Parvalbumin-positive neurons in the basal forebrain projecting to thalamic reticular nucleus modulate sleep and wakefulness

Abstract

Introduction: The parvalbumin (PV) neurons in the basal forebrain (BF) play pivotal roles in controlling sleep and wakefulness. Optogenetic stimulation of BF PV neurons in mice regulates the gamma band oscillations in the cortex via their cortical projection and also increases wakefulness. Interestingly, BF PV neurons have dense projections to the thalamic reticular nucleus (TRN), of which intermittent stimulation at 8 Hz generates sleep spindles and protects NREM sleep. However, the function of this anatomical connection in sleep-wake control is still unclear. Therefore, I hypothesized that the projections of BF PV neurons toward TRN (PVBF→TRN) might modulate sleep and wakefulness and sought to investigate their roles by projection-specific optogenetic stimulation. Method: First, I bilaterally injected viral vector (AAV5-DIO-ChR2-eYFP) into the BF region of PV-Cre transgenic mice. After two weeks, electrodes for electroencephalography (EEG) were implanted on the frontal and parietal areas of the skull and fiber optic cannula into the BF or TRN. The spontaneous 24-hour EEG was recorded on baseline day, and I performed the optogenetic stimulation at 8 Hz and 40 Hz on separate days with 6-hour EEG recordings including 2-hour pre-, during-, and post-stimulation periods. EEG was analyzed for the duration, bouts, and power spectrum and sleep spindles. To validate the transduction and optogenetic stimulation, I completed immunohistochemistry against parvalbumin and c-fos; and to confirm the afferent and efferent projections of the BF, histological analysis using antero- and retrograde tracing with different serotypes of AAV viral vectors were performed. Results: I confirmed that the bilateral stimulation showed significantly higher evoked EEG power at gamma frequency than unilateral stimulations, e.i., both ipsilateral and contralateral stimulation. The two-hour intermittent optogenetic stimulation on BF PV neurons at 8 Hz during dark period increased NREM duration and NREM bout counts, whereas no significant changes were observed during light period. Sleep spindle density was not changed. To stimulate a branch of projection to TRN specifically, the optogenetic stimulation of axon terminals of PV(BF→TRN) neurons in TRN at 8 Hz showed various responses depending on the timing of stimulation. Slow wave activity was increased at early and mid-dark period, but the slow wave energy was increased during early dark and mid-light period. The number of NREM bouts increased during late dark period. However, sleep spindle density was not significantly affected by the stimulation. Conclusion: I found that driving BF PV neurons at 8Hz intermittently could modulate NREM sleep including continuity of NREM sleep, SWA, and SWE. A novel role of PV(BF→TRN) neurons in modulating NREM sleep may be associated with increasing SWA or SWE rather than changing sleep spindles. These findings suggest that PV(BF→TRN) subpopulation among BF PV neurons could operate as sleep-promoting neurons in addition to their well-known wake-promoting function, depending on their firing frequency and pattern. Further investigation about the regulation of BF PV neurons in different vigilant status is warranted.