Poster Session 2 - B2
1Jasmin A. Aggarwal, 2Hattie Liu, 2,3Gaspard Montandon, 1,2Richard L. Horner
1 Dept. of Physiology, University of Toronto; 2 Dept. of Medicine, University of Toronto; 3 Keenan Research Centre for Biomedical Science, St. Michael’s Hospital, Toronto, Ontario, Canada
SIGNIFICANCE STATEMENT: Motoneurons are the brain’s final common output pathway controlling motor behavior. Measuring and modulating the excitability of a central motoneuron pool in-vivo to identify mechanisms of control is, however, difficult and studies are few. Here we apply ‘optogenetics’ to optically control genetically targeted motoneurons and determine their net excitability from measures of motor output from the electromyogram. We apply this tool to a motor circuit that is critical to the pathogenesis of a major clinical problem. RATIONALE AND FOCIUS OF STUDY: Obstructive sleep apnea (OSA) is a common and serious clinical problem affecting up to 25% of adults. OSA is caused by closure of the air passage in the throat in sleep due to relaxation of the tongue muscles whose activity normally keeps the airway open. The hypoglossal motor nucleus is the source of motor output to the tongue, and strategies to measure and modulate its activity are needed to identify, develop and test new pharmacological treatments for OSA. HYPOTHESES: (1) The endogenous excitability of the hypoglossal motoneuron pool can be measured in-vivo using an optogenetic protocol. (2) Excitability can be measured during general anesthesia and the behavioral states of wakefulness, non-rapid eye movement (non-REM) and REM sleep. METHODS: Two studies were performed on genetically modified mice expressing a light sensitive cation channel exclusively on cholinergic neurons (ChAT-ChR2(H134R)-eYFP). Mice were obtained from the Jackson Laboratory and have been described and validated (Zhao et al., Nat Methods, 8: 745-52, 2011). Study 1: Mice were anesthetized with isoflurane and instrumented with electrodes to record tongue and diaphragm muscle activities. Optical fibres were placed above the hypoglossal motor pool (mean=0.60mm, SEM=0.12mm). Blue light pulses (λ=473nm, 2s duration) were applied in one of two protocols: (a) Frequency Protocol (n=8 mice): 0, 5, 10, 15, 20 and 25Hz stimuli (20ms pulse durations at 20mW as measured from the fiber tip) were applied in random order in blocks of 7; (b) Power Protocol (n=7 mice): 0, 5, 10, 15, and 20mW stimuli (as measured from the laser tip, 10ms pulse durations, 10Hz) were also applied in random order in blocks of 7. Study 2: Mice are chronically implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and tongue and diaphragm muscle activities. Optical fibres are also placed above the hypoglossal motor pool. At least two weeks after recovery, the optical stimuli are applied at 0, 5, 10, 15, or 20mW in random order across states of wakefulness, non-REM and REM sleep. RESULTS: Histology: Histological sections (40μm, n=3 from 1 mouse) showed that 100% of channelrhodopsin (ChR2) expressing cells (n=93) were cholinergic, as identified by GFP and ChAT immunohistochemistry. Greater than 99% of ChR2-expressing cells (n=434 cells, n=3 sections from 3 mice) also expressed c-fos, suggesting their neural activation during the experiments. Study 1: Each individual pulse of the optical stimulus trains was associated with increased motor output, identified from the electromyogram as compound action potentials. (a) Frequency Protocol: The magnitude of the motor output for the first and last pulse of each stimulus train did not vary significantly with frequency of stimulation (P = 0.06 and 0.982 respectively). (b) Power Protocol: In contrast, motor output increased as a function of power for the first and last pulse of each stimulus train (both P<0.001), with increased responses at 10-20mW compared to 5mW (each P<0.002, post-hoc test). Stimulations also spanned the endogenous rhythmic bursts of motor activity that occurred with each inspiratory drive to this respiratory motoneuron pool. The stimulations that occurred during an inspiratory drive led to an increased peak magnitude of the burst activity as a function of both frequency and power compared to the inspiratory drives before and after stimulation for each of 5-20mW (range of P<0.001 to <0.05, post hoc paired t-tests vs. 0mW) and 25Hz (P<0.001 vs. 0Hz). Study 2: Stimulations across the behavioral states of wakefulness, non-REM and REM sleep are ongoing. DISCUSSION AND SIGNIFICANCE: This study identifies that an optogenetic protocol can be used for the measurement and modulation of the excitability of a central motoneuron pool in-vivo. The hypoglossal motoneuron pool is critical to the pathogenesis of OSA, a common and serious clinical problem. Further studies performed in wakefulness and sleep will identify and test new pharmacological strategies to increase motor activity as future treatments for OSA. Our previous discoveries have led others to manipulate those mechanisms as potential OSA pharmacotherapy (e.g., trial IDs: NCT02428478, NCT02656160, NCT02908529 and ACTRN12614000364673). We will further drive new treatment strategies from the fundamental mechanisms identified by this research.