Figure 4: Administration of CNO activates GABAergic BF hM4Di⁺ neurons, and promoted slow-wave sleep (SWS) and an increase in slow EEG power density.

(a) Experimental design: bilateral injections of DIO-hM4Di-AAV were placed into the BF of Vgat-IRES-Cre mice, resulting in expression of hM4Di on BF GABAergic neurons. (b,c) hM4Di-expressing BF GABAergic neurons (left) visualized under IR-DIC (right) during whole-cell recordings (scale bar, 20 μm) showed a strong membrane hyperpolarization and reduction in firing to bath application of CNO (500 nM; c). (d) coronal section from injected Vgat-IRES-Cre mouse showing hM4Di-expressing BF GABAergic (mCherry⁺) neurons (scale bar, 1 mm). (e) Heat map generated from seven injection cases in the Vgat-IRES-Cre mouse; white colour=area of maximum overlap of hM4Dq-AAV transduction/expression across seven injection cases. (f–h) Hourly sleep–wake amounts (±s.e.m.) following injection of CNO (0.3 mg kg⁻¹, IP, ZT12=1900 hours, n=7 mice) or vehicle; inset in f shows ∼30% decrease in wake during the 3-h post CNO injection period. (i–k) Power spectrum changes (±s.e.m.) over baseline during the 3-h post-injection period for vehicle injection as compared with the 3-h post-injection period for CNO (0.3 mg kg⁻¹, IP, ZT3=1900 hours, n=5 mice) administration and the quantitative changes (±s.e.m.) in power for the δ (0.5–3 Hz for W and 0.5–5 Hz for SWS and REM sleep), θ (3–9 or 5–9 Hz), α (9–15 Hz), β (15–30 Hz), low γ (30–60 Hz) and high γ (60–120 Hz) frequency bands following vehicle or CNO administrations. (l–n) EEG/EMG examples of wake (W), SWS and REM sleep (RS) from the first hour post injection of saline (top) or CNO (bottom) in a mouse with bilateral hM4Di receptor expression in BF GABAergic neurons. The raw EEG and EMG traces following CNO injection provide unambiguous evidence of the existence of the three sleep–wake stages while BF GABAergic neurons are inhibited. (o,p) Example compressed spectral array (CSA; 0–30 Hz and 0–150 Hz), EMG activity and hypnogram over 12 h following vehicle (o) or CNO (0.3 mg kg⁻¹, IP; ZT12; (p)) administration in a mouse with bilateral hM4Di receptor expression in BF GABAergic neurons. A black bar (4–6 bins=1–1.5 Hz large) was inserted to mask the 60 Hz contamination of the EEG recordings. AC, anterior commissure; CPu, caudate putamen; MPO, medial preoptic area; SI, substantia innominata; ox, optic chiasm. A two-way analysis of variance using the between-subjects factor of injection (vehicle or CNO) and the within-subjects factors of time of day or frequency band was used to analyse the percentage(s) of time spent in W, SWS and REM sleep or the frequency bands during W and SWS; *P<0.05, **P<0.01 and ***P<0.001; P≥0.05=not significant (NS).