Arcuate Nucleus Of Mice Research Articles

Voluntary exercise suppresses inflammation and improves insulin resistance in the arcuate nucleus and ventral tegmental area in mice on a high-fat diet

A high-fat diet (HFD) causes inflammation with an increase in microglial activity in the hypothalamic arcuate nucleus (ARC) and ventral tegmental area (VTA), resulting in insulin resistance in both regions. This leads to a deterioration in glucose and energy metabolism. The effect of voluntary exercise on HFD-induced inflammation in the central nervous system (CNS) remains unclear. To clarify the effects of voluntary exercise on the CNS, 8-week-old male C57BL6 mice were fed a chow diet (CHD) or HFD for 4 weeks; each group was further divided into running exercise (EX+) on a wheel and no exercise (EX-) groups. The expression of the inflammatory cytokine, tumor necrosis factor alpha (TNFα), in the ARC and VTA was significantly increased in the HFD/EX- group, with an increase of microglial activity noted, compared to the CHD/EX- group. The expression of TNFα was significantly suppressed, with a decrease of microglial activity, in the HFD/EX+ compared to HFD/EX- group. Insulin resistance in the ARC and VTA was improved with the suppression of TNFα expression. The HFD/EX- group showed significant weight gain and impaired glucose metabolism compared to the CHD/EX- group. The HFD/EX+ group showed an improvement in glucose and energy metabolism compared to the HFD/EX- group. In addition, voluntary wheel running suppressed HFD-induced inflammation in the ARC, with a decrease in microglial activity observed independently of weight changes. Our data suggest that voluntary exercise prevents obesity and improves glucose metabolism by suppressing inflammation in the ARC and VTA under HFD conditions.

Reduced voluntary wheel running behaviour in Kiss1r knockout mice

Kisspeptin and its receptor, Kiss1r, are novel players in the central balance of energy intake and expenditure. Recent evidence also indicates that kisspeptin signalling is important in thermoregulation and generation of the circadian rhythm. We used global Kiss1r knockout mice (Kiss1r KO), which are hypogonadal and develop obesity, to determine the impact of kisspeptin on circadian related behaviour. Voluntary wheel running was examined in Kiss1r KO and wild-type (WT) mice, using gonad intact and gonadectomised (GDX) mice to account for the effects of kisspeptin on gonadal sex steroids. Intact male and female Kiss1r KO mice covered only 10% and 30% of the distance travelled each day by their respective WT controls. In all mice, most of the running activity occurred during the dark phase. GDX WT mice ran significantly less during dark periods than the intact WT. GDX Kiss1r KO male mice ran significantly less than the GDX WT male mice, but the decrease was attenuated compared to intact mice. There was no difference between the female GDX Kiss1r KO and GDX WT. In contrast to the obese phenotype that develops in Kiss1r KO mice, body mass at the end of the study was significantly lower in the GDX Kiss1r KO than it was in the GDX WT mice. The difference in wheel running activity was not associated with any histological change in WAT, BAT, or muscle diameter. No difference in immunohistochemistry expression was seen in lateral hypothalamic orexin neurons or dopamine neurons in the ventral tegmental area / substantia nigra. We observed increased Iba1 expression (activation of microglia) in the arcuate nucleus of male Kiss1r KO mice. Overall, the circadian locomotor activity in male Kiss1r KO mice appears dependent on kisspeptin signalling and the obese phenotype does not develop in Kiss1r KO mice when they engage in voluntary activity.

Alx3 deficiency disrupts energy homeostasis, alters body composition, and impairs hypothalamic regulation of food intake

The coordination of food intake, energy storage, and expenditure involves complex interactions between hypothalamic neurons and peripheral tissues including pancreatic islets, adipocytes, muscle, and liver. Previous research shows that deficiency of the transcription factor Alx3 alters pancreatic islet-dependent glucose homeostasis. In this study we carried out a comprehensive assessment of metabolic alterations in Alx3 deficiency. We report that Alx3-deficient mice exhibit decreased food intake without changes in body weight, along with reduced energy expenditure and altered respiratory exchange ratio. Magnetic resonance imaging reveals increased adiposity and decreased muscle mass, which was associated with markers of motor and sympathetic denervation. By contrast, Alx3-deficient mice on a high-fat diet show attenuated weight gain and improved insulin sensitivity, compared to control mice. Gene expression analysis demonstrates altered lipogenic and lipolytic gene profiles. In wild type mice Alx3 is expressed in hypothalamic arcuate nucleus neurons, but not in major peripheral metabolic organs. Functional diffusion-weighted magnetic resonance imaging reveals selective hypothalamic responses to fasting in the arcuate nucleus of Alx3-deficient mice. Additionally, altered expression of proopiomelanocortin and melanocortin-3 receptor mRNA in the hypothalamus suggests impaired regulation of feeding behavior. This study highlights the crucial role for Alx3 in governing food intake, energy homeostasis, and metabolic nutrient partitioning, thereby influencing body mass composition.

Conditional Deletion of β-Catenin in the Mediobasal Hypothalamus Impairs Adaptive Energy Expenditure in Response to High-Fat Diet and Exacerbates Diet-Induced Obesity.

β-Catenin is a bifunctional molecule that is an effector of the wingless-related integration site (Wnt) signaling to control gene expression and contributes to the regulation of cytoskeleton and neurotransmitter vesicle trafficking. In its former role, β-catenin binds transcription factor 7-like 2 (TCF7L2), which shows strong genetic associations with the pathogenesis of obesity and type-2 diabetes. Here, we sought to determine whether β-catenin plays a role in the neuroendocrine regulation of body weight and glucose homeostasis. Bilateral injections of adeno-associated virus type-2 (AAV2)-mCherry-Cre were placed into the arcuate nucleus of adult male and female β-catenin flox mice, to specifically delete β-catenin expression in the mediobasal hypothalamus (MBH-β-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-β-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-β-cat KO mice were significantly heavier than the control mice in both sexes (p < 0.05). This deficit seemed to be due to a failure to show an adaptive increase in energy expenditure seen in controls, which served to offset the increased calories by HFD. Both male and female MBH-β-cat KO mice were highly glucose intolerant when on HFD and displayed a significant reduction in both leptin and insulin sensitivity compared with controls. This study highlights a critical role for β-catenin in the hypothalamic circuits regulating body weight and glucose homeostasis and reveals potential mechanisms by which genetic variation in this pathway could impact on development of metabolic disease.

Postweaning Social Isolation Alters Puberty Onset by Suppressing Electrical Activity of Arcuate Kisspeptin Neurons

Introduction: Postweaning social isolation (PWSI) in rodents is an advanced psychosocial stress model in early life. Some psychosocial stress, such as restrain and isolation, disrupts reproductive physiology in young and adult periods. Mechanisms of early-life stress effects on central regulation of reproduction need to be elucidated. We have investigated the effects of PWSI on function of arcuate kisspeptin (ARCKISS1) neurons by using electrophysiological techniques combining with monitoring of puberty onset and estrous cycle in male and female Kiss1-Cre mice. Methods: Female mice were monitored for puberty onset with vaginal opening examination during social isolation. After isolation, the estrous cycle of female mice was monitored with vaginal cytology. Anxiety-like behavior of mice was determined by an elevated plus maze test. Effects of PWSI on electrophysiology of ARCKISS1 neurons were investigated by the patch clamp method after intracranial injection of AAV-GFP virus into arcuate nucleus of Kiss1-Cre mice after the isolation period. Results: We found that both male and female isolated mice showed anxiety-like behavior. PWSI caused delay in vaginal opening and extension in estrous cycle length. Spontaneous-firing rates of ARCKISS1 neurons were significantly lower in the isolated male and female mice. The peak amplitude of inhibitory postsynaptic currents to ARCKISS1 neurons was higher in the isolated mice, while frequency of excitatory postsynaptic currents was higher in group-housed mice. Conclusion: These findings demonstrate that PWSI alters pre- and postpubertal reproductive physiology through metabolic and electrophysiological pathways.

Musclin prevents depression-like behavior in male mice by activating urocortin 2 signaling in the hypothalamus.

Physical activity is recommended as an alternative treatment for depression. Myokines, which are secreted from skeletal muscles during physical activity, play an important role in the skeletal muscle-brain axis. Musclin, a newly discovered myokine, exerts physical endurance, however, the effects of musclin on emotional behaviors, such as depression, have not been evaluated. This study aimed to access the anti-depressive effect of musclin and clarify the connection between depression-like behavior and hypothalamic neuropeptides in mice. We measured the immobility time in the forced swim (FS) test, the time spent in open arm in the elevated-plus maze (EPM) test, the mRNA levels of hypothalamic neuropeptides, and enumerated the c-Fos-positive cells in the paraventricular nucleus (PVN), arcuate nucleus (ARC), and nucleus tractus solitarii (NTS) in mice with the intraperitoneal (i.p.) administration of musclin. Next, we evaluated the effects of a selective corticotropin-releasing factor (CRF) type 1 receptor antagonist, selective CRF type 2 receptor antagonist, melanocortin receptor (MCR) agonist, and selective melanocortin 4 receptor (MC4R) agonist on changes in behaviors induced by musclin. Finally we evaluated the antidepressant effect of musclin using mice exposed to repeated water immersion (WI) stress. We found that the i.p. and i.c.v. administration of musclin decreased the immobility time and relative time in the open arms (open %) in mice and increased urocortin 2 (Ucn 2) levels but decreased proopiomelanocortin levels in the hypothalamus. The numbers of c-Fos-positive cells were increased in the PVN and NTS but decreased in the ARC of mice with i.p. administration of musclin. The c-Fos-positive cells in the PVN were also found to be Ucn 2-positive. The antidepressant and anxiogenic effects of musclin were blocked by central administration of a CRF type 2 receptor antagonist and a melanocortin 4 receptor agonist, respectively. Peripheral administration of musclin also prevented depression-like behavior and the decrease in levels of hypothalamic Ucn 2 induced by repeated WI stress. These data identify the antidepressant effects of musclin through the activation of central Ucn 2 signaling and suggest that musclin and Ucn 2 can be new therapeutic targets and endogenous peptides mediating the muscle-brain axis.

Nonphotic entrainment of central and peripheral circadian clocks in mice by scheduled voluntary exercise under constant darkness.

In mammals, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) entrains to an environmental light-dark (LD) cycle and organizes the temporal order of circadian rhythms in physiology and behavior. Previously, some studies have demonstrated that scheduled exercise could entrain the free-running behavior rhythm in nocturnal rodents. However, it remains unknown whether entrainment by scheduled exercise alters the internal temporal order of the behavioral circadian rhythms or clock gene expression in the SCN, extra-SCN brain regions, and peripheral organs when mice are entrained to the scheduled exercise under constant darkness (DD). In the present study, we examined circadian rhythms in locomotor activity and clock gene Per1 expression by bioluminescence reporter (Per1-luc) in the SCN, arcuate nucleus (ARC), liver, and skeletal muscle of mice entrained to an LD cycle, mice free-running under DD, and mice entrained to daily exposure to a new cage with a running wheel (NCRW) under DD. All mice showed a steady-state entrainment of behavioral circadian rhythms to NCRW exposure under DD in parallel with shortening of the α when compared with that under DD. The temporal order of behavioral circadian rhythms and the Per1-luc rhythms in the SCN and peripheral tissues but not in the ARC were maintained in the mice entrained to the NCRW and LD cycles; in contrast, the temporal order was altered in the mice under DD. The present findings reveal that the SCN entrains to daily exercise, and daily exercise reorganizes the internal temporal order of behavioral circadian rhythms and clock gene expression in the SCN and peripheral tissues.

Unaltered Tonic Inhibition in the Arcuate Nucleus of Diet-induced Obese Mice.

The principal inhibitory transmitter, γ-aminobutyric acid (GABA), is critical for maintaining hypothalamic homeostasis and released from neurons phasically, as well as from astrocytes tonically. Although astrocytes in the arcuate nucleus (ARC) of the hypothalamus are shown to transform into reactive astrocytes, the tonic inhibition by astrocytic GABA has not been adequately investigated in diet-induced obesity (DIO). Here, we investigated the expression of monoamine oxidase- B (MAOB), a GABA-synthesizing enzyme, in reactive astrocytes in obese mice. We observed that a chronic high-fat diet (HFD) significantly increased astrocytic MAOB and cellular GABA content, along with enhanced hypertrophy of astrocytes in the ARC. Unexpectedly, we found that the level of tonic GABA was unaltered in chronic HFD mice using whole-cell patch-clamp recordings in the ARC. Furthermore, the GABA-induced current was increased with elevated GABAA receptor α5 (GABRA5) expression. Surprisingly, we found that a nonselective GABA transporter (GAT) inhibitor, nipecotic acid (NPA)-induced current was significantly increased in chronic HFD mice. We observed that GAT1 inhibitor, NO711-induced current was significantly increased, whereas GAT3 inhibitor, SNAP5114-induced current was not altered. The unexpected unaltered tonic inhibition was due to an increase of GABA clearance in the ARC by neuronal GAT1 rather than astrocytic GAT3. These results imply that increased astrocytic GABA synthesis and neuronal GABAA receptor were compensated by GABA clearance, resulting in unaltered tonic GABA inhibition in the ARC of the hypothalamus in obese mice. Taken together, GABA-related molecular pathways in the ARC dynamically regulate the tonic inhibition to maintain hypothalamic homeostasis against the HFD challenge.

Transcriptome profiling of kisspeptin neurons from the mouse arcuate nucleus reveals new mechanisms in estrogenic control of fertility

Kisspeptin neurons in the mediobasal hypothalamus (MBH) are critical targets of ovarian estrogen feedback regulating mammalian fertility. To reveal molecular mechanisms underlying this signaling, we thoroughly characterized the estrogen-regulated transcriptome of kisspeptin cells from ovariectomized transgenic mice substituted with 17β-estradiol or vehicle. MBH kisspeptin neurons were harvested using laser-capture microdissection, pooled, and subjected to RNA sequencing. Estrogen treatment significantly (p.adj. < 0.05) up-regulated 1,190 and down-regulated 1,139 transcripts, including transcription factors, neuropeptides, ribosomal and mitochondrial proteins, ion channels, transporters, receptors, and regulatory RNAs. Reduced expression of the excitatory serotonin receptor-4 transcript (Htr4) diminished kisspeptin neuron responsiveness to serotonergic stimulation. Many estrogen-regulated transcripts have been implicated in puberty/fertility disorders. Patients (n = 337) with congenital hypogonadotropic hypogonadism (CHH) showed enrichment of rare variants in putative CHH-candidate genes (e.g., LRP1B, CACNA1G, FNDC3A). Comprehensive characterization of the estrogen-dependent kisspeptin neuron transcriptome sheds light on the molecular mechanisms of ovary-brain communication and informs genetic research on human fertility disorders.

ASB4 modulates central melanocortinergic neurons and calcitonin signaling to control satiety and glucose homeostasis.

Variants in the gene encoding ankyrin repeat and SOCS box-containing 4 (ASB4) are linked to human obesity. Here, we characterized the pathways underlying the metabolic functions of ASB4. Hypothalamic Asb4 expression was suppressed by fasting in wild-type mice but not in mice deficient in AgRP, which encodes Agouti-related protein (AgRP), an appetite-stimulating hormone, suggesting that ASB4 is a negative target of AgRP. Many ASB4 neurons in the brain were adjacent to AgRP terminals, and feeding induced by AgRP neuronal activation was disrupted in Asb4-deficient mice. Acute knockdown of Asb4 in the brain caused marked hyperphagia due to increased meal size, and Asb4 deficiency led to increased meal size and food intake at the onset of refeeding, when very large meals were consumed. Asb4-deficient mice were resistant to the meal-terminating effects of exogenously administered calcitonin and showed decreased neuronal expression of Calcr, which encodes the calcitonin receptor. Pro-opiomelanocortin (POMC) neurons in the arcuate nucleus in mice are involved in glucose homeostasis, and Asb4 deficiency specifically in POMC neurons resulted in glucose intolerance that was independent of obesity. Furthermore, individuals with type 2 diabetes showed reduced ASB4 abundance in the infundibular nuclei, the human equivalent of the arcuate nucleus. Together, our results indicate that ASB4 acts in the brain to improve glucose homeostasis and to induce satiety after substantial meals, particularly those after food deprivation.

Astaxanthin Exerts Anabolic Effects via Pleiotropic Modulation of the Excitable Tissue.

Astaxanthin is a lipid-soluble carotenoid influencing lipid metabolism, body weight, and insulin sensitivity. We provide a systematic analysis of acute and chronic effects of astaxanthin on different organs. Changes by chronic astaxanthin feeding were analyzed on general metabolism, expression of regulatory proteins in the skeletal muscle, as well as changes of excitation and synaptic activity in the hypothalamic arcuate nucleus of mice. Acute responses were also tested on canine cardiac muscle and different neuronal populations of the hypothalamic arcuate nucleus in mice. Dietary astaxanthin significantly increased food intake. It also increased protein levels affecting glucose metabolism and fatty acid biosynthesis in skeletal muscle. Inhibitory inputs innervating neurons of the arcuate nucleus regulating metabolism and food intake were strengthened by both acute and chronic astaxanthin treatment. Astaxanthin moderately shortened cardiac action potentials, depressed their plateau potential, and reduced the maximal rate of depolarization. Based on its complex actions on metabolism and food intake, our data support the previous findings that astaxanthin is suitable for supplementing the diet of patients with disturbances in energy homeostasis.

Prenatal androgenization causes expression changes of progesterone and androgen receptor mRNAs in the arcuate nucleus of female mice across development.

Prenatal exposure to excess androgens is associated with the development of polycystic ovary syndrome (PCOS). In prenatally androgenised (PNA) mice, a model of PCOS, progesterone receptor (PR) protein expression is reduced in arcuate nucleus (ARC) GABA neurons. This suggests a mechanism for PCOS-related impaired steroid hormone feedback and implicates androgen excess with respect to inducing transcriptional repression of the PR-encoding gene Pgr in the ARC. However, the androgen sensitivity of ARC neurons and the relative gene expression of PRs over development and following prenatal androgen exposure remain unknown. Here, we used a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) of microdissected ARC to determine the relative androgen receptor (Ar) and progesterone receptor (Pgr) gene expression in PNA and control mice at five developmental timepoints. In a two-way analysis of variance, none of the genes examined showed expression changes with a statistically significant interaction between treatment and age, although PgrA showed a borderline interaction. For all genes, there was a statistically significant main effect of age on expression levels, reflecting a general increase in expression with increasing age, regardless of treatment. For PgrB and Ar, there was a statistically significant main effect of treatment, indicating a change in expression following PNA (increased for PgrB and decreased for Ar), regardless of age. For PgrA, there was a borderline main effect of treatment, suggesting a possible change in expression following PNA, regardless of age. PgrAB gene expression changes showed no significant main effect of treatment. We additionally examined androgen and progesterone responsiveness specifically in P60 ARC GABA neurons using RNAScope® (Advanced Cell Diagnostics, Inc.) in situ hybridization. This analysis revealed that Pgr and Ar were expressed in the majority of ARC GABA neurons in normal adult females. However, our RNAScope® analysis did not show significant changes in Pgr or Ar expression within ARC GABA neurons following PNA. Lastly, because GABA drive to gonadotropin-releasing hormone neurons is increased in PNA, we hypothesised that PNA mice would show increased expression of glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA production. However, the RT-qPCR showed that the expression of GAD encoding genes (Gad1 and Gad2) was unchanged in adult PNA mice compared to controls. Our findings indicate that PNA treatment can impact Pgr and Ar mRNA expression in adulthood. This may reflect altered circulating steroid hormones in PNA mice or PNA-induced epigenetic changes in the regulation of Pgr and Ar gene expression in ARC neurons.

Expression of glucagon-like peptide 1 receptor in neuropeptide Y neurons of the arcuate nucleus in mice.

Glucagon-like peptide 1 (GLP-1) and its agonists exert anorexigenic effect at least partly via acting on GLP-1 receptors (GLP-1R) in the arcuate nucleus (ARC). While the anorexigenic, proopiomelanocortin (POMC) neurons of the ARC were shown previously to express GLP-1R, the putative GLP-1R-content of the orexigenic, neuropeptide Y (NPY) neurons remained so far undetected. As GLP-1R is abundant in the ventromedial ARC, where NPY neurons are located; here, we address the possibility that GLP-1 can act directly on the orexigenic NPY system via GLP-1R. Double-labeling immunocytochemistry and in situ hybridization were performed on tissues of adult male mice to detect GLP-1R in NPY neurons. In double-immunolabeled preparations, GLP-1R-immunoreactivity was observed in NPY neurons and in axons ensheathing the majority of NPY neurons. Ultrastructural studies confirmed that GLP-1R-immunoreactivity is associated with the outer membrane of NPY perikarya as well as with axons forming symmetric type, inhibitory synapses on NPY-containing neurons. Double-labeling in situ hybridization experiments demonstrated the expression of GLP-1R mRNA in approximately 20% of NPY mRNA-containing neurons of the ARC. In summary, our data demonstrate the presence of GLP-1R protein and mRNA in NPY neurons of ARC and also reveal the innervation of NPY neurons by GLP-1R-containing inhibitory neurons. These observations suggest that GLP-1 signaling can influence NPY neurons both directly and indirectly. Furthermore, GLP-1 signaling on energy homeostasis appears to involve both direct and indirect effects of GLP-1 on the orexigenic NPY neurons, in addition to the previously known effects via the anorexigenic POMC neuronal system.

Sex-specific pubertal and metabolic regulation of Kiss1 neurons via Nhlh2.

Hypothalamic Kiss1 neurons control gonadotropin-releasing hormone release through the secretion of kisspeptin. Kiss1 neurons serve as a nodal center that conveys essential regulatory cues for the attainment and maintenance of reproductive function. Despite this critical role, the mechanisms that control kisspeptin synthesis and release remain largely unknown. Using Drop-Seq data from the arcuate nucleus of adult mice and in situ hybridization, we identified Nescient Helix-Loop-Helix 2 (Nhlh2), a transcription factor of the basic helix-loop-helix family, to be enriched in Kiss1 neurons. JASPAR analysis revealed several binding sites for NHLH2 in the Kiss1 and Tac2 (neurokinin B) 5' regulatory regions. In vitro luciferase assays evidenced a robust stimulatory action of NHLH2 on human KISS1 and TAC3 promoters. The recruitment of NHLH2 to the KISS1 and TAC3 promoters was further confirmed through chromatin immunoprecipitation. In vivo conditional ablation of Nhlh2 from Kiss1 neurons using Kiss1Cre:Nhlh2fl/fl mice induced a male-specific delay in puberty onset, in line with a decrease in arcuate Kiss1 expression. Females retained normal reproductive function albeit with irregular estrous cycles. Further analysis of male Kiss1Cre:Nhlh2fl/fl mice revealed higher susceptibility to metabolic challenges in the release of luteinizing hormone and impaired response to leptin. Overall, in Kiss1 neurons, Nhlh2 contributes to the metabolic regulation of kisspeptin and NKB synthesis and release, with implications for the timing of puberty onset and regulation of fertility in male mice.

Sirtuin 6 supra-physiological overexpression in hypothalamic pro-opiomelanocortin neurons promotes obesity via the hypothalamus-adipose axis.

Sirtuin 6 (Sirt6), a member of the Sirtuin family, has important roles in maintaining glucose and lipid metabolism. Our previous studies demonstrated that the deletion of Sirt6 in pro-opiomelanocortin (POMC)-expressing cells by the loxP-Cre system resulted in severe obesity and hepatic steatosis. However, whether overexpression of Sirt6 in hypothalamic POMC neurons could ameliorate diet-induced obesity is still unknown. Thus, we generated mice specifically overexpressing Sirt6 in hypothalamic POMC neurons (PSOE) by stereotaxic injection of Cre-dependent adeno-associated viruses into the arcuate nucleus of Pomc-Cre mice. PSOE mice showed increased adiposity and decreased energy expenditure. Furthermore, thermogenesis of BAT and lipolysis of WAT were both impaired, caused by reduced sympathetic nerve innervation and activity in adipose tissues. Mechanistically, Sirt6 overexpression decreasing STAT3 acetylation, thus lowering POMC expression in the hypothalamus underlined the observed phenotypes in PSOE mice. These results demonstrate that Sirt6 overexpression specifically in the hypothalamic POMC neurons exacerbates diet-induced obesity and metabolic disorders via the hypothalamus-adipose axis.

Antagonism for NPY signaling reverses cognitive behavior defects induced by activity-based anorexia in mice

Patients with AN often express psychological symptoms such as body image distortion, cognitive biases, abnormal facial recognition, and deficits in working memory. However, the molecular mechanisms underlying the impairment of cognitive behaviors in AN remain unknown. In the present study, we measured cognitive behavior using novel object recognition (NOR) tasks and mRNA expressions in hypothalamic neuropeptides in female C57BL/6J mice with activity-based anorexia (ABA). Additionally, we evaluated the effects of antagonists with intracerebroventricular (icv) administration on the impairment of cognitive behavior in NOR tasks. Our results showed that NOR indices were lowered, subsequently increasing mRNA levels of agouti-related peptide (AgRP) and neuropeptide Y (NPY), and c-Fos- and AgRP- or NPY-positive cells in the hypothalamic arcuate nucleus in ABA mice. We also observed that icv administration of anti-NPY antiserum (2 µl), anti-AgRP antibody (0.1 μg), and Y5 receptor antagonist CPG71683 (15 nmol) significantly reversed the decreased NOR indices. Therefore, our results suggest that increased NPY and AgRP signaling in the brain might contribute to the impairment of cognitive behavior in AN.

Characterization of Genome-Wide DNA Methylation and Hydroxymethylation in Mouse Arcuate Nucleus of Hypothalamus During Puberty Process.

Background: Pulsatile pituitary gonadotropin secretion governed by hypothalamic gonadotropin-releasing hormone (GnRH) is essential for the pubertal onset. The epigenetic mechanism underlying the activation of GnRH-dependent regulatory axis in hypothalamus remains elusive. This study aims to explore the potential correlation between the signature of DNA (hydroxyl)methylation and pubertal process.Methods: Hypothalamic arcuate nucleus (ARC) of mouse at early (4-weeks) and late pubertal (8-weeks) stages underwent RNA-, RRBS-, and RRHP-seq to investigate the genome-wide profiles of transcriptome, differential DNA methylation and hydroxymethylation.Results: A series of differential expressed genes (DEGs) involved in sexual development could be separated into three subgroups with the significant difference of DNA methylation or hydroxymethylation or both in promoter regions. Compared to DNA methylation, DNA hydroxymethylation partook in more signaling pathways including synapse morphology, channel activity and glial development, which could enhance transsynaptic change and glia-to-neuron communication to faciliate GnRH release. The correlation between transcription and these epigenetic modifications indicated that DNA hydroxymethylation impacted with gene transcription independently of DNA methylation spanning puberty.Conclusion: Our results characterized the hydroxymethylation pattern and provided an insight into the novel epigenetic regulation on gene expression during pubertal process.

ERα Signaling in GHRH/Kiss1 Dual-Phenotype Neurons Plays Sex-Specific Roles in Growth and Puberty.

Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRHΔERα), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRHΔERα females. Lack of AR in GHRH cells (GHRHΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRHtdTom neurons minimally colocalize with Kiss1hrGFP in prepubertal mice, ∼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition.SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition.

Postnatal Overnutrition Induces Changes in Synaptic Transmission to Leptin Receptor-Expressing Neurons in the Arcuate Nucleus of Female Mice.

The adipocyte-derived hormone leptin is a potent neurotrophic factor that contributes to the neural plasticity and development of feeding circuitry, particularly in the arcuate nucleus of the hypothalamus (ARH). Postnatal overnutrition affects leptin secretion and sensitivity, but whether postnatal overnutrition produces changes in the development of the synaptic transmission to ARH neurons is currently unknown. We evaluated the excitatory and inhibitory currents to ARH leptin receptor (LepR)-expressing neurons in prepubertal, pubertal and adult female mice. The effects of postnatal overnutrition in the expression of genes that code ion channels subunits in the ARH were also evaluated. We observed that the transition from prepubertal to pubertal stage is characterized by a rise in both excitatory and inhibitory transmission to ARH LepR-expressing neurons in control mice. Postnatal overnutrition induces a further increase in the excitatory synaptic transmission in pubertal and adult animals, whereas the amplitude of inhibitory currents to ARH LepR-expressing cells was reduced. Postnatal overnutrition also contributes to the modulation of gene expression of N-methyl-D-aspartate, GABAB and ATP-sensitive potassium channel subunits in ARH. In summary, the synaptic transmission to ARH cells is profoundly influenced by postnatal overnutrition. Thus, increased adiposity during early postnatal period induces long-lasting effects on ARH cellular excitability.

Mitochondrial dysfunction in GnRH neurons impaired GnRH production

The onset establishment and maintenance of gonadotropin-releasing hormone (GnRH) secretion is an important phenomenon regulating pubertal development and reproduction. GnRH neurons as well as other neurons in the hypothalamus have high-energy demands and require a constant energy supply from their mitochondria machinery to maintain active functioning. However, the involvement of mitochondrial function in GnRH neurons is still unclear. In this study, we examined the role of NADH Dehydrogenase (Ubiquinone) Fe–S protein 4 (Ndufs4), a member of the mitochondrial complex 1, on GnRH neurons using Ndufs4-KO mice and Ndufs4-KO GT1-7 cells. Ndufs4 was highly expressed in GnRH neurons in the medial preoptic area (MPOA) and NPY/AgRP and POMC neurons in the arcuate (ARC) nucleus in WT mice. Conversely, there was a significant decrease in GnRH expression in MPOA and median eminence of Ndufs4-KO mice, followed by impaired peripheral endocrine system. In Ndufs4-KO GT1-7 cells, Gnrh1 expression was significantly decreased with or without stimulation with either kisspeptin or NGF, whereas, stimulation significantly increased Gnrh1 expression in control cells. In contrast, there was no difference in cell signaling activity including ERK and CREB as well as the expression of GPR54, TrkA and p75NTR, suggesting that Ndufs4 is involved in the transcriptional regulation system for GnRH production. These findings may be useful in understanding the mitochondrial function in GnRH neuron.