Diminished REV-ERBα levels in the TMN of HDC-ΔBmal1 mice ( Figure S1G) might derepress the hdc gene. SCN neurons show cell-intrinsic circadian regulation of their electrophysiological parameters, partly determining when these neurons fire [30, 31 and 32]. We made whole-cell current-clamp recordings PI3K inhibition of histaminergic neurons from littermate and HDC-ΔBmal1 mice during night and day ( Figure S3C). Resting membrane potential, input conductance, current injection to threshold of action potential firing, capacitance, and membrane time constant were unaffected by time of day or the absence

of BMAL1 ( Figure S3C). We expect that HDC-ΔBmal1 histaminergic neurons will fire action potentials normally but release more histamine. HDC-ΔBmal1

knockout mice had an unchanged behavioral circadian rhythm and phase, compared with littermate controls, as assessed by wheel running in free-running conditions of constant darkness (DD) (unpaired two-tailed t test, p > 0.05) ( Figures 2A and 2B) [ 25]. In free-running constant light (LL), both genotypes were more variable in period length than in LD or DD ( Figure 2A). However, the amplitude of the peak period was lower and more variable in LL than in LD and DD, indicating the mice were equally less active in LL than in LD or DD, regardless of genotype ( Figures 2A and 2B). Within the SCN, the circadian variation in BMAL1 and PER2 proteins was unchanged between HDC-ΔBmal1 knockout mice and littermate controls ( Figures 2C and 2D); there was little variation in BMAL1 staining intensity in the SCN between ZT6 and ZT18 ( Figure 2C), highlighting RAD001 molecular weight that although BMAL1 is the core component of the clock, its levels change little during the circadian cycle. CLOCK and BMAL1 are often constitutively bound to E boxes. The

critical rhythm for BMAL1-CLOCK activity arises from PER-CRY, which arrives PRKACG to inhibit, and then dissociates from, the BMAL1-CLOCK complex [ 33]. PER2 staining in the SCN of both groups of mice increased at ZT18 compared with ZT6 ( Figure 2D). Thus, the HDC-ΔBmal mice had an unaffected SCN molecular clock and circadian pace making. Mice unable to synthesize histamine (HDC knockouts) show normal sleep-wake behavior throughout most of the 24 hr cycle, except they are significantly less awake just before, and for the first few hours after, the start of the night [ 10]. It is intriguing that HDC knockout mice have a selective deficit in anticipating lights off, further suggesting a circadian involvement of histaminergic neurons. In contrast to HDC knockout mice, the HDC-ΔBmal1 mice have a gain of function in the histaminergic system. We looked at the consequences for the sleep-wake cycle ( Figure 3; Figure S4). Sleep experiments and nontethered electroencephalogram (EEG) analysis were performed using Neurologger2 devices [ 22 and 34].