Orexin-Driven GAD65 Network of the Lateral Hypothalamus Sets Physical Activity in Mice
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Orexin-driven GAD65 network of the lateral hypothalamus sets physical activity in mice Christin Kossea, Cornelia Schöneb, Edward Braceya, and Denis Burdakova,c,1 aThe Francis Crick Institute, London NW1 1AT, United Kingdom; bCentre for Experimental Neurology, Inselspital University Hospital, Bern 3010, Switzerland; and cDepartment of Developmental Neurobiology, King’s College London, London WC2R 2LS, United Kingdom Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved March 23, 2017 (received for review November 30, 2016) Damage to the lateral hypothalamus (LH) causes profound physical orexin-ChR2 cells (22). We then examined resulting optically in- inactivity in mammals. Several molecularly distinct types of LH neurons duced postsynaptic responses in GAD65LH neurons (Fig. 1A;group have been identified, including orexin cells and glutamic acid decar- data and/or statistics for findings illustrated as figures are described boxylase 65 (GAD65) cells, but their interplay in orchestrating physical in the figure legends). We found that GAD65LH cells received time- activity is not fully understood. Here, using optogenetic circuit analysis locked excitatory glutamatergic inputs when orexinLH cells were and cell type-specific deep-brain recordings in behaving mice, we stimulated (Fig. 1A). To determine if orexin peptide signaling drives show that orexin cell activation rapidly recruits GAD65LH neurons. We the GAD65LH network, we also investigated the effect of applying demonstrate that internally initiated GAD65LH cell bursts precede and exogenous orexin peptide. We used confocal network imaging of a accompany spontaneous running bouts, that selective chemogenetic Cre-dependent calcium indicator, GCaMP6s (23), expressed in LH silencing of natural GAD65LH cell activity depresses voluntary locomo- slices from GAD65-Ires-Cre mice (Fig. 1B). Orexin-A peptide ro- tion, and that GAD65 cell overactivation leads to hyperlocomotion. LH bustly excited the GAD65LH network, and this excitation persisted These results thus identify a molecularly distinct, orexin-activated (but was slightly reduced) in the presence of a mix of synaptic LH submodule that governs physical activity in mice. blockers (Fig. 1B). In parallel, electrophysiological recordings showed that orexin excites individual GAD65LH cells by activating a hypothalamus | GAD65 | orexin | hypocretin | locomotion | stress current with a reversal potential consistent with a nonselective cationic conductance (Fig. 1C). Overall, >98% of GAD65LH cells he lateral hypothalamus (LH) is thought to provide an es- (64 of 65 cells) were activated by orexin peptide in vitro. Tsential drive for diverse vital behaviors, including locomotion. If orexin cells also activate GAD65LH cells in vivo, then stress Peri-LH lesions in mammals disrupt context-appropriate physical stimuli that elicit orexinLH cell activity should also activate activity, and are associated with the human disorder encephalitis GAD65LH cells in vivo. Acute immobilization stress rapidly and “ lethargica, which has been noted to make humans sit motion- robustly activates orexinLH cells in awake mice (13). We therefore less...all day” (1–6). The LH is not a homogeneous entity, but used this paradigm, combined with deep-brain fiber photometry contains many molecularly distinct classes of neurons that are recording of GAD65LH cell activity and pharmacological anta- thought to have different physiological roles (7). However, the gonism of orexin receptors, to determine if stress and orexin interrelations of distinct LH cell classes in computing and driving drive GAD65LH cell activity in vivo. We expressed Cre-dependent context-appropriate physical activity are not fully understood. GCaMP6s, or control protein EGFP, in the LH of GAD65-Ires- For example, LH neurons expressing orexins/hypocretins (8, 9) Cre mice, and recorded fluorescence intensity using a fiberoptic become activated in diverse stressful contexts, including acute au- probe implanted in the LH (Fig. 2A). We observed bursts of ac- ditory stimulation, hypoglycemia, hypercapnia, and physical capture tivity in GAD65LH-GCaMP6s cells, but not in GAD65LH-EGFP (3, 9–13). OrexinLH cell activity may thus represent an important cells, of freely moving mice (Fig. 2A). These bursts were similar input variable for computing context-appropriate locomotor outputs (≈5–30% ΔF/F) to the bursts recorded using photometry in (3, 14), and orexin peptides have been proposed to increase arousal and locomotion by actions on extrahypothalamic projections to areas Significance such as the locus coeruleus (15, 16). However, hypoactivity pheno- types caused by orexinLH cell deletion are milder than the hypo- A century ago, it was noted that damage to the lateral hypotha- activity phenotypes caused by broader LH lesions (1–3, 5, 12, 13, – lamic (LH) brain area caused people to be motionless most of the 17 19), whereas melanin-concentrating hormone LH (MCHLH)cell time. Therefore, it is thought that this brain area emits essential deletion causes hyperactivity, implying that MCHLH cells suppress signals for promoting physical activity. However, it remained a locomotion (20). These findings suggest that additional drivers mystery what these signals are. Using newly developed genetic of physical activity may exist in the LH. and deep-brain recording methods, we found that, unexpectedly, a It was recently found that LH neurons expressing glutamic acid critical signal for movement comes from a subset of LH cells that decarboxylase 65 (GAD65) are distinct from orexin and MCH are molecularly distinct from neurons previously implicated in lo- neurons (21). We hypothesized that these cells may be a source of comotion control. These movement-promoting cells were switched natural LH signals underlying normal levels of physical activity. on by the local peptide orexin, a key signal of stress and hunger. Here, we investigate this hypothesis by using a combination of cell These findings shed new light on deep-brain signals that maintain type-specific manipulation and recording techniques. We find that healthy levels of physical activity. NEUROSCIENCE GAD65LH cells operate as a stress- and orexin-activated LH module whose physiological activity is essential for normal loco- Author contributions: C.K. and D.B. designed research; C.K., C.S., and E.B. performed motion, and whose hyperactivity causes hyperlocomotion. research; C.K. and D.B. analyzed data; and D.B. wrote the paper. The authors declare no conflict of interest. Results This article is a PNAS Direct Submission. Orexin and Stress Rapidly Recruit GAD65LH Neurons. To examine Freely available online through the PNAS open access option. whether orexin cells communicate with GAD65LH cells, we tar- 1To whom correspondence should be addressed. Email: [email protected]. geted the light-activated excitatory actuator channelrhodpsin-2 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (ChR2) to orexin cells, enabling selective optical activation of 1073/pnas.1619700114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1619700114 PNAS | April 25, 2017 | vol. 114 | no. 17 | 4525–4530 Downloaded by guest on September 30, 2021 Cre-inducible Orexin-ChR2-mCherry Orexin-ChR2- h A mCherry ChR2-mCherry 400 m GAD65-GFP L 3V GAD65-GFP LH CNQX DMH D-AP5 LH control VMH Orexin-Cre x GAD65-GFP 20 pA = -90 mV 50 m Vhold 10 ms orexin B Cre-inducible 950 1 0.0439 GCaMP6s cell number 200 < 0.0001 100 LH < 0.0001 -50 44 50 GAD65-Ires-Cre 40 m 0 5 min C B orexin baseline Orexin evoked current (pA) C syn.blockers membrane potential (mV) 150 orexin 20 -100 -60 100 0 50 -20 30 mV -40 1 min 0 -50 -60 membrane current (pA) -80 -60 -40 -20 -80 20 mV 1 s membrane potential (mV) Fig. 1. Evidence for orexinLH → GAD65LH excitatory circuit. (A) Optogenetic interrogation of orexinLH → GAD65LH signaling. (Left)Targetingstrategy.(Center, Left and Right) Localization of orexin-ChR2-mCherry and GAD65-GFP expression (representative example of five brains). (Right) Orexin cell photostimulation (vertical blue line) evokes a CNQX/D-AP5–sensitive (i.e., glutamate receptor-mediated) inward current in a GAD65LH cell. Gray lines are individual trials, and colored lines are trial averages [representative example of five cells; ≈80% cells (n = 5/6 cells) were connected]. (B) Effects of orexin peptide on GAD65LH network activity. Scheme for targeting GCaMP6s (Left), example of GCaMP6s expression in GAD65LH cells (representative example of five brains) (Center Left), GCaMP6s response of GAD65LH cells to 300 nM orexin-A with corresponding mean ± SEM plot (n = 44) (Center Right), and data summary without (C) and with (B)synaptic blockers (n = 44 and n = 13 cells, respectively, mean ± SEM responses of cells during 2–20 min after orexin-A infusion) (Right). P values (italics) are from sign tests of whether the response within each group is different from zero, and P value (regular font) is from a two-tailed Mann–Whitney test comparing response amplitude between groups. (C) Effects of orexin peptide on GAD65LH cell electrical activity. (Left) Membrane potential effect of 300 nM orexin-A on a GAD65LH cell (representative example of six cells). syn., synaptic. (Center) Example of whole-cell current–voltage relation showing effect of orexin-A (representative example of five cells). (Right) Net orexin-activated current (mean and SEM of n = 5 cells). other deep networks, and are thought to represent