Wake-promoting effects of glutamatergic pedunculopontine tegmental neurons
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The pedunculopontine tegmental (PPT) nucleus is a brainstem structure thought to be important in the regulation of sleep/wake states. The PPT is comprised of three distinct types of neurons (cholinergic, GABAergic, and glutamatergic), and each may serve different functions. It remains unknown how PPT neurons affect specific sleep/wake states and which of their axonal projections mediate their effect. Therefore, we used optogenetics to selectively activate glutamatergic PPT (PPTglut) neurons at both the cell soma and axon terminals in a temporally and spatially precise manner. The purpose of these experiments was to determine the role of PPTglut neurons during wake, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep, and to identify the key projections through which PPTglut neurons produce their effects. Using transgenic mice, we transfected PPTglut neurons with an adeno-associated viral vector to induce expression of a light-dependent ion channel, channelrhodopsin-2 (ChR2). While recording electroencephalography (EEG), electromyography (EMG), and video feed, we photostimulated the transfected PPTglut neurons and measured the effects on sleep/wake states of the mice. Stimulation of the PPTglut soma during NREM sleep produced a frequency-dependent wake response. With increasing frequency of stimulation, we observed an increase in the speed of the wake response, as well as the amplitude and duration of the wake response. Stimulating the PPTglut soma increased time spent in wake, decreased NREM sleep, and slightly decreased REM sleep. We also noticed that mice did not exhibit spontaneous body movements during stimulation of the PPTglut soma. Stimulating individual PPTglut axon terminal fields partially recapitulated the phenotype observed with stimulation of the cell soma. Photostimulation of axon terminals in the basal forebrain, lateral hypothalamus, and thalamus elicited a fast wake response, stimulation of both the basal forebrain and lateral hypothalamus terminal fields produced a strong wake response, but long-lasting wakefulness was observed only with high-frequency stimulation of axon terminals in the lateral hypothalamus. In summary, photostimulation of PPTglut neurons promotes wake, and slightly decreases REM sleep. Our experiments strongly support the role of PPTglut neurons in promoting wakefulness from NREM sleep, and this wake response is carried out through several axonal projections which, in sum, recapitulate the wake phenotype observed with stimulation of the cell soma in the PPT itself. Further exploration of the axonal projections of PPTglut neurons is warranted to elucidate the neuronal targets through which this response is carried out.