(A) Schematic illustrating the bilateral stereotaxic injection of either AAV8-GFAP-Cre-GFP or GFAP-eGFP virus into the NAc of Bmal1 floxed mutant mice; injection at AP: +1.5, ML: ± 1.5, and DV: −4.4 microns resulted in a region-specific expression of the virus in the NAc. (B) GFAP-Cre-GFP virus resulted in a NAc-specific loss of BMAL1 detection by immunofluorescence (IF), specifically in GFAP+ cells (i.e., astrocytes). Arrows indicate cells with co-expression. (C) In the locomotor motor response to novelty (LRN) task, mice with a loss of BMAL1 function in NAc astrocytes (GFAP-Cre) show a significantly greater LRN relative to control mice during the day (Virus: F(1, 36) = 5.68, * p=0.02; Time: F(11, 396) = 40.41, p<0.0001; Time × Virus: F(11, 396) = 0.92, p=0.51; inset, t(35)=2.32, * p=0.02). (D) This increase in LRN is not driven by differences in habituation or overall locomotor activity across the full task (inset) Virus: F(1, 36) = 0.0003, n.s. p=0.98; Time: F(11, 396) = 16.66, p<0.0001; Time × Virus:
F(11, 396) = 0.26, p=0.99; inset, t(35) = 0.46, p=0.64. (E) However, during the night, mice show no significant differences in locomotor response to novelty (Virus: F(1, 22) = 1.017, p=0.32; Time: F(11, 242) = 44.68, p<0.0001; Time × Virus: F(11, 242) = 0.62, p=0.8), (F) habituation (Virus: F(1, 22) = 2.19, p=0.15; Time: F(11, 242) = 3.28, p=0.0003; Time × Virus: F(11, 242) = 1.91, p=0.03), or (G) overall locomotor differences (top: t(21)=0.58, p=0.56; bottom: t(21)=0.91, p=0.36). White background indicates behavior run during the day (ZT 2–6). Blue background indicates behavior run during the night (ZT 14–18). Mean ± SEM; n=12–22; * p<0.05, n.s. = not significant.