Abstract
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.
Keywords: Chemogenetics, DREADD, Energy expenditure, Food intake, Indirect calorimetry, TSE
Background
The POA is a central hub for body temperature homeostasis, which receives thermosensory information from the periphery and tunes the degree of sympathetic output to brown adipose tissue (BAT), cutaneous blood vessels, and heart to control the amount of heat generation and dissipation (Nakamura, 2011). We discovered that LepRb neurons in the POA are stimulated by warm ambient temperature and mediate warm-adaptive responses that include suppression of BAT thermogenesis and food intake (Yu et al., 2016). This discovery was made mainly through chemogenetic stimulation of POA LepRb neurons by virally expressing Gq-coupled DREADD, hM3Dq, in POA LepRb neurons and injecting CNO in mice. A single IP injection of CNO can stimulate target neurons up to 10 h (Krashes et al., 2011; Rezai-Zadeh et al., 2014). This long-lasting CNO effect allows researchers to modulate target neuron activity chronically with two IP injections of CNO per day. Optogenetics and chemogenetics have revolutionized the field of neuroscience by providing tools to selectively manipulate target neuron activity with its own unique advantages and disadvantages. For studying neurons that modulate energy balance, researchers often use an indirect calorimetry system to simultaneously measure energy expenditure and food intake for an extended period of time (Rezai-Zadeh et al., 2014; Correa et al., 2015; Qualls-Creekmore et al., 2017). Chemogenetics is best suited for this type of study thanks to slow clearance of CNO from the body and no requirement of optical devices as in optogenetics. In our study, we investigated consequences of chronic stimulation of POA LepRb neurons by DREADD with the PhenoMaster indirect calorimetry system. CNO was injected at 0.3 mg/kg twice per day for six days, and energy expenditure and food intake were continuously measured every 25 min. Body weight was measured once every morning to monitor how changed energy expenditure and food intake affected body weight. In this protocol, we describe a step-by-step procedure of using the TSE PhenoMaster indirect calorimetry system to measure energy expenditure and food intake during chronic stimulation of POA LepRb neurons by DREADD in mice.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Homozygous LepRb-Cre mice were used to maximize Cre expression for more efficient viral gene expression.
Recipes
Acknowledgments
This work was supported by AHA053298N, P/F DK020572-30, R01DK092587 (HM), P20GM103528 (HM and SY), and 2P30-DK072476 (HM and SY). Partial support was provided through the Animal Phenotyping Core funded by NIDDK NORC Center Grant P30 DK072476. The authors declare no competing financial interests. This protocol is adapted from procedures published in Yu et al. (2016).
References
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