Figure 2.

Pregnenolone and TSPO oscillations are dependent on a functional circadian clock in mouse brains. (A) P5 concentration was quantified in brain homogenates from non-fasted wild-type mice kept in constant darkness using a direct pregnenolone ELISA test. Circadian times from CT0 to CT24 are shown. All data are normalized to 1 and presented as mean ± SEM (one experiment, seven time points, n = 3–4 for each time point). (B) Variations in P5 concentration are abolished in PER1/PER2 (P1P2) knock-out (P1P2 KO) mice. P5 levels at peak and trough corresponding to CT4 and CT16 from brain homogenates of wild-type (WT) mice compared to P1P2 KO mice. All data are normalized to 1 and presented as mean ± SEM (WT and P1P2 KO: one experiment, two time points, n_WT = 8 and n_{P1P2 KO} = 3–4 for each time point). *** p < 0.001 (unpaired Student’s t-test). (C) TSPO protein levels were analyzed in brain homogenates from non-fasted wild-type mice kept in constant darkness by immunoblotting. Circadian times from CT0 to CT24 are shown. All data are normalized to 1 and presented as mean ± SEM (one experiment, seven time points, n = 2–3 for each time point). (D) Variations in TSPO protein levels are abolished in PER1/PER2 (P1P2) knock-out (P1P2 KO) mice. TSPO levels at peak and trough corresponding to CT4 and CT16 from brain homogenates of WT mice compared to P1P2 KO mice. All data are normalized to 1 and presented as mean ± SEM (WT and P1P2 KO: one experiment, two time points, n_WT = 8 and n_{P1P2 KO} = 3–4 for each time point). *** p < 0.001 (unpaired Student’s t-test). (E,F) Representative immunoblots of brain lysates from non-fasted wild-type mice kept in constant darkness stained with antibodies specific for TSPO and VDAC (control). (E) Circadian times from CT0 to CT24 are shown for wild-type animals (WT). (F) Circadian times CT4 and CT 16 (corresponding to peak and trough) are shown for WT and PER1/PER2 knock-out (P1P2 KO) animals.