Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs

Leptin Receptor Signaling and Action in the Central Nervous System

The increasing incidence of obesity in developed nations represents an ever‐growing challenge to health care by promoting diabetes and other diseases. The discovery of the hormone, leptin, a decade ago has facilitated the acquisition of new knowledge regarding the regulation of energy balance. A great deal remains to be discovered regarding the molecular and anatomic actions of leptin, however. Here, we discuss the mechanisms by which leptin activates intracellular signals, the roles that these signals play in leptin action in vivo, and sites of leptin action in vivo. Using “reporter” mice, in which LRb‐expressing (long form of the leptin receptor) neurons express the histological marker, β‐galactosidase, coupled with the detection of LRb‐mediated signal transducer and activator of transcription 3 signaling events, we identified LRb expression in neuronal populations both within and outside the hypothalamus. Understanding the regulation and physiological function of these myriad sites of central leptin action will be a crucial next step in the quest to understand mechanisms of leptin action and energy balance.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93692/1/oby.2006.310.pd

Testing Effects of Chronic Chemogenetic Neuronal Stimulation on Energy Balance by Indirect Calorimetry

The fundamental of neuroscience is to connect the firing of neurons to physiological and behavioral outcomes. Chemogenetics enables researchers to control the activity of a genetically defined population of neurons in vivo through the expression of designer receptor exclusively activated by designer drug (DREADD) in specific neurons and the administration of its synthetic ligand clozapine N-oxide (CNO) (Sternson and Roth, 2014). Using stimulatory Gq-coupled DREADD (hM3Dq) in mice, we showed that leptin receptor (LepRb)-expressing neurons in the preoptic area (POA) of the hypothalamus are warm-sensitive neurons that mediate warm-responsive metabolic and behavioral adaptations by reducing energy expenditure and food intake (Yu et al., 2016). We also used DREADD technology to test effects of chronic stimulation of POA LepRb neurons on energy expenditure, food intake, and body weight with the TSE indirect calorimetry system. Here we describe the detailed protocol of how we used indirect calorimetry to study the outcome of chronic stimulation of POA LepRb neurons. This protocol can be adapted to study long-term metabolic and behavioral consequences of other neuronal modulations, with possible modifications to the type of DREADD, duration of CNO treatment, or method of CNO delivery

Regulation of hypothalamic prohormone convertases 1 and 2 and effects on processing of prothyrotropin-releasing hormone

Regulation of energy balance by leptin involves regulation of several neuropeptides, including thyrotropin-releasing hormone (TRH). Synthesized from a larger inactive precursor, its maturation requires proteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). Since this maturation in response to leptin requires prohormone processing, we hypothesized that leptin might regulate hypothalamic PC1 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Using hypothalamic neurons, we found that leptin stimulated PC1 and PC2 mRNA and protein expression and also increased PC1 and PC2 promoter activities in transfected 293T cells. Starvation of rats, leading to low serum leptin levels, decreased PC1 and PC2 gene and protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Exogenous administration of leptin to fasted animals restored PC1 levels in the median eminence (ME) and the PVN to approximately the level found in fed control animals. Consistent with this regulation of PCs in the PVN, concentrations of TRH in the PVN and ME were substantially reduced in the fasted animals relative to the fed animals, and leptin reversed this decrease. Further analysis showed that proteolytic cleavage of pro–thyrotropin-releasing hormone (proTRH) at known PC cleavage sites was reduced by fasting and increased in animals given leptin. Combined, these findings suggest that leptin-dependent stimulation of hypothalamic TRH expression involves both activation of trh transcription and stimulation of PC1 and PC2 expression, which lead to enhanced processing of proTRH into mature TRH

Body Composition, Food Intake, and Energy Expenditure in a Murine Model of Roux-en-Y Gastric Bypass Surgery

BACKGROUND: The mechanisms by which Roux-en-Y gastric bypass surgery (RYGB) so effectively lowers body weight and improves glycemic control are not well understood and murine models are essential for identifying the crucial signaling pathways involved. AIMS: To characterize the time course of RYGB on body weight, body composition, food intake, and energy expenditure in diet-induced obese mice, and establish a tissue bank for global “omics” or targeted biochemical and structural analyses. METHODS: High-fat diet-induced obese mice were subjected to RYGB using an improved surgical technique with a small gastric pouch. The effects on body weight, body composition, food intake, and energy expenditure were compared to sham surgery, high-fat diet-restricted weight-matched controls, and never obese chow-fed controls. RESULTS: Without mortality or complications, RYGB surgery in high-fat diet-induced obese mice gradually decreased body weight to a plateau that was more or less sustained for up to 12 weeks (33 g, −18%, p<0.01) and significantly lower compared with sham-operated mice (51 g, +25%, p <0.01), but higher (+18%, p<0.01) than age-matched, chow-fed control mice (27 g). Energy intake after RYGB was significantly suppressed compared to sham only for the first 10 days, but significantly higher compared to weight-matched mice. Energy expenditure after RYGB was higher throughout the study compared with weight-matched, but not sham animals. CONCLUSIONS: RYGB surgery in diet-induced obese mice results in similar body weight and body composition changes as observed in humans, but in contrast to humans, this is achieved mainly through increased energy expenditure rather than decreased food intake