Fig. 1.

Translational switching of Cry1 expression controls molecular circadian rhythms in organotypic Cry-null SCN slices. (A) Schematic views of the AAV constructs used to deliver the orthogonal aminoacyl tRNA synthetase/tRNA pair and the target Cry1(_177TAG)::EGFP. (B) Phase (Left) and fluorescence (Right) views of control (Upper) and AAV-transduced (Lower) SCN slices to show activity of the mCherry reporter. (Scale bar: 250 μm.) (C) Representative photomicrographs of AAV-transduced SCN slices taken before (−AlkK) and 34 h after treatment with 1 mM AlkK (+AlkK). The yellow signal in the AlkK-treated slice reveals the colocalization of the mCherry and Cry1::EGFP signals. (Scale bar: 100 μm.) (D) Representative plot of Per2::Luc bioluminescence from a Cry1,2-null SCN slice cotransduced with AAVs as in A and then treated with two sequences of AlkK addition followed by washout (w/o) with fresh medium. (E) Detrended plot of data in D. (F) FFT-determined periods, amplitude, RAE, and GOF of bioluminescence rhythms of SCN slices before and during the addition of AlkK. *P < 0.05, **P < 0.01, ***P < 0.001, paired t test. (G) As in F, for SCN slices during and after withdrawal of AlkK. *P < 0.05, **P < 0.01 paired t tests. (H) Dose–response curve showing the increase in period with increasing concentration of AlkK. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0.1 mM AlkK, one-way ANOVA.