Abstract
To determine how a waking brain falls asleep researchers have monitored and manipulated activity of neurons and glia in various brain regions. While imaging GABA neurons in the zona incerta (ZI) we found a subgroup that anticipates onset of NREM sleep 1. To differentiate the GABA subtype we now image and optogenetically manipulate the ZI neurons containing the transcription factor, Lhx6. In the first study Lhx6-cre mice (n=5; female=4) were given rAAV-DJ-EF1a-DIO-GCaMP6M into the ZI (isofluorane anesthesia), a GRIN lens implanted, and 21d later sleep and fluorescence in individual Lhx6 neurons were recorded for 4h. Calcium fluorescence was detected in 132 neurons. 45.5% of the Lhx6 neurons were REM-max; 30.3% were Wake-max; 11.4% were wake+REM max; 9% were NREM-max; and 3.8% had no change. The NREM-max group of neurons fluoresced 30s ahead of sleep onset. The second study tested the effects of unilateral optogenetic stimulation of the ZI Lhx6 neurons (n=14 mice) (AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato]. Stimulation at 1 and 5Hz (1 minute on- 4 minute off) significantly increased percent REM sleep during the 4h stimulation period (last half of day cycle). The typical experimental approach is to stimulate neurons in both hemispheres, but here we found that low frequency stimulation of ZI Lhx6 neurons in one hemisphere is sufficient to shift states of consciousness. Detailed mapping combined with mechanistic testing is necessary to identify local nodes that can shift the brain between wake-sleep states.