Melanin-concentrating hormone neurons specifically promote rapid eye movement sleep in mice

Abstract

Currently available evidence indicates that neurons containing melanin-concentrating hormone (MCH) in the lateral hypothalamus are critical modulators of sleep-wakefulness, but their precise role in this function is not clear. Studies employing optogenetic stimulation of MCH neurons have yielded inconsistent results, presumably due to differences in the optogenetic stimulation protocols, which do not approximate normal patterns of cell firing. In order to resolve this discrepancy, we (1) selectively activated the MCH neurons using a chemogenetic approach (Cre-dependent hM3Dq expression) and (2) selectively destroyed MCH neurons using a genetically targeted diphtheria toxin deletion method, and studied the changes in sleep-wake in mice. Our results indicate that selective activation of MCH neurons causes specific increases in rapid eye movement (REM) sleep without altering wake or non-REM (NREM) sleep. On the other hand, selective deletions of MCH neurons altered the diurnal rhythm of wake and REM sleep without altering their total amounts. These results indicate that activation of MCH neurons primarily drives REM sleep and their presence may be necessary for normal expression of diurnal variation of REM sleep and wake.

Keywords: chemogenetics; conditional deletion; feeding; lateral hypothalamus; paradoxical sleep; sleep–wake.

Figure 1. Specific expression of AAV-hM3Dq-mCherry in MCH neurons in MCH-Cre mice and their activation by CNO

A. Photomicrograph of a brain section labeled for mCherry (red cells) and MCH (green cells) by immunofluorescence from a mouse injected with AAV-hM3Dq into the lateral hypothalamus indicating DREADD expression in the MCH neurons (white arrows indicate doubly labeled neurons). The percentage of MCH neurons transfected ranged from 21–60%. B- Representative whole cell, current clamp recording from a MCH neuron (identified by mCherry fluorescence) indicating bath application of CNO (500 nM) induced depolarization and increased the firing rate of these neurons. Intraperitoneal (IP) injections of CNO induced cFos expression (black nuclei) in DREADD-expressing neurons (brown cytoplasm) in the lateral hypothalamus (C). Double labelling of sections for MCH (brown) and cFos (black) indicated cFos expression in MCH neurons after IP CNO (black arrows in D). Brains were obtained for cFos analysis 180 minutes after IP CNO (0.3 mg/kg of body weight). Activation of MCH neurons by IP CNO caused specific increases in REM sleep in mice. REM sleep amounts (in minutes) during the first two 4 hr periods following IP saline or CNO injections either during the light (E; n=11) or dark period (F; n=9) in MCH-Cre mice injected with AAV-hM3Dq into the LH. Data are mean ± SEM. **P<0.01; ***P<0.001; paired t test.

Figure 2. Changes in sleep-wake following specific deletion of MCH neurons

Photomicrographs from brain sections double-labelled for MCH (black) and orexin (Brown) from a WT mouse (A, A′) and a MCH-Cre/+;iDTR mouse (B, B′). Injections of diphtheria toxin resulted in complete elimination of MCH neurons only in the MCH-Cre/+; iDTR mouse (B, B′) but not in WT mice (A, A′). Orexin neurons remained intact in both groups of mice ruling out any non-specific cell loss (A, B). Percentages of Wake, non-REM and REM sleep during dark (D) light (E) or the entire 24-hr period (C) in MCH-Cre/+;iDTR mice (n=9) before (white bars) and at 3 weeks after (grey bars) the diphtheria toxin injections. Circadian index (amount of behavior in light phase minus dark phase, over total amount of behavior) of sleep-wake stages expressed as percentage of control mice are shown in F.

Figure 3. Metabolic changes following MCH neuronal deletions

Food intake and body weight gain in MCH-Cre/+;iDTR mice (n=9) before (white bar in A and white square in B) and 1, 2 and 3 weeks after diphtheria toxin (DT) injections (grey bars in A and grey squares in B), showing dramatic reduction following deletion of MCH neurons. Total locomotor activity (LMA; C) and mean body temperature (Tb; D) during the light and dark phases in MCH-Cre/+;iDTR mice before (white bars) and 3 weeks (grey bars) after DT injections show the increase in both LMA and Tb during the dark phase after deletion of MCH neurons.