Sleep State Dependence of Optogenetically evoked Responses in Neuronal Nitric Oxide Synthase-positive Cells of the Cerebral Cortex

Slow-wave activity (SWA) in the electroencephalogram during slow-wave sleep (SWS) varies as a function of sleep-wake history. A putative sleep-active population of neuronal nitric oxide synthase (nNOS)-containing interneurons in the cerebral cortex, defined as such by the expression of Fos in animals euthanized after protracted deep sleep, may be a local regulator of SWA. We investigated whether electrophysiological responses to activation of these cells are consistent with their role of a local regulator of SWA. Using a Cre/loxP strategy, we targeted the population of nNOS interneurons to express the light-activated cation channel Channelrhodopsin2 and the histological marker tdTomato in mice. We then performed histochemical and optogenetic studies in these transgenic mice. Our studies provided histochemical evidence of transgene expression and electrophysiological evidence that the cerebral cortex was responsive to optogenetic manipulation of these cells in both anesthetized and behaving mice. Optogenetic stimulation of the cerebral cortex of animals expressing Channelrhodopsin2 in nNOS interneurons triggered an acute positive deflection of the local field potential that was followed by protracted oscillatory events only during quiet wake and slow wave sleep. The response during wake was maximal when the electroencephalogram (EEG) was in a negative polarization state and abolished when the EEG was in a positive polarization state. Since the polarization state of the EEG is a manifestation of slow-wave oscillations in the activity of underlying pyramidal neurons between the depolarized (LFP negative) and hyperpolarized (LFP positive) states, these data indicate that sleep-active cortical neurons expressing nNOS function in sleep slow-wave physiology.