0126 Zona incerta Lhx6 neurons are most active during NREM and REM sleep and after prolonged waking. (25th May 2022) - Blanco-Centurion, Carlos
Abstract: Introduction: Emerging data shows that GABA neurons in the zona incerta (ZI) play a prominent role in regulating sleep. Transfer of the orexin gene into ZI neurons blocks cataplexy in narcoleptic orexin-knockout mice (Liu et al., JNeurosci, 2011) and vGAT GABA neurons in the ZI anticipate onset of NREM (Blanco-Centurion et al., SLEEP, 2021). To identify the subtype of GABA neurons regulating sleep, we examined the activity of Lhx6 neurons. This study tests the hypothesis that Lhx6 neurons show peak calcium fluorescence during sleep, and that the fluorescence is further increased after prolonged waking. Methods: In Lhx6-cre mice (mice=4; all females; 4-5 mo), rAAV-DIO-GCaMP6s was delivered stereotaxically to the ZI (isofluorane anesthesia) and a GRIN lens, along with EEG and EMG electrodes were implanted. 21d later a miniscope (INSCOPIX) was attached, and after 3d of adaptation, sleep and fluorescence in individual Lhx6 neurons were recorded for 4h (baseline). On another day, the mice were kept awake for 6h (gentle handling; 9a-3p) and fluorescence in Lhx6 neurons was recorded for 2h during recovery sleep. The imaged data from the two recording periods (baseline and recovery sleep) was combined into a single data file and the change in fluorescence was determined against the mean image frame (F0). Previously, we and others found that the GCamP calcium fluorescence is a direct measure of action potentials and serves as a marker of activity. Results: 97 neurons were automatically extracted (PCA-ICA analysis; blinded without knowledge of sleep state). In 66 neurons (68%) the average fluorescence was significantly higher during REM, NREM or both, compared to waking (Mixed Model ANOVA; SPSS25; P<0.01). In this population the fluorescence was significantly higher during recovery sleep compared to baseline (P<0.001) indicating increased activity of the sleep-active neurons during recovery sleep. With ensuing sleep, the increase in fluorescence gradually returned to baseline levels, attesting to the fluorescence as a marker of homeostatic sleep pressure. In 14 neurons (14%), fluorescence was highest in waking as compared to the other states during baseline, and in these neurons, fluorescence did not increase after sleep loss. Interestingly, six neurons (6%) were most active in waking and NREM but silent in REM (REM-off). Conclusion: This is the first study to measure fluorescence in individual neurons after sleep loss. We find that the fluorescence in two-thirds of ZI Lhx6 neurons is tightly linked to sleep, and that the average fluorescence is further increased after prolonged waking. Microendoscopy is superior to indirect measures such as c-FOS or photometry in gauging sleep pressure in individual neurons. Support (If Any): VA BX000798, 1K6BX004216.