Anterior hypothalamic parvalbumin neurons are glutamatergic and promote escape behavior.

The anterior hypothalamic area (AHA) is a critical structure for defensive responding. Here, we identified a cluster of parvalbumin-expressing neurons in the AHA (AHAPV) that are glutamatergic with fast-spiking properties and send axonal projections to the dorsal premammillary nucleus (PMD). Using in vivo functional imaging, optogenetics, and behavioral assays, we determined the role of these AHAPV neurons in regulating behaviors essential for survival. We observed that AHAPV neuronal activity significantly increases when mice are exposed to a predator, and in a real-time place preference assay, we found that AHAPV neuron photoactivation is aversive. Moreover, activation of both AHAPV neurons and the AHAPV → PMD pathway triggers escape responding during a predator-looming test. Furthermore, escape responding is impaired after AHAPV neuron ablation, and anxiety-like behavior as measured by the open field and elevated plus maze assays does not seem to be affected by AHAPV neuron ablation. Finally, whole-brain metabolic mapping using positron emission tomography combined with AHAPV neuron photoactivation revealed discrete activation of downstream areas involved in arousal, affective, and defensive behaviors including the amygdala and the substantia nigra. Our results indicate that AHAPV neurons are a functional glutamatergic circuit element mediating defensive behaviors, thus expanding the identity of genetically defined neurons orchestrating fight-or-flight responses. Together, our work will serve as a foundation for understanding neuropsychiatric disorders triggered by escape such as post-traumatic stress disorder (PTSD).

Regulation of body weight and food intake by AGRP neurons during opioid dependence and abstinence in mice.

Dysregulation of body weight maintenance and opioid dependence are often treated as independent disorders. Here, we assessed the effects of both acute and long-term administration of morphine with and without chemogenetic activation of agouti-related peptide (AGRP)-expressing neurons in the arcuate nucleus (ARCAGRP neurons) to elucidate whether morphine and neuronal activation affect feeding behavior and body weight. First, we characterized interactions of opioids and energy deficit in wild-type mice. We observed that opioid administration attenuated both fasting-induced refeeding and ghrelin-stimulated feeding. Moreover, antagonism of opioid receptors blocked fasting-induced refeeding behavior. Next, we interfaced chemogenetics with opioid dependence. For chemogenetic experiments of ARCAGRP neurons, we conducted a priori behavioral qualification and post-mortem FOS immunostaining verification of arcuate activation following ARCAGRP chemogenetic activation. We administered clozapine during short-term and long-term morphine administration paradigms to determine the effects of dependence on food intake and body weight. We found that morphine occluded feeding behavior characteristic of chemogenetic activation of ARCAGRP neurons. Notably, activation of ARCAGRP neurons attenuated opioid-induced weight loss but did not evoke weight gain during opioid dependence. Consistent with these findings, we observed that morphine administration did not block fasting-induced activation of the ARC. Together, these results highlight the strength of opioidergic effects on body weight maintenance and demonstrate the utility of ARCAGRP neuron manipulations as a lever to influence energy balance throughout the development of opioid dependence.

Fluorescence microendoscopy for in vivo deep-brain imaging of neuronal circuits.

Imaging neuronal activity in awake, behaving animals has become a groundbreaking method in neuroscience that has rapidly enhanced our understanding of how the brain works. In vivo microendoscopic imaging has enabled researchers to see inside the brains of experimental animals and thus has emerged as a technology fit to answer many experimental questions. By combining microendoscopy with cutting edge targeting strategies and sophisticated analysis tools, neuronal activity patterns that underlie changes in behavior and physiology can be identified. However, new users may find it challenging to understand the techniques and to leverage this technology to best suit their needs. Here we present a background and overview of the necessary components for performing in vivo optical calcium imaging and offer some detailed guidance for current recommended approaches.

Centrally circulating α-klotho inversely correlates with human obesity and modulates arcuate cell populations in mice.

OBJECTIVE: Our laboratory recently identified the centrally circulating α-klotho protein as a novel hypothalamic regulator of food intake and glucose metabolism in mice. The current study aimed to investigate novel molecular effectors of central α-klotho in the arcuate nucleus of the hypothalamus (ARC), while further deciphering its role regulating energy balance in both humans and mice. METHODS: Cerebrospinal fluid (CSF) was collected from 22 adults undergoing lower limb orthopedic surgeries, and correlations between body weight and α-klotho were determined using an α-klotho enzyme-linked immunosorbent assay (ELISA) kit. To investigate the effects of α-klotho on energy expenditure (EE), 2-day intracerebroventricular (ICV) treatment was performed in diet-induced obesity (DIO) mice housed in TSE Phenomaster indirect calorimetry metabolic cages. Immunohistochemical staining for cFOS and patch clamp electrophysiology were used to determine the effects of central α-klotho on proopiomelanocortin (POMC) and tyrosine hydroxylase (TH) neurons. Additional stainings were performed to determine novel roles for central α-klotho to regulate non-neuronal cell populations in the ARC. Lastly, ICV pretreatment with fibroblast growth factor receptor (FGFR) or PI3kinase inhibitors was performed to determine the intracellular signaling involved in α-klotho-mediated regulation of ARC nuclei. RESULTS: Obese/overweight human subjects had significantly lower CSF α-klotho concentrations compared to lean counterparts (1,044 ± 251 vs. 1616 ± 218 pmol/L, respectively). Additionally, 2 days of ICV α-klotho treatment increased EE in DIO mice. α-Klotho had no effects on TH neuron activity but elicited varied responses in POMC neurons, with 44% experiencing excitatory and 56% experiencing inhibitory effects. Inhibitor experiments identified an α-klotho→FGFR→PI3kinase signaling mechanism in the regulation of ARC POMC and NPY/AgRP neurons. Acute ICV α-klotho treatment also increased phosphorylated ERK in ARC astrocytes via FGFR signaling. CONCLUSION: Our human CSF data provide the first evidence that impaired central α-klotho function may be involved in the pathophysiology of obesity. Furthermore, results in mouse models identify ARC POMC neurons and astrocytes as novel molecular effectors of central α-klotho. Overall, the current study highlights prominent roles of α-klotho→FGFR→PI3kinase signaling in the homeostatic regulation of ARC neurons and whole-body energy balance.

ARCAgRP/NPY Neuron Activity Is Required for Acute Exercise-Induced Food Intake in Un-Trained Mice.

While much is known about the role of agouti-regulated peptide/neuropeptide Y (AgRP/NPY) and pro-opiomelanocortin (POMC) neurons to regulate energy homeostasis, little is known about how forced energy expenditure, such as exercise, modulates these neurons and if these neurons are involved in post-exercise feeding behaviors. We utilized multiple mouse models to investigate the effects of acute, moderate-intensity exercise on food intake and neuronal activity in the arcuate nucleus (ARC) of the hypothalamus. NPY-GFP reporter mice were utilized for immunohistochemistry and patch-clamp electrophysiology experiments investigating neuronal activation immediately after acute treadmill exercise. Additionally, ARCAgRP/NPY neuron inhibition was performed using the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system in AgRP-Cre transgenic mice to investigate the importance of AgRP/NPY neurons in post-exercise feeding behaviors. Our experiments revealed that acute moderate-intensity exercise significantly increased food intake, ARCAgRP/NPY neuron activation, and PVNSim1 neuron activation, while having no effect on ARCPOMC neurons. Strikingly, this exercise-induced refeeding was completely abolished when ARCAgRP/NPY neuron activity was inhibited. While acute exercise also increased PVNSim1 neuron activity, inhibition of ARCAgRP/NPY neurons had no effect on PVNSim1 neuronal activation. Overall, our results reveal that ARCAgRP/NPY activation is required for acute exercise induced food intake in mice, thus providing insight into the critical role of ARCAgRP/NPY neurons in maintaining energy homeostasis in cases of exercise-mediated energy deficit.

Central α-Klotho Suppresses NPY/AgRP Neuron Activity and Regulates Metabolism in Mice.

α-Klotho is a circulating factor with well-documented antiaging properties. However, the central role of α-klotho in metabolism remains largely unexplored. The current study investigated the potential role of central α-klotho to modulate neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing neurons, energy balance, and glucose homeostasis. Intracerebroventricular administration of α-klotho suppressed food intake, improved glucose profiles, and reduced body weight in mouse models of type 1 and 2 diabetes. Furthermore, central α-klotho inhibition via an anti-α-klotho antibody impaired glucose tolerance. Ex vivo patch clamp electrophysiology and immunohistochemical analysis revealed that α-klotho suppresses NPY/AgRP neuron activity, at least in part, by enhancing miniature inhibitory postsynaptic currents. Experiments in hypothalamic GT1-7 cells observed that α-klotho induces phosphorylation of AKTser473, ERKthr202/tyr204, and FOXO1ser256 as well as blunts AgRP gene transcription. Mechanistically, fibroblast growth factor receptor 1 (FGFR1) inhibition abolished the downstream signaling of α-klotho, negated its ability to modulate NPY/AgRP neurons, and blunted its therapeutic effects. Phosphatidylinositol 3 kinase (PI3K) inhibition also abolished α-klotho's ability to suppress food intake and improve glucose clearance. These results indicate a prominent role of hypothalamic α-klotho/FGFR1/PI3K signaling in the modulation of NPY/AgRP neuron activity and maintenance of energy homeostasis, thus providing new insight into the pathophysiology of metabolic disease.

Activation of a lateral hypothalamic-ventral tegmental circuit gates motivation.

Across species, motivated states such as food-seeking and consumption are essential for survival. The lateral hypothalamus (LH) is known to play a fundamental role in regulating feeding and reward-related behaviors. However, the contributions of neuronal subpopulations in the LH have not been thoroughly identified. Here we examine how lateral hypothalamic leptin receptor-expressing (LHLEPR) neurons, a subset of GABAergic cells, regulate motivation in mice. We find that LHLEPR neuronal activation significantly increases progressive ratio (PR) performance, while inhibition decreases responding. Moreover, we mapped LHLEPR axonal projections and demonstrated that they target the ventral tegmental area (VTA), form functional inhibitory synapses with non-dopaminergic VTA neurons, and their activation promotes motivation for food. Finally, we find that LHLEPR neurons also regulate motivation to obtain water, suggesting that they may play a generalized role in motivation. Together, these results identify LHLEPR neurons as modulators within a hypothalamic-ventral tegmental circuit that gates motivation.

Administration of alpha klotho reduces liver and adipose lipid accumulation in obese mice.

α-Klotho, a known anti-aging protein, exerts diverse physiological effects including: maintenance of phosphate and calcium homeostasis, modulation of cell proliferation, and enhanced buffering of reactive oxygen species. However, the role of α-Klotho in the regulation of energy metabolism is complex and poorly understood. Here we investigated the effects of 5 weeks peripheral administration of α-Klotho in high fat diet induced obese mice. Food intake, blood glucose, and body weight were measured daily. Energy expenditure was determined with indirect calorimetry and body composition with magnetic resonance imaging. Liver and adipose tissue were collected for lipid content measurements and gene expression analysis. α-Klotho-treated mice experienced reduced adiposity, increased lean mass, and elevated energy expenditure, despite no changes in food intake, body weight, or fed blood glucose levels. Lipid accumulation in liver and adipose tissue was also reduced compared to controls. Furthermore, Real-time quantitative PCR showed reduced expression of key lipogenic genes in α-Klotho treated mice in these organs. Taken together, these data suggest encouraging therapeutic potential of α-Klotho and highlight a need for further research into the specific mechanisms explaining improved body composition, elevated energy expenditure, and reduced lipid content in both liver and adipose tissue in α-Klotho-treated mice.

Vertical sleeve gastrectomy improves liver and hypothalamic functions in obese mice.

Vertical sleeve gastrectomy (VSG) is an effective surgery to treat obesity and diabetes. However, the direct effect of VSG on metabolic functions is not fully understood. We aimed to investigate if alterations in hypothalamic neurons were linked with perturbations in liver metabolism after VSG in an energy intake-controlled obese mouse model. C57BL/6 and hrNPY-GFP reporter mice received HFD for 12 weeks and were then divided into three groups: Sham (ad lib), sham (pair-fed) with VSG, and VSG. Food intake was measured daily, and blood glucose levels were measured before and after the study. Energy expenditure and body composition were determined. Serum parameters, liver lipid and glycogen contents were measured, and gene/protein expression were analyzed. Hypothalamic POMC, AgRP/NPY, and tyrosine hydroxylase expressing neurons were counted. As results, we found that VSG reduced body weight gain and adiposity induced by HFD, increased energy expenditure independent of energy intake. Fed and fasted blood glucose levels were reduced in the VSG group. While serum active GLP-1 level was increased, the active ghrelin and triglycerides levels were decreased along with improved insulin resistance in VSG group. Liver lipid accumulation, glycogen content, and gluconeogenic gene expression were reduced in the VSG group. In the hypothalamus, TH expressing neuron population was decreased, and the POMC-expressing neuron population was increased in the VSG group. Our data suggests that VSG improves metabolic symptoms by increasing energy expenditure and lowering lipid and glycogen contents in the liver. These physiological alterations are possibly related to changes in hypothalamic neuron populations.

Phospholipid methylation regulates muscle metabolic rate through Ca2+ transport efficiency.

The biophysical environment of membrane phospholipids affects structure, function, and stability of membrane-bound proteins.1,2 Obesity can disrupt membrane lipids, and in particular, alter the activity of sarco/endoplasmic reticulum (ER/SR) Ca2+-ATPase (SERCA) to affect cellular metabolism.3-5 Recent evidence suggests that transport efficiency (Ca2+ uptake / ATP hydrolysis) of skeletal muscle SERCA can be uncoupled to increase energy expenditure and protect mice from diet-induced obesity.6,7 In isolated SR vesicles, membrane phospholipid composition is known to modulate SERCA efficiency.8-11 Here we show that skeletal muscle SR phospholipids can be altered to decrease SERCA efficiency and increase whole-body metabolic rate. The absence of skeletal muscle phosphatidylethanolamine (PE) methyltransferase (PEMT) promotes an increase in skeletal muscle and whole-body metabolic rate to protect mice from diet-induced obesity. The elevation in metabolic rate is caused by a decrease in SERCA Ca2+-transport efficiency, whereas mitochondrial uncoupling is unaffected. Our findings support the hypothesis that skeletal muscle energy efficiency can be reduced to promote protection from obesity.

AgRP/NPY Neuron Excitability Is Modulated by Metabotropic Glutamate Receptor 1 During Fasting.

The potential to control feeding behavior via hypothalamic AgRP/NPY neurons has led to many approaches to modulate their excitability-particularly by glutamatergic input. In the present study using NPY-hrGFP reporter mice, we visualize AgRP/NPY neuronal metabotropic glutamate receptor 1 (mGluR1) expression and test the effect of fasting on mGluR1 function. Using the pharmacological agonist dihydroxyphenylglycine (DHPG), we demonstrate the enhanced capacity of mGluR1 to drive firing of AgRP/NPY neurons after overnight fasting, while antagonist 3-MATIDA reduces firing. Further, under synaptic blockade we demonstrate that DHPG acts directly on AgRP/NPY neurons to create a slow inward current. Using an in vitro approach, we show that emulation of intracellular signals associated with fasting by forskolin enhances DHPG induced phosphorylation of extracellularly regulated-signal kinase (1/2) in GT1-7 cell culture. We show in vivo that blocking mGluR1 by antagonist 3-MATIDA lowers fasting induced refeeding. In summary, this study identifies a novel layer of regulation on AgRP/NPY neurons integrated with whole body energy balance.

The effects of exercise on hypothalamic neurodegeneration of Alzheimer's disease mouse model.

Alzheimer's disease is a neurodegenerative disorder that affects the central nervous system. In this study, we characterized and examined the early metabolic changes in the triple transgenic mouse AD model (3xtg-AD), and their relationship with the hypothalamus, a key regulator of metabolism in the central nervous system. We observed that the 3xtg-AD model exhibited significantly higher oxygen consumption as well as food intake before reported amyloid plaque formation, indicating that metabolic abnormalities occurred at early onset in the 3xtg-AD model compared with their counterparts. Analysis of gene expression in the hypothalamus indicated increased mRNA expression of inflammation- and apoptosis-related genes, as well as decreased gene expression of Agouti-related protein (AgRP) and Melanocortin 4 receptor (MC4R) at 12 weeks of age. Immunofluorescence analysis revealed that pro-opiomelanocortin (POMC) and NPY-expressing neurons decreased at 24 weeks in the 3xtg-AD model. Four weeks of voluntary exercise were sufficient to reverse the gene expression of inflammation and apoptotic markers in the hypothalamus, six weeks of exercise improved glucose metabolism, moreover, 8 weeks of voluntary exercise training attenuated apoptosis and augmented POMC and NPY-expressing neuronal populations in the hypothalamus compared to the control group. Our results indicated that early onset of metabolic abnormalities may contribute to the pathology of AD, which is associated with increased inflammation as well as decreased neuronal population and key neuropeptides in the hypothalamus. Furthermore, early intervention by voluntary exercise normalized hypothalamic inflammation and neurodegeneration as well as glucose metabolism in the 3xtg-AD model. The data, taken as a whole, suggests a hypothalamic-mediated mechanism where exercise prevents the progression of dementia and of Alzheimer's disease.

Voluntary exercise improves hypothalamic and metabolic function in obese mice.

Exercise plays a critical role in regulating glucose homeostasis and body weight. However, the mechanism of exercise on metabolic functions associated with the CNS has not been fully understood. C57BL6 male mice (n=45) were divided into three groups: normal chow diet, high-fat diet (HFD) treatment, and HFD along with voluntary running wheel exercise training for 12 weeks. Metabolic function was examined by the Comprehensive Lab Animal Monitoring System and magnetic resonance imaging; phenotypic analysis included measurements of body weight, food intake, glucose and insulin tolerance tests, as well as insulin and leptin sensitivity studies. By immunohistochemistry, the amount changes in the phosphorylation of signal transducer and activator of transcription 3, neuronal proliferative maker Ki67, apoptosis positive cells as well as pro-opiomelanocortin (POMC)-expressing neurons in the arcuate area of the hypothalamus was identified. We found that 12 weeks of voluntary exercise training partially reduced body weight gain and adiposity induced by an HFD. Insulin and leptin sensitivity were enhanced in the exercise training group verses the HFD group. Furthermore, the HFD-impaired POMC-expressing neuron is remarkably restored in the exercise training group. The restoration of POMC neuron number may be due to neuroprotective effects of exercise on POMC neurons, as evidenced by altered proliferation and apoptosis. In conclusion, our data suggest that voluntary exercise training improves metabolic symptoms induced by HFD, in part through protected POMC-expressing neuron from HFD and enhanced leptin signaling in the hypothalamus that regulates whole-body energy homeostasis.

Across time and space: effects of urbanization on corticosterone and body condition vary over multiple years in song sparrows (Melospiza melodia).

Animals inhabiting urban areas must simultaneously cope with the unique challenges presented by this novel habitat type while exploiting the distinctive opportunities it offers. The costs and benefits of urban living are often assumed to be consistent across time, but may in fact vary depending on the habitat features influencing them. Here we examine the glucocorticoid levels and body condition of song sparrows (Melospiza melodia) resident at urban and rural sites over four consecutive years to determine whether these traits, which may be linked to the relative costs and benefits of these respective habitats, are consistent over time. Glucocorticoid levels and body condition varied by year in both habitat types. While habitat alone did not influence glucocorticoid levels, there was a significant interaction between year and habitat, indicating that glucocorticoids differ between habitats in some years but not others. There was no discernable effect of habitat alone on body condition. Overall, these data suggest that the costs and benefits of inhabiting urban versus rural habitats differ substantially from year to year.