Optogenetic and pharmacogenetic probing of rapid eye movement (REM) sleep circuitry

Abstract

Introduction Rapid eye movement (REM) sleep is characterized by the activation of cortical electroencephalogram (EEG) and loss of muscle tone (atonia). The exact neuronal circuit mediating the generation and timing of this state is not fully understood. The subcoeruleus (Sub-C) neurons are hypothesized to generate REM sleep and its characteristics. Here we aimed to determine how optogenetics and pharmacogenetic stimulation impacts REM sleep expression. Materials and methods Study#1: To precisely control the neuronal activity of the Sub-C region, we bilaterally infused 200 nL of an adeno-associated viral vector (AAV) containing a light-sensitive opsin (AAV-hsyn- hChR2(H134R)-eYFP) virus into the Sub-C of 4 mice. Animals were instrumented for EEG and EMG recordings. Neurons were stimulated with short blue light pulses (5 ms) at 1 and 10 Hz either independently of behavioral state or specifically during REM sleep. Study#2: To stimulate the Sub-C population for longer time periods, we bilaterally microinjected 400 nL of an AAV harboring a modified muscarinic G-protein coupled receptor (AAV-HSYN-HA-hm3D(Gq)- IRES-mCitrine) into the Sub-C of 3 mice. Administration of clozapine-N-oxide (CNO, 5 mg/kg) activated neurons in the Sub-C. Only animals that had histological verification of ChR2 and hm3D(Gq) receptor expression in the Sub-C region were used for analysis. Results We found that semi-chronic bilateral light activation of Sub-C neurons at 10 Hz, but not 1 Hz, triggered REM sleep-like EEG activity (theta, 4–8 Hz) during light stimulation, and increased EEG Theta power by 64 ± 16% compared to baseline. Under some conditions, light stimulation prolonged the duration of REM sleep episodes by almost 2-fold. Pharmacogenetic manipulation of the neurons in the Sub-C caused a change in behavioural phenotype where the distribution of EEG frequencies shifted towards a REM sleep-like pattern (theta, 4–8 Hz) independent of the behavioural state. Moreover, the average duration of REM sleep-like periods were greater than REM sleep periods under control condition (i.e., saline). Conclusion These results support the hypothesis that the Sub-C region is involved in controlling REM sleep and its associated phenomena. Acknowledgements This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) , the Canadian Institutes of Health Research (CIHR) and the CIHR Sleep and Biological Rhythms Toronto .