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RGS (regulator of G protein signaling) family members catalyze the termination of G protein signaling cascades. Single nucleotide polymorphisms in theTo study cell-specific functions of RGS2, a novel gene-targeted mouse harboring a conditional allele for theWhereasThese results demonstrate the development of a novel mouse with conditional expression of

Low-renin hypertension is common among the elderly, subjects of black African ancestry, those with heart or renal failure, and women with preeclampsia. Increased understanding of the causes and consequences of low-renin hypertension is necessary for the development of new efficacious and personalized therapeutics. One commonly employed preclinical model is “DOCA-salt” (DOCA), which causes robust hypertension, proteinuria, and organ damage. The arcuate nucleus of the hypothalamus (ARC) has been implicated in control of metabolic rate and blood pressure responses to DOCA. To characterize the DOCA related changes within the ARC, we used single nucleus RNA sequencing (snRNA-Seq) to individually interrogate the transcriptomes of numerous mouse ARC cell types. Male C57BL/6J mice 7 weeks-of-age underwent sham surgery or subcutaneous implantation of a 50mg DOCA pellet. All mice had water; DOCA mice were also provided 0.15 M NaCl drink ad libitum. Body composition was assessed by nuclear magnetic resonance. After 3 weeks, tissues were weighed and blood chemistries assessed. Features of DOCA treatment were consistent with previous studies, such as suppressed growth trajectory (sham n=7, DOCA n=8; -0.8±0.4g/3wk; p

Most of the animal studies using inflammation-induced cognitive change have relied on behavioral testing without objective and biologically solid methods to quantify the severity of cognitive disturbances. We have developed a bispectral EEG (BSEEG) method using a novel algorithm in clinical study. This method effectively differentiates between patients with and without delirium, and predict long-term mortality. In the present study, we aimed to apply our bispectral EEG (BSEEG) method, which can detect patients with delirium, to a mouse model of delirium with systemic inflammation induced by lipopolysaccharides (LPS) injection. We recorded EEG after LPS injection using wildtype early adulthood mice (2~3-month-old) and aged mice (18-19-month-old). Animal EEG recordings were converted for power spectral density to calculate BSEEG score using the similar BSEEG algorithm previously developed for our human study. The BSEEG score was relatively stable and slightly high during the day. Alternatively, the BSEEG score was erratic and low in average during the night. LPS injection increased the BSEEG score dose-dependently and diminished the diurnal changes. The mean BSEEG score increased much more in the aged mice group as dosage increased. Our results suggest that BSEEG method can objectively "quantify" level of neuro-Inflammation induced by systemic inflammation (LPS), and that this BSEEG method can be useful as a model of delirium in mice.

The brain renin-angiotensin system (RAS) is implicated in control of blood pressure (BP), fluid intake, and energy expenditure (EE). Angiotensin II (ANG II) within the arcuate nucleus of the hypothalamus contributes to control of resting metabolic rate (RMR) and thereby EE through its actions on Agouti-related peptide (AgRP) neurons, which also contribute to EE control by leptin. First, we determined that although leptin stimulates EE in control littermates, mice with transgenic activation of the brain RAS (sRA) exhibit increased EE and leptin has no additive effect to exaggerate EE in these mice. These findings led us to hypothesize that leptin and ANG II in the brain stimulate EE through a shared mechanism. Because AgRP signaling to the melanocortin MC4R receptor contributes to the metabolic effects of leptin, we performed a series of studies examining RMR, fluid intake, and BP responses to ANG II in mice rendered deficient for expression of MC4R via a transcriptional block ( Mc4r-TB). These mice were resistant to stimulation of RMR in response to activation of the endogenous brain RAS via chronic deoxycorticosterone acetate (DOCA)-salt treatment, whereas fluid and electrolyte effects remained intact. These mice were also resistant to stimulation of RMR via acute intracerebroventricular (ICV) injection of ANG II, whereas BP responses to ICV ANG II remained intact. Collectively, these data demonstrate that the effects of ANG II within the brain to control RMR and EE are dependent on MC4R signaling, whereas fluid homeostasis and BP responses are independent of MC4R signaling.

Estrogen receptor alpha (ERα)-mediated estrogen signaling play a pivotal role in both reproductive and non-reproductive functions. Transcriptional regulation of ERα gene is highly complex, with multiple transcript variants being differentially produced across the tissues. However, tissue-specific variation and physiological specificity of the ERα variants are not yet fully understood. In an attempt to generate a Cre-dependently restorable ERα-null mice for functional investigation of the genetic sufficiency, we unexpectedly produced ERα hypomorphic mice with biased downregulation of a previously unappreciated long ERα isoform that is enriched in the female reproductive organs (uterus and ovaries) and the pituitary but minimally expressed in the brain. Female homozygous mutant mice were capable of pregnancy but displayed irregular estrus cycle and rarely maintained alive newborns without significant morphological and pathological changes in reproductive system and the disruption of body weight homeostasis, indicating the vital role of this long isoform in female reproductive function. Collectively, our results define a tissue-specifically enriched long ERα isoform and its preferential role in female reproductive function over body weight homeostasis.

Prolonged obesity is associated with blunted feeding and thermogenic autonomic responses to leptin, but cardiovascular responses to leptin are maintained. This state of selective leptin resistance is, therefore, proposed to contribute to the pathogenesis and maintenance of obesity-associated hypertension. Cells of the arcuate nucleus of the hypothalamus detect leptin, and although the cellular and molecular mechanisms remain unclear, altered arcuate nucleus biology is hypothesized to contribute to selective leptin resistance. Male C57BL/6J mice were fed a high-fat diet (HFD) or chow from 8 to 18 weeks of age, as this paradigm models selective leptin resistance. Nuclei were then isolated from arcuate nucleus for single-nucleus RNA sequencing. HFD caused expected gains in adiposity and circulating leptin. Twenty-three unique cell-type clusters were identified, and Ingenuity Pathway Analysis was used to explore changes in gene expression patterns due to chronic HFD within each cluster. Notably, gene expression signatures related to leptin signaling exhibited suppression predominantly in neurons identified as the Agouti-related peptide ( Agrp ) subtype. Ingenuity Pathway Analysis results were also consistent with alterations in CREB (cAMP response element-binding protein) signaling in Agrp neurons after HFD, and reduced phosphorylated CREB was confirmed in arcuate nucleus after prolonged HFD by capillary electrophoresis-based Western blotting. These findings support the concept that prolonged HFD-induced obesity is associated with selective changes in Agrp neuron biology, possibly secondary to altered CREB signaling.

Objective The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input–output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. Methods Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. Results We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. Conclusions Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation., Highlights • PVNMC4R+ neurons innervate brain regions involved in feeding and autonomic regulation. • Activation of PVNMC4R+ neurons increases BAT thermogenesis. • Activation of PVNMC4R+ neurons increases blood pressure and heart rate. • Activation of PVNMC4R+ neurons lead to transient hyperactivity followed by hypoactivity and resting-like behavior.

ObjectiveThe paraventricular nucleus of hypothalamus (PVN) is an integrative center in the brain orchestrating a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is undoubtedly involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ pathway and its role in diverse physiological and behavioral regulations have not been fully understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and further test their potential functional roles beyond feeding.MethodsUsing a combination of viral tools, we performed a comprehensive mapping of PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermogenesis, cardiovascular control and other behavioral regulations beyond feeding.ResultsWe found that PVNMC4R+ neurons broadly innervate many different brain regions known to be important not only for feeding but also for neuroendocrine and autonomic control of thermogenesis and cardiovascular function, including but not limited to preoptic area, median eminence, parabrachial nucleus, locus coeruleus, nucleus of solitary tract, ventrolateral medulla and thoracic spinal cord. Contrary to broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs from limited brain regions, including medial preoptic nucleus, arcuate and dorsomedial hypothalamic nuclei, and supraoptic nucleus. Consistent with broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure and brown adipose tissue thermogenesis. Strikingly, these physiological changes accompanied an unexpected repetitive bedding-removing behavior followed by hypoactivity and resting-like behavior.ConclusionsOur results clarify the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermogenesis, cardiovascular function and other behavioral regulations.

The arcuate nucleus of the hypothalamus (ARC) plays a key role in linking peripheral metabolic status to the brain melanocortin system, which influences a wide range of physiological processes including the sympathetic nervous system and blood pressure. The importance of the activity of agouti-related peptide (AgRP)- and proopiomelanocortin (POMC)-expressing neurons, two molecularly distinct populations of ARC neurons, for metabolic regulation is well established, but their relevance for sympathetic and cardiovascular control remains unclear. We used designer receptors exclusively activated by designer drug (DREADD) technology to study how activation of AgRP and POMC neurons affect renal sympathetic nerve traffic and blood pressure. In addition to the drastic feeding-stimulatory effect, DREADD-mediated activation of AgRP, but not POMC neurons, induced an acute reduction in renal sympathetic nerve activity in conscious mice. Paradoxically, however, DREADD-mediated chronic activation of AgRP neurons caused a significant increase in blood pressure specifically in the inactive light phase. On the other hand, chronic activation of POMC neurons led to a significant reduction in blood pressure. These results bring new insights to a previously unappreciated role of ARC AgRP and POMC neuronal activity in autonomic and cardiovascular regulation. NEW & NOTEWORTHY Agouti-related peptide (AgRP)- and proopiomelanocortin (POMC)-expressing neurons of the arcuate nucleus are essential components of the brain melanocortin system that controls various physiological processes. Here, we tested the metabolic and cardiovascular effects of direct activation of these two populations of neurons. Our findings show that, in addition to stimulation of food intake, chemogenetic mediated activation of hypothalamic arcuate nucleus AgRP, but not POMC, neurons reduce renal sympathetic traffic. Despite this, chronic activation of AgRP neurons increased blood pressure. However, chronic activation of POMC neurons led to a significant reduction in blood pressure. Our findings highlight the importance of arcuate nucleus AgRP and POMC neuronal activity in autonomic and cardiovascular regulation.

Glucose is the essential energy source for the brain, whose deficit, triggered by energy deprivation or therapeutic agents, can be fatal. Increased appetite is the key behavioral defense against hypoglycemia; however, the central pathways involved are not well understood. Here, we describe a glucoprivic feeding pathway by tyrosine hydroxylase (TH)-expressing neurons from nucleus of solitary tract (NTS), which project densely to the hypothalamus and elicit feeding through bidirectional adrenergic modulation of agouti-related peptide (AgRP)- and proopiomelanocortin (POMC)-expressing neurons. Acute chemogenetic inhibition of arcuate nucleus (ARC)-projecting NTSTH neurons or their target, AgRP neurons, impaired glucoprivic feeding induced by 2-Deoxy-D-glucose (2DG) injection. Neuroanatomical tracing results suggested that ARC-projecting orexigenic NTSTH neurons are largely distinct from neighboring catecholamine neurons projecting to parabrachial nucleus (PBN) that promotes satiety. Collectively, we describe a circuit organization in which an ascending pathway from brainstem stimulates appetite through key hunger neurons in the hypothalamus in response to hypoglycemia. EMBO IG FOEDRC

Eating disorders (EDs) are serious mental illnesses thought to arise from the complex gene-environment interactions. DNA methylation patterns in histone deacetylase 4 (HDAC4) locus have been associated with EDs and we have previously identified a missense mutation in the HDAC4 gene (HDAC4 A786T ) that increases the risk of developing an ED. In order to evaluate the biological consequences of this variant and establish a useful mouse model of EDs, here we performed behavioral characterization of mice homozygous for Hdac4 A778T (corresponding to human HDAC4 A786T ) that were further backcrossed onto C57BL/6 background. When fed high-fat diet, male, but not female, homozygous mice showed a trend toward decreased weight gain compared to their wild-type littermates. Behaviorally, male, but not female, homozygous mice spent less time in eating and exhibited reduced motivation to work for palatable food and light phase-specific decrease in locomotor activity. Additionally, homozygous Hdac4 A778T female, but not male, mice display social subordination when subjected to a tube dominance test. Collectively, these results reveal a complex sex- and circadian-dependent role of ED-associated Hdac4 A778T mutation in affecting mouse behaviors. Homozygous Hdac4 A778T mice could therefore be a useful animal model to gain insight into the neurobiological basis of EDs.

Objectives: The autonomic nervous system is critically involved in mediating the control by leptin of many physiological processes. Here, we examined the role of the leptin receptor (LepR) in proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in mediating the effects of leptin on regional sympathetic and parasympathetic nerve activity. Methods: We analyzed how deletion of the LepR in POMC neurons (POMCCre/LepRfl/fl mice) or AgRP neurons (AgRPCre/LepRfl/fl mice) affects the ability of leptin to increase sympathetic and parasympathetic nerve activity. We also studied mice lacking the catalytic p110α or p110β subunits of phosphatidylinositol-3 kinase (PI3K) in POMC neurons. Results: Leptin-evoked increase in sympathetic nerve activity subserving thermogenic brown adipose tissue was partially blunted in mice lacking the LepR in either POMC or AgRP neurons. On the other hand, loss of the LepR in AgRP, but not POMC, neurons interfered with leptin-induced sympathetic nerve activation to the inguinal fat depot. The increase in hepatic sympathetic traffic induced by leptin was also reduced in mice lacking the LepR in AgRP, but not POMC, neurons whereas LepR deletion in either AgRP or POMC neurons attenuated the hepatic parasympathetic nerve activation evoked by leptin. Interestingly, the renal, lumbar and splanchnic sympathetic nerve activation caused by leptin were significantly blunted in POMCCre/LepRfl/fl mice, but not in AgRPCre/LepRfl/fl mice. However, loss of the LepR in POMC or AgRP neurons did not interfere with the ability of leptin to increase sympathetic traffic to the adrenal gland. Furthermore, ablation of the p110α, but not the p110β, isoform of PI3K from POMC neurons eliminated the leptin-elicited renal sympathetic nerve activation.Finally, we show trans-synaptic retrograde tracing of both POMC and AgRP neurons from the kidneys. Conclusions: POMC and AgRP neurons are differentially involved in mediating the effects of leptin on autonomic nerve activity subserving various tissues and organs. Keywords: Leptin, Autonomic nervous system, Energy homeostasis, Cardiovascular regulation

Remote and selective spatiotemporal control of the activity of neurons to regulate behavior and physiological functions has been a long-sought goal in system neuroscience. Identification and subsequent bioengineering of light-sensitive ion channels (e.g., channelrhodopsins, halorhodopsin, and archaerhodopsins) from the bacteria have made it possible to use light to artificially modulate neuronal activity, namely optogenetics. Recent advance in genetics has also allowed development of novel pharmacological tools to selectively and remotely control neuronal activity using engineered G protein-coupled receptors, which can be activated by otherwise inert drug-like small molecules such as the designer receptors exclusively activated by designer drug, a form of chemogenetics. The cutting-edge optogenetics and pharmacogenetics are powerful tools in neuroscience that allow selective and bidirectional modulation of the activity of defined populations of neurons with unprecedented specificity. These novel toolboxes are enabling significant advances in deciphering how the nervous system works and its influence on various physiological processes in health and disease. Here, we discuss the fundamental elements of optogenetics and chemogenetics approaches and some of the applications that yielded significant advances in various areas of neuroscience and beyond.

Obesity is commonly associated with sympathetic overactivity and elevated blood pressure, and accumulating evidence suggests that an adipocyte-derived metabolic hormone, leptin, may plays an important role in these pathological processes, likely through a mechanism of so-called ‘selective leptin resistance’. However, underlying neural circuits through which leptin causes sympathoexcitation and increases blood pressure remain incompletely understood. Here we hypothesized that leptin may acts on a subset of lateral hypothalamic area (LHA) neurons to affect cardiovascular functions. We show that stereotaxic microinfusion of leptin into the LHA dose-dependently increases renal sympathetic nerve activity (RSNA) (% changes from baseline at 4 th hour: vehicle -25.03 ± 7.09 % vs leptin 100.23 ± 26.94 %, p

Selective leptin resistance (SLR) is a pathological state caused by prolonged high fat diet (HFD) feeding in which cardiovascular responses to leptin are preserved, but metabolic effects are attenuated. Thus, SLR is hypothesized to contribute to obesity-associated hypertension, but the mechanisms causing SLR are unclear. Desensitization of leptin signaling in cells of the arcuate nucleus (ARC) of the hypothalamus has been proposed as one of potential mechanisms contributing to SLR. Tanycytes, located along the walls of the third ventricle, play critical roles in the regulation of energy balance through the transport of blood-borne metabolic hormones such as leptin and ghrelin into the cerebrospinal fluid. Previous studies have reported that 10 weeks of 45% HFD (D12451) feeding is sufficient to cause SLR in wildtype C57BL/6J mice. To examine the transcriptomic changes induced by prolonged HFD exposure in tanycytes, we performed single-nucleus RNA-sequencing on nuclei isolated from ARC of ad libitum fed male C57BL/6J mice fed chow diet (7013; CD, n=7) or HFD (n=8) from 8 to 18 weeks of age. At 18 weeks, fat mass (+10.1±1.4 vs +3.2±0.4g, pAgrp ) neurons (z-score -1.6, p=0.009) and unchanged in neurons expressing proopiomelanocortin ( Pomc ) (p=0.2). Collectively, these results lead us to hypothesize that SLR is associated with normal transport of circulating leptin into the arcuate nucleus, while the primary dysfunction that causes SLR instead occurs within selected neuronal populations such as Agrp neurons.

Leptin resistance is a hallmark of obesity with unclear etiology. Celastrol, a compound found in the roots of the Tripterygium wilfordii and known to reduce endoplasmic reticulum (ER) stress, has recently emerged as a promising candidate to treat obesity by improving leptin sensitivity. However, the underlying neural mechanisms by which celastrol reduces obesity remain unclear. Using three different mouse models of obesity—diet-induced obesity (DIO), leptin receptor (LepR)-null, and melanocortin 4 receptor (MC4R)-null mice—in this study, we show that systemic celastrol administration substantially reduces food intake and body weight in MC4R-null comparable to DIO, proving the MC4R-independent antiobesity effect of celastrol. Body weight reduction was due to decreases in both fat and lean mass, and modest but significant body weight reduction was also observed in nonobese wild-type and LepR-null mice. Unexpectedly, celastrol upregulated proinflammatory cytokines without affecting genes involved in ER stress. Importantly, celastrol steadily increased sympathetic nerve activity to the brown fat and kidney with concordant increases of resting metabolic rate and arterial pressure. Our results suggest a previously unappreciated mechanism of action of celastrol in the regulation of energy homeostasis and highlight the need for careful consideration of its development as a safe antiobesity medication.

Eating disorders (EDs) are severe, life-threatening mental illnesses characterized by marked disturbances in body image and eating patterns. Attempts to understand the neurobiological basis of EDs have been hindered by the perception that EDs are primarily socially reinforced behaviors and not the result of a pathophysiologic process. This view is reflected by the diagnostic criteria of anorexia nervosa and bulimia nervosa, which emphasize intrapsychic conflicts such as “inability to maintain body weight,” “undue influence of body weight or shape on self-evaluation,” and “denial of the seriousness of low body weight” over neuropsychological measures. The neuropsychological constructs introduced within the research domain criteria (RDoC) matrix offer new hope for determining the neural substrate underlying the biological predisposition to EDs. We present selected studies demonstrating deficits in patients with EDs within each domain of the RDoC and propose a set of behavioral tasks in model systems that reflect aspects of that deficit. Finally, we propose a battery of tasks to examine comprehensively the function of neural circuits relevant to the development of EDs.

The brain melanocortin-4 receptor (MC4R) plays an important in the control of food intake and energy expenditure by increasing thermogenic sympathetic nerve activity (SNA). In addition, brain MC4R-mediated activation of SNA subserving cardiovascular organs such as the kidneys increase blood pressure. The melanocortin receptor accessory protein 2 (MRAP2) regulates the activity of the MC4R and other G-protein coupled receptors, but its specific role in MC4R-containing neurons is not clear . We hypothesized that MRAP2 in MC4R neurons is essential for the regulation of SNA, energy homeostasis and blood pressure. We generated mice (MRAP2 MC4R ) that lack MRAP2 in MC4R neurons by crossing MRAP2 fl/fl mice with MC4R Cre mice and confirmed Cre recombinase activity in MC4R neurons using td-Tomato reporter mice. Interestingly, the MRAP2 MC4R mice displayed obesity as indicated by the increased (pMC4R relative to littermate controls. Food intake was increased in the MRAP2 MC4R mice relative to littermate controls (males: 4.7±0.3 vs 3.9±0.3g and females: 3.5±0.2 vs 2.8±0.2g). Glucose and insulin tolerance tests revealed impaired glucose tolerance and insulin-induced glucose clearance in MRAP2 MC4R mice compared to littermate controls. Baseline brown adipose tissue (BAT) SNA tended to be lower in MRAP2 MC4R mice relative to controls. Moreover, intracerebroventricular (icv) injection of MTII (MC4R agonist, 1 μg) increased BAT SNA in control mice (107±31%) but not in MRAP2 MC4R mice (18±24%, p=0.02). On the other hand, icv the MTII (1 and 2 μg)-induced increase in renal SNA (p=0.2) and blood pressure (p=0.4) was not different in MRAP2 MC4R (100±32% and 15±8mmHg, respectively) relative to controls (74±24% and 12±6mmHg). These findings support a critical role for MRAP2 in uncoupling neuronal MC4R-mediated control of regional sympathetic traffic impacting energy metabolism versus blood pressure.

We recently demonstrated that leptin stimulates resting metabolic rate (RMR) through a mechanism that requires angiotensin (ANG) type 1A receptors (AT 1A ) localized to neurons of the arcuate nucleus (ARC) which express Agouti-related peptide (AgRP). Genetic disruption of AT 1A in AgRP neurons results in the loss of RMR responses to leptin, high fat diet, and various other stimuli which correlates with the disinhibition of AgRP, neuropeptide Y (NPY), and production enzymes and transporters (GAD1, GAD2, VGAT) for γ-aminobutyric acid (GABA) within the ARC. We hypothesize that AT 1A activation in AgRP neurons causes disinhibition of AgRP, NPY, GAD1, GAD2 and VGAT expression and thus increased inhibitory neurotransmission to pre-autonomic nuclei, and we seek to understand the second-messenger network activated by AT 1A which mediates transcriptional control of these genes. Intracellular calcium ([Ca 2+ ] i , determined by FURA-2 fluorescence), cyclic AMP (cAMP, by LANCE assay), receptor surface localization (by HiBiT-tagging), and gene expression responses (by qPCR) to ANG were examined in immortalized mouse hypothalamic cells (N43/5 and N39 cells) that express typical markers of AgRP neurons. ANG (0.1 and 1 μM, n = 3) had no effect to modulate [Ca 2+ ] i , but caused a dose-dependent reduction in forskolin-stimulated cAMP (EC 50 = 43 nM) which could be blocked by losartan (1 μM). ANG increased GTP-bound Gαi (PBS vehicle = 0.57±0.08, ANG 1 μM = 0.94±0.07 ratio vs IgG, p < 0.05, n = 3), reduced cell-surface localization of HiBiT-tagged AT 1A (PBS vehicle = 0.22±0.02, ANG 0.1 μM = 0.15±0.01, p < 0.05, n = 4), and significantly (p < 0.05, n = 6) reduced AgRP and GAD1 expression by 43% and 27%, respectively. Lastly, preliminary studies suggest that pretreatment with pertussis toxin (PTX, 100 ng/mL), an inhibitor of Gαi, abrogated AgRP and GAD1 suppression by ANG (0.01 μM). Collectively these findings support the novel concept that within immortalized cells that express markers of AgRP neurons, AT 1A couples to Gαi to reduce cAMP, which suppresses AgRP, NPY and GABA. This should disinhibit pre-motor, pre-autonomic circuits within the hypothalamus, which results in increased thermogenic sympathetic nerve activity, ultimately increasing RMR and energy expenditure.

It is well established that the central melanocortin system is critical for autonomic functions and energy homeostasis mainly via signaling at melanocortin-4 receptor (MC4R). Importantly, while obesity is commonly associated with elevated sympathetic tone and blood pressure, severely obese humans and rodents due to genetic MC4R deficiency exhibit normal to low sympathetic tone and blood pressure, suggesting a significant role of MC4R pathway in mediating obesity-associated sympathoexcitation and hypertension. MC4R is widely expressed in the brain including hypothalamic paraventricular nucleus (PVN) which regulates feeding and sympathetic traffic. However, the neuroanatomical basis of PVN MC4R neurons for sympathetic regulation is unclear. The goal of current study is to map the PVN MC4R neural circuits affecting sympathetic tone in mice. To this end, we injected Cre-dependent AAV driving eYFP-fused channel rhodopsin-2 expression into the PVN of MC4R-t2a-Cre knock-in mice, which allow targeted anterograde tract-tracing of PVN MC4R neurons throughout the brain. In addition to known brain region for feeding behavior (i.e. parabrachial nucleus), we found broad innervations of PVN MC4R neurons to various brain regions important for autonomic-cardiovascular control, including, but not limited to, nucleus of solitary tractus, dorsomotor nucleus of vagus, and ventrolateral medulla. Considerable innervation was also evident in spinal cord, which is further confirmed by Fluoro-gold (FG)-mediated retrograde tracing in the spinal cord (thoracic T6-10) of MC4R-GFP transgenic mouse. Double immunofluorescence labeling of GFP and FG revealed that ~50% (58 out of 116) MC4R neurons in the posterior parvocellular subdivision of PVN project to thoracic spinal cord. Furthermore, microinjection of synthetic MC4R agonist (MTII) into the PVN evokes ~42% increase (from baseline) in renal sympathetic nerve activity in anesthetized mice. These results provide important insights into understanding the divergent neural circuits by which PVN MC4R signaling differentially regulates metabolic and cardiovascular functions. Functional dissection of these diverging neural pathways using optogenetic/chemogenetic approaches is ongoing.

The estrogen-related receptor alpha (Esrra) is an orphan nuclear receptor with strong homology to estrogen receptor alpha (ERα), whereas it exhibits estrogen-independent constitutive transcriptional activity to regulate a number of genes involving in cellular energy metabolism. Esrra is broadly expressed throughout the body including those metabolically active tissues such as skeletal muscles, heart, brown adipose tissues, kidney, and brain. While previous reports showed that Esrra knockout (KO) mice were hypotensive and resistant to high fat diet-induced obesity (DIO), systemic understanding of the roles of Esrra in cardiometabolic control is limited. We therefore performed a variety of metabolic and cardiovascular measures to evaluate the cardiometabolic consequences of mice lacking Esrra globally. Our results revealed that Esrra KO mice were hypoactive and were resistant to DIO mainly due to decreased food intake. Despite of lower body weight and plasma leptin level, female Esrra KO mice tend to have elevated blood glucose level (p=0.08) without notable changes of insulin and glucagon levels. Non-invasive blood pressure measurement by tail-cuff sphygmomanometer showed that male Esrra KO mice had significantly lower blood pressure when the measurement was performed during light period (WT vs. KO: 99.8±1.4 vs. 93.9±1.8 mmHg, p

PURPOSE OF REVIEW: Here we review the current understanding of the functional neuroanatomy of neurons expressing Agouti-related peptide (AgRP) and the angiotensin 1A receptor (AT(1A)) within the arcuate nucleus (ARC) in the control of energy balance. RECENT FINDINGS: The development and maintenance of obesity involves suppression of resting metabolic rate (RMR). RMR control is integrated via AgRP and proopiomelanocortin neurons within the ARC. Their projections to other hypothalamic and extrahypothalamic nuclei contribute to RMR control, though relatively little is known about the contributions of individual projections and the neurotransmitters involved. Recent studies highlight a role for AT(1A), localized to AgRP neurons, but the specific function of AT(1A) within these cells remains unclear. SUMMARY: AT(1A) functions within AgRP neurons to control RMR, but additional work is required to clarify its role within subpopulations of AgRP neurons projecting to distinct second-order nuclei, and the molecular mediators of its signaling within these cells.

SUMMARY Eating disorders, such as anorexia nervosa and bulimia nervosa, are common and severe mental illnesses of unknown etiology. Recently, we identified a rare missense mutation in the transcription factor estrogen-related receptor alpha (ESRRA) that is associated with the development of eating disorders. However, little is known about ESRRA function in the brain. Here, we report that Esrra is expressed in the mouse brain and demonstrate that Esrra levels are regulated by energy reserves. Esrra-null female mice display a reduced operant response to a high-fat diet, compulsivity/behavioral rigidity, and social deficits. Selective Esrra knockdown in the prefrontal and orbitofrontal cortices of adult female mice recapitulates reduced operant response and increased compulsivity, respectively. These results indicate that Esrra deficiency in the mouse brain impairs behavioral responses in multiple functional domains., Graphical Abstract

Studies in both humans and rodents have clearly established a role for melanocortin-4 receptor (MC4R) in energy homeostasis, sympathetic and cardiovascular function. Both MC4R-deficient humans and rodents develop severe obesity while keeping the sympathetic tone and blood pressure below or within the normal range, indicating that MC4R is required for obesity-associated sympathetic overdrive and hypertension. Indeed, sympathetic overdrive and cardiovascular side effect have been a major problem in developing anti-obesity drugs that target MC4R. Thus, better understanding the neural substrates that mediate distinct metabolic and sympathetic actions of MC4R is needed to provide a more specific target to treat obesity and associated hypertension. We have recently mapped the pattern of co-expression of MC4R and leptin receptor (LepR) and found that, throughout entire mouse brain, these two metabolically important receptors are uniquely co-expressed only in neurons of two brain regions, including the lateral hypothalamic area (LHA) and the periaqueductal gray (PAG). To specifically test the role of MC4R in LepR-expressing neurons, we generated mice with MC4R expression only in LepR-positive neurons by breeding Cre-dependently “reactivatable” MC4R-null mice (MC4R-TB) to LepR-Cre mice. We found that specific re-expression of MC4Rs only in LepR-positive neurons partially suppresses body weight gain especially in female mice (MC4R-TB 31.73 ± 2.2g vs. MC4R-TB::LepR-Cre 27.97 ± 1.8 g, p=0.0013). Direct multi-fiber recording of renal nerve activity (RSNA), however, revealed that neither baseline activity nor MC4R agonist-induced RSNA is significantly altered by restoration of MC4R signaling in LepR-positive neurons (change of RSNA in MC4R-TB 8.844 ± 3.05 vs. MC4R-TB::LepR-Cre 11.95 ± 2.7, p>0.05). Our results suggest that MC4R signaling in leptin-responsive neurons is sufficient to suppress body weight in female mice, but seemingly has no effect on sympathetic traffic to the kidney. Further investigation of the effect of MC4Rs in LepR-positive neurons on blood pressure regulation is underway.

While neurons in the lateral hypothalamic area (LHA) are clearly involved in feeding, metabolism, and cardiovascular regulation, neurochemically and neuroanatomically heterogeneous nature of LHA neurons has been a challenge in understanding their function in physiological regulation. We have recently identified a unique subset of LHA GABAergic neurons, which are distinct from well-known orexin and MCH neurons, that co-express two metabolically important leptin receptor (LepR) and melanocortin-4 receptor (MC4R), suggesting that these neurons might be the important targets of leptin and melanocortin for metabolic and cardiovascular regulation. Here we show that LHA LepR-positive neurons innervate broadly to intra- and extra-hypothalamic brain regions important for feeding, sympathetic nervous activity, and cardiovascular function, including but not limited to arcuate nucleus, paraventricular nucleus of hypothalamus, parabrachial nucleus, and nucleus of the solitary tract. Stereotaxic microinfusion of leptin into the LHA increases renal sympathetic nerve activity (RSNA) (% changes from baseline at 4 th hour: vehicle -25.03 ± 7.09 % vs leptin 100.23 ± 26.94 %, p

Obesity, a major risk factor for the development of diabetes mellitus, cardiovascular diseases and certain types of cancer, arises from a chronic positive energy balance that is often due to unlimited access to food and an increasingly sedentary lifestyle on the background of a genetic and epigenetic vulnerability. Our understanding of the humoral and neuronal systems that mediate the control of energy homeostasis has improved dramatically in the past few decades. However, our ability to develop effective strategies to slow the current epidemic of obesity has been hampered, largely owing to the limited knowledge of the mechanisms underlying resistance to the action of metabolic hormones such as leptin and ghrelin. The development of resistance to leptin and ghrelin, hormones that are crucial for the neuroendocrine control of energy homeostasis, is a hallmark of obesity. Intensive research over the past several years has yielded tremendous progress in our understanding of the cellular pathways that disrupt the action of leptin and ghrelin. In this Review, we discuss the molecular mechanisms underpinning resistance to leptin and ghrelin and how they can be exploited as targets for pharmacological management of obesity.