetrate the environment and living organisms, negatively affecting, among others, the nervous, immune, endocrine, and cardiovascular systems. Knowledge of the impact of BPA on the urinary bladder is extremely scarce. This study investigated the influence of two doses of BPA (0.05 mg/kg body weight (b.w.)/day and 0.5 mg/kg b.w./day) given orally for 28 days on the neurons situated in the ganglia located in the urinary bladder trigone using the typical double immunofluorescence method. In the study, an increase in the percentage of neurons containing substance P (SP), galanin (GAL), a neuronal isoform of nitric oxide synthase (nNOS—used as the marker of nitrergic neurons), and/or cocaine- and amphetamine-regulated transcript (CART) peptide was noted after BPA administration. The severity of these changes depended on the dose of BPA and the type of neuronal factors studied. The most visible changes were noted in the cases of SP- and/or GAL-positive neurons after administering a higher dose of BPA. The results have shown that oral exposure to BPA, lasting even for a short time, affects the intramural neurons in the urinary bladder wall, and changes in the neurochemical characterisation of these neurons may be the first signs of BPA-induced pathological processes in this organ.

Peptidergic modulation of motor neuron output via CART signaling at C bouton synapses

The intensity of muscle contraction, and therefore movement vigor, needs to be adaptable to enable complex motor behaviors. This can be achieved by adjusting the properties of motor neurons, which form the final common pathway for all motor output from the central nervous system. Here, we identify roles for a neuropeptide, cocaine- and amphetamine-regulated transcript (CART), in the control of movement vigor. We reveal distinct but parallel mechanisms by which CART and acetylcholine, both released at C bouton synapses on motor neurons, selectively amplify the output of subtypes of motor neurons that are recruited during intense movement. We find that mice with broad genetic deletion of CART or selective elimination of acetylcholine from C boutons exhibit deficits in behavioral tasks that require higher levels of motor output. Overall, these data uncover spinal modulatory mechanisms that control movement vigor to support movements that require a high degree of muscle force.

Mate calling alters expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain of male frog Microhyla nilphamariensis

Croaking is a unique component of reproductive behaviour in amphibians which plays a key role in intraspecies communication and mate evaluation. While gonadal hormones are known to induce croaking, central regulation of sound production is less studied. Croaking is a dramatic, transient activity that sets apart an animal from non-croaking individuals. Herein, we aim at examining the profile of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in actively croaking and non-croaking frog Microhyla nilphamariensis. In anurans, this peptide is widely expressed in the areas inclusive of acoustical nuclei as well as areas relevant to reproduction. CART immunoreactivity was far more in the preoptic area (POA), anteroventral tegmentum (AV), ventral hypothalamus (vHy), pineal (P) and pituitary gland of croaking frog compared to non-croaking animals. On similar lines, tissue fragments collected from the mid region of the brain inclusive of POA, vHy, AV, pineal and pituitary gland of croaking frog showed upregulation of CART mRNA. However, CART immunoreactivity in the neuronal perikarya of raphe (Ra) was completely abolished during croaking activity. The data suggest that CART signaling in the brain may be an important player in mediating croaking behaviour in the frog.

Cocaine and amphetamine regulated transcript (CART): a newly characterized neuropeptide in human prostate

ABSTRACT Cocaine and amphetamine regulated transcript (CART) is a somatostatin-like polypeptide. CART has been localized in the CNS, hypothalamo-pituitary-adrenocortical (HPA) axis, pancreatic islets and enteric nervous system. We investigated the cellular localization of CART in normal human prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia and acinar adenocarcinoma. CART was assessed using immunohistochemistry (IHC) and in situ hybridization (ISH), and its gene expression was identified by RTqPCR. We found cellular expression of CART in both normal prostatic luminal secretory epithelial cells neuroendocrine cells (NEC) of both ducts and acini. The cellular appearance indicated a cycle of neuropeptide synthesis and secretion as validated by ISH/IHC concordance. RTqPCR analysis also validated the immunohistochemical data and gene expression, which both indicated low to moderate expression in prostatic tissues. CART expression also was increased in both neuroendocrine and glandular epithelial cell populations from samples of benign prostatic hyperplasia as validated by IHC, ISH and RTqPCR. CART expression was markedly diminished and, in some cases, entirely absent in tissues of prostatic intraepithelial neoplasia and adenocarcinoma. Owing to loss of CART expression in adenocarcinoma and its increase in benign prostatic hyperplasia, CART may prove to be an important prostate marker.

Cocaine and amphetamine regulated transcript (CART) mediates sex differences in binge drinking through central taste circuits

The neuropeptide cocaine- and amphetamine-regulated transcript (CART) has been implicated in alcohol consumption and reward behaviours, yet mechanisms mediating these effects have yet to be identified. Using a transgenic CART knockout (KO) mouse line we uncovered a sexually dimorphic effect of CART in binge drinking, with male CART KO mice increasing intake, whilst female CART KO mice decreased their alcohol intake compared to controls. Female CART KO mice show greater sensitivity to bitter solutions that can be overshadowed through addition of a sweetener, implicating taste as a factor. Further we identify that this is not driven through peripherally circulating sex hormones, but the central nucleus of the amygdala (CeA) is a locus where CART contributes to the regulation of alcohol consumption, with CeA CART neutralisation specifically reducing plain alcohol, but not sweetened alcohol consumption in female mice. These findings may have implications for the development of sex-specific treatment options for alcohol use disorders through targeting the CART system.

Influence of bisphenol A and its analog bisphenol S on cocaine- and amphetamine-regulated transcript peptide-positive enteric neurons in the mouse gastrointestinal tract.

Bisphenol A (BPA) is used in large quantities for the production of plastics and is present in various everyday objects. It penetrates living organisms and shows multidirectional adverse influence on many internal organs. For this reason, BPA is often replaced in plastic production by other substances. One of them is bisphenol S (BPS), whose effects on the enteric nervous system (ENS) have not been explained. Therefore, the present study compares the influence of BPA and BPS on the number of enteric neurons immunoreactive to cocaine-and amphetamine-regulated transcript (CART) peptide located in the ENS of the stomach, jejunum and colon with the use of double immunofluorescence method. The obtained results have shown that both bisphenols studied induced an increase in the number of CART-positive enteric neurons, and the severity of changes depended on the type of enteric ganglion, the dose of bisphenols and the segment of the digestive tract. The most visible changes were noted in the myenteric ganglia in the colon. Moreover, in the colon, the changes submitted by BPS are more noticeable than those observed after BPA administration. In the stomach and jejunum, bisphenol-induced changes were less visible, and changes caused by BPS were similar or less pronounced than those noted under the impact of BPA, depending on the segment of the gastrointestinal tract and ganglion type studied. The results show that BPS affects the enteric neurons containing CART in a similar way to BPA, and the BPS impact is even stronger in the colon. Therefore, BPS is not neutral for the gastrointestinal tract and ENS.

Obesity wars: hypothalamic sEVs a new hope.

There are currently several pharmacological therapies available for the treatment of obesity, targeting both the central nervous system (CNS) and peripheral tissues. In recent years, small extracellular vesicles (sEVs) have been shown to be involved in many pathophysiological conditions. Because of their special nanosized structure and contents, sEVs can activate receptors and trigger intracellular pathways in recipient cells. Notably, in addition to transferring molecules between cells, sEVs can also alter their phenotypic characteristics. The purpose of this review is to discuss how sEVs can be used as a CNS-targeted strategy for treating obesity. Furthermore, we will evaluate current findings, such as the sEV-mediated targeting of hypothalamic AMP-activated protein kinase (AMPK), and discuss how they can be translated into clinical application.

Sanguinarine-based isoquinoline alkaloids modulated the gut-brain axis and enhanced growth performance and gut integrity in natural heat stress broiler chickens

Persistent hot and humid tropical climate induces heat stress (HS), negatively impacting gut integrity with inflammation and tight junction degradation in broilers. As integrating operations of the digestive tract and hypothalamus in avian are regulated by feed intake, the effect of HS may be eased by feed supplementation. Herein, the effect of isoquinoline alkaloids (IQ) supplementation from Macleaya cordata on integrating gut-brain axis and gut integrity was assessed. 720 male broilers (Ross 308) were treated with IQ at null (control), 60 (IQ60), and 100 mg/kg (IQ100). They were reared in open production house between April and May in Southern Thailand with 33.0–38.0°C (average 35.0°C), 75–80% relative humidity (RH), and 160–200 heat stress index (HI). From day 1 to 42, IQ supplementation positively affected growth performance where body weight (BW), body weight gain (BWG), and feed intake (FI) were significantly higher in birds fed IQs than in the control group (P<0.05), and feed conversion ratio (FCR) was significantly lower in the IQ group (P<0.05). IQ improved gut integrity by reducing inflammation with significant down-regulation of nitric oxide synthase (iNOS), cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), and nuclear factor κB (NF-κB) in the ileum (P<0.05). It strengthened tight junction by upregulates occludin (OCLD), claudin-1 (CLDN1), tight junction proteins-1 (TJP-1) and mucin-2 (MUC-2) in the jejunum (P<0.05). As a result, serum fluorescein isothiocyanate-dextran (FITC-d) and corticosterone levels were significantly lower (P<0.05). In terms of the gut-brain axis, IQ supplementation reduces ghrelin and cholecystokinin (CCK) mRNA expression in the jejunum and hypothalamus (P<0.05), which downregulated the expression of pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), corticotropin-releasing hormone (CRH), and melanocortin four receptor (MC4R). It was the first evidence that IQ influences on modulating the gut-brain axis to suppress anorexigenic regulation in broilers with HS exposure.

Caudal DMN neurons innervate the spleen and release CART peptide to regulate neuroimmune function

BackgroundInflammation is a fundamental biological response to injury and infection, which if unregulated can contribute to the pathophysiology of many diseases. The vagus nerve, which primarily originates from the dorsal motor nucleus (DMN), plays an important role in rapidly dampening inflammation by regulating splenic function. However, direct vagal innervation of the spleen, which houses the majority of immune and inflammatory cells, has not been established. As an alternative to direct innervation, an anti-inflammatory reflex pathway has been proposed which involves the vagus nerve, the sympathetic celiac ganglion, and the neurotransmitter norepinephrine. Although sympathetic regulation of inflammation has been shown, the interaction of the vagus nerve and the celiac ganglia requires a unique interaction of parasympathetic and sympathetic inputs, making this putative mechanism of brain–spleen interaction controversial.BodyAs neuropeptides can be expressed at relatively high levels in neurons, we reasoned that DMN neuropeptide immunoreactivity could be used to determine their target innervation. Employing immunohistochemistry, subdiaphragmatic vagotomy, viral tract tracing, CRISPR-mediated knock-down, and functional assays, we show that cocaine and amphetamine-regulated transcript (CART) peptide-expressing projection neurons in the caudal DMN directly innervate the spleen. In response to lipopolysaccharide (LPS) stimulation, CART acts to reduce inflammation, an effect that can be augmented by intrasplenic administration of a synthetic CART peptide. These in vivo effects could be recapitulated in cultured splenocytes, suggesting that these cells express the as yet unidentified CART receptor(s).ConclusionOur results provide evidence for direct connections between the caudal DMN and spleen. In addition to acetylcholine, these neurons express the neuropeptide CART that, once released, acts to suppress inflammation by acting directly upon splenocytes.

Cocaine- and Amphetamine-Regulated Transcript (CART) Peptide Is Co-Expressed with Parvalbumin, Neuropeptide Y and Somatostatin in the Claustrum of the Chinchilla.

Although for many years, researchers have been working on understanding the function of the cocaine- and amphetamine-regulated transcript (CART) peptide at the central- and peripheral-nervous-system level, data describing the presence of CART in the claustrum are still missing. Therefore, the aim of the present study was to immunohistochemically investigate the CART expression in the claustrum neurons in chinchillas as well as the CART co-localization with somatostatin (SOM), parvalbumin (PV), and neuropeptide Y (NPY) using double-immunohistochemical staining. The claustrum is divided into two main parts: the dorsal segment (CL), which is located above the rhinal fissure, and the ventral segment (EN), located below the rhinal fissure. The presence of HU C/D-IR CART-IR-positive neurons was detected in both the insular claustrum (CL) and the endopiriform nucleus (EN). The vast majority of CART-IR neurons were predominantly small and medium in size and were evenly scattered throughout the claustrum. CART co-localization with selected neurotransmitters/neuromodulators (SOM, NPY, and PV) showed the presence of a CART-IR reaction only in the neurons, while the nerve fibers were, in all cases, devoid of the CART-IR response. Our research supplements missing knowledge about the distribution and co-localization pattern of CART with SOM, NPY, and PV in the chinchilla claustrum, and also provides a better understanding of the similarities and differences compared to other species of rodents and other mammals.

GPR-160 Receptor Signaling in the Dorsal Vagal Complex of Male Rats Modulates Meal Microstructure and CART-Mediated Hypophagia.

The g-protein coupled receptor GPR-160, recently identified as a putative receptor for the cocaine and amphetamine-regulated transcript (CART) peptide, shows abundant expression in the energy-balance control nuclei, including the dorsal vagal complex (DVC). However, its physiological role in the control of food intake has yet to be fully explored. Here, we performed a virally mediated, targeted knockdown (KD) of Gpr160 in the DVC of male rats to evaluate its physiological role in control of feeding. Our results indicate that DVC Gpr160 KD affects meal microstructure. Specifically, DVC Gpr160 KD animals consumed more frequent, but shorter meals during the dark phase and showed decreased caloric intake and duration of meals during the light phase. Cumulatively, however, these bidirectional effects on feeding resulted in no difference in body weight gain. We next tested the role of DVC GPR-160 in mediating the anorexigenic effects of exogenous CART. Our results show that DVC Gpr160 KD partially attenuates CART's anorexigenic effects. To further characterize Gpr160+ cells in the DVC, we utilized single-nucleus RNA sequencing data to uncover abundant GPR-160 expression in DVC microglia and only minimal expression in neurons. Altogether, our results suggest that DVC CART signaling may be mediated by Gpr160+ microglia, which in turn may be modulating DVC neuronal activity to control food intake.

A potential role for GPR160 and its ligand, CART, in the modulation of feeding behavior in Prader-Willi Syndrome

Hyperphagia is a hallmark of Prader-Willi syndrome (PWS). Managing the extreme hunger and insatiability associated with PWS remains a considerable challenge to PWS patients and their caregivers. Appetite and feeding behavior are governed by numerous neural circuits, several of which are potentially disrupted when some or all of the Prader-Willi critical region on chromosome 15 is lost. Previous research has focused on well-established feeding circuits in an effort to uncover the physiology underlying PWS hyperphagic behavior; however, due to the complex interplay between these circuits, much is left to be explored. We have previously demonstrated that interactions between GPR160 and its ligand cocaine and amphetamine regulated transcript (CART) are important in the control of feeding behavior. Several groups have reported that CART exerts anorexigenic properties when injected into areas of the brain that are important to feeding behavior, including regions of the brainstem and hypothalamus. The prohormone convertase PSCK1, which is responsible for post-translational processing of the CART prohormone, appears to be reduced in PWS patients, suggesting that CART could play a role in the pathophysiology of PWS. Using a rat model of PWS supplied by the Foundation for Prader-Willi Research (Magel2- deficient), we seek to investigate this potential role of CART and GPR160 in PWS-associated hyperphagia. Through the use of continuous monitoring of food and water intake, we were able to define meal patterning differences between wild type and paternally Magel2-deficient rats. In addition to evaluating baseline meal patterning, we also administered CART centrally and recorded CART-induced changes in feeding behavior. Lastly, we determined physiologic changes in levels of GPR160, CART, and PSCK1 in the pituitary, hypothalamus, amygdala, and brainstem from WT and paternally-deficient Magel2 rats in the setting of metabolic stress (fasting). Although additional studies must be performed to better define the role of CART and GPR160 in the feeding behavior of PWS patients, we anticipate that this ligand-receptor pair has the potential to serve as a novel therapeutic target for the treatment of PWS-associated hyperphagia. Foundation for Prader-Willi Research This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alteration in the neurochemical profile of ENS neurons in the porcine ileum after oral exposure of glyphosate

Glyphosate-based herbicides are one of the most intensively used pollutants worldwide. The main goal of present study was to determine the changes in the neurochemical phenotype of enteric nervous system (ENS) neurons in the porcine ileum after oral glyphosate administration.Study was performed on 15 immature gilts divided into 3 groups: control (C) - animals receiving empty gelatin capsules; G1 - animals receiving a low dose of glyphosate- corresponding to the theoretical maximum daily intake (TMDI) - 0.05 mg/kg of b.w./day; G2- animals receiving a higher dose of glyphosate - corresponding to the acceptable daily intake (ADI) - 0.5 mg/kg of b.w./day) in gelatin capsules orally for 28 days. After this time, the animals were euthanized and ileum samples were collected. Frozen sections (12μm thick) were then subjected to the double immunofluorescent staining technique using primary antibody directed towards Hu C/D (used as a pan-neuronal marker) and pituitary adenylate cyclase-activating polypeptide (PACAP), neuronal nitric oxide synthase (nNOS), vesicular acetylcholine transporter (VAChT), cocaine and amphetamine-regulated transcript (CART) and appropriate secondary antibodies.Supplementation of lower dose of glyphosate increased the population of PACAP- like immunoreactive (LI) neurons in the outer submucous plexus (OSP) (from 6.34±1.24 to 14.12±1.68%), nNOS-LI neurons in the myenteric plexus (MP) (from 29.96±2.40 to 37.57±1.17%) and the inner submucous plexus (ISP) (from 12.64±1.10 to 19.76±1.34%), CART-LI neurons in the MP (from 20.44±0.81 to 29.77±1.16%) and the ISP (from 6.07±1.00 to 10.44±0.94%) and decreased the number of VAChT-LI neurons in both submucous plexuses (from 30.69±1.22 to 24.84±1.20% in the OSP and from 53.03±1.67 to 46.68±1.18% in the ISP). The higher dose of glyphosate evoke marked changes in the immunoreactivity of all neuroactive substance studied and type of enteric plexus analyzed. An increased number of PACAP-LI (from 13.75±to 40.20 to 40.20±1.62% in the MP, to 21.12 ±1.40% in the OSP and from 11.22±1.26 to 34.94±1.61% in the ISP), nNOS-LI (to 49.92±1.73% in the MP, from 19.53±1.29 to 27.69±1.29% in the OSP and to 27.92±1.26% in the ISP), CART-LI (to 35.03±1.67% in the MP, from 10.60±1.38 to 22.23±1.27% in the OSP and to14.78±1.53% in the ISP) neurons as well as a decrease in the population of VAChT-LI neurons (from 44.58±1.82 to 29.03±1.74% in the MP, to 22.24±1.96% in the OSP and to 35.87±1.42% in the ISP) were noted.Observed changes may result from neurotoxic effect of glyphosate on the ENS neurons and be part of adaptive processes of these neurons to ileum dysfunction. the National Science Centre in Poland [grant no. 2020/04/X/NZ7/00338]. Project financially supported by the Minister of Education and Science under the program entitled “Regional Initiative of Excellence” for the years 2019-2023, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bone adaptation and osteoporosis prevention in hibernating mammals

Hibernating bears and rodents have evolved mechanisms to prevent disuse osteoporosis during the prolonged physical inactivity that occurs during hibernation. Serum markers and histological indices of bone remodeling in bears indicate reduced bone turnover during hibernation, which is consistent with organismal energy conservation. Calcium homeostasis is maintained by balanced bone resorption and formation since hibernating bears do not eat, drink, urinate, or defecate. Reduced and balanced bone remodeling protect bear bone structure and strength during hibernation, unlike the disuse osteoporosis that occurs in humans and other animals during prolonged physical inactivity. Conversely, some hibernating rodents show varying degrees of bone loss such as osteocytic osteolysis, trabecular loss, and cortical thinning. However, no negative effects of hibernation on bone strength in rodents have been found. More than 5000 genes in bear bone tissue are differentially expressed during hibernation, highlighting the complexity of hibernation induced changes in bone. A complete picture of the mechanisms that regulate bone metabolism in hibernators still alludes us, but existing data suggest a role for endocrine and paracrine factors such as cocaine- and amphetamine-regulated transcript (CART) and endocannabinoid ligands like 2-arachidonoyl glycerol (2-AG) in decreasing bone remodeling during hibernation. Hibernating bears and rodents evolved the capacity to preserve bone strength during long periods of physical inactivity, which contributes to their survival and propagation by allowing physically activity (foraging, escaping predators, and mating) without risk of bone fracture following hibernation. Understanding the biological mechanisms regulating bone metabolism in hibernators may inform novel treatment strategies for osteoporosis in humans.

Spexin2 Is a Novel Food Regulator in Gallus gallus

Spexin2 (SPX2), a paralog of SPX1, is a newly identified gene in non-mammalian vertebrates. Limited studies in fish have evidenced its important role in food intake and energy balance modulation. However, little is known about its biological functions in birds. Using the chicken (c-) as a model, we cloned the full-length cDNA of SPX2 by using RACE-PCR. It is 1189 base pair (bp) in length and predicted to generate a protein of 75 amino acids that contains a 14 amino acids mature peptide. Tissue distribution analysis showed that cSPX2 transcripts were detected in a wide array of tissues, with abundant expression in the pituitary, testis, and adrenal gland. cSPX2 was also observed to be ubiquitously expressed in chicken brain regions, with the highest expression in the hypothalamus. Its expression was significantly upregulated in the hypothalamus after 24 or 36 h of food deprivation, and the feeding behavior of chicks was obviously suppressed after peripheral injection with cSPX2. Mechanistically, further studies evidenced that cSPX2 acts as a satiety factor via upregulating cocaine and amphetamine regulated transcript (CART) and downregulating agouti-related neuropeptide (AGRP) in hypothalamus. Using a pGL4-SRE-luciferase reporter system, cSPX2 was demonstrated to effectively activate a chicken galanin II type receptor (cGALR2), a cGALR2-like receptor (cGALR2L), and a galanin III type receptor (cGALR3), with the highest binding affinity for cGALR2L. Collectively, we firstly identified that cSPX2 serves as a novel appetite monitor in chicken. Our findings will help clarify the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.

Functionally active TRPA1 ion channel is downregulated in peptidergic neurons of the Edinger-Westphal nucleus upon acute alcohol exposure.

Introduction: The centrally projecting Edinger-Westphal nucleus (EWcp) contributes to the control of alcohol consumption by its urocortin 1 (UCN1) and cocaine- and amphetamine-regulated transcript (CART) co-expressing peptidergic neurons. Our group recently showed that the urocortinergic centrally projecting EWcp is the primary seat of central nervous system transient receptor potential ankyrin 1 (TRPA1) cation channel mRNA expression. Here, we hypothesized that alcohol and its metabolites, that pass through the blood-brain barrier, may influence the function of urocortinergic cells in centrally projecting EWcp by activating TRPA1 ion channels. We aimed to examine the functional activity of TRPA1 in centrally projecting EWcp and its possible role in a mouse model of acute alcohol exposure. Methods: Electrophysiological measurements were performed on acute brain slices of C57BL/6J male mice containing the centrally projecting EWcp to prove the functional activity of TRPA1 using a selective, potent, covalent agonist JT010. Male TRPA1 knockout (KO) and wildtype (WT) mice were compared with each other in the morphological studies upon acute alcohol treatment. In both genotypes, half of the animals was treated intraperitoneally with 1g/kg 6% ethanol vs. physiological saline-injected controls. Transcardial perfusion was performed 2h after the treatment. In the centrally projecting EWcp area, FOS immunohistochemistry was performed to assess neuronal activation. TRPA1, CART, and urocortin 1 mRNA expression as well as urocortin 1 and CART peptide content was semi-quantified by RNAscope in situ hybridization combined with immunofluorescence. Results: JT010 activated TRPA1 channels of the urocortinergic cells in acute brain slices. Alcohol treatment resulted in a significant FOS activation in both genotypes. Alcohol decreased the Trpa1 mRNA expression in WT mice. The assessment of urocortin 1 peptide immunoreactivity revealed lower basal urocortin 1 in KO mice compared to WTs. The urocortin 1 peptide content was affected genotype-dependently by alcohol: the peptide content decreased in WTs while it increased in KO mice. Alcohol exposure influenced neither CART and urocortin 1 mRNA expression nor the centrally projecting EWcp/CART peptide content. Conclusion: We proved the presence of functional TRPA1 receptors on urocortin 1 neurons of the centrally projecting EWcp. Decreased Trpa1 mRNA expression upon acute alcohol treatment, associated with reduced neuronal urocortin 1 peptide content suggesting that this cation channel may contribute to the regulation of the urocortin 1 release.

Convergent Energy State-Dependent Antagonistic Signaling by Cocaine- and Amphetamine-Regulated Transcript (CART) and Neuropeptide Y (NPY) Modulates the Plasticity of Forebrain Neurons to Regulate Feeding in Zebrafish.

Dynamic reconfiguration of circuit function subserves the flexibility of innate behaviors tuned to physiological states. Internal energy stores adaptively regulate feeding-associated behaviors and integrate opposing hunger and satiety signals at the level of neural circuits. Across vertebrate lineages, the neuropeptides cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) have potent anorexic and orexic functions, respectively, and show energy-state-dependent expression in interoceptive neurons. However, how the antagonistic activities of these peptides modulate circuit plasticity remains unclear. Using behavioral, neuroanatomical, and activity analysis in adult zebrafish of both sexes, along with pharmacological interventions, we show that CART and NPY activities converge on a population of neurons in the dorsomedial telencephalon (Dm). Although CART facilitates glutamatergic neurotransmission at the Dm, NPY dampens the response to glutamate. In energy-rich states, CART enhances NMDA receptor (NMDAR) function by protein kinase A/protein kinase C (PKA/PKC)-mediated phosphorylation of the NR1 subunit of the NMDAR complex. Conversely, starvation triggers NPY-mediated reduction in phosphorylated NR1 via calcineurin activation and inhibition of cAMP production leading to reduced responsiveness to glutamate. Our data identify convergent integration of CART and NPY inputs by the Dm neurons to generate nutritional state-dependent circuit plasticity that is correlated with the behavioral switch induced by the opposing actions of satiety and hunger signals.SIGNIFICANCE STATEMENT Internal energy needs reconfigure neuronal circuits to adaptively regulate feeding behavior. Energy-state-dependent neuropeptide release can signal energy status to feeding-associated circuits and modulate circuit function. CART and NPY are major anorexic and orexic factors, respectively, but the intracellular signaling pathways used by these peptides to alter circuit function remain uncharacterized. We show that CART and NPY-expressing neurons from energy-state interoceptive areas project to a novel telencephalic region, Dm, in adult zebrafish. CART increases the excitability of Dm neurons, whereas NPY opposes CART activity. Antagonistic signaling by CART and NPY converge onto NMDA-receptor function to modulate glutamatergic neurotransmission. Thus, opposing activities of anorexic CART and orexic NPY reconfigure circuit function to generate flexibility in feeding behavior.