Fig. 5. AVP in the graft SCN is responsible for the rescue of circadian PER2::LUC rhythms in the adult Cry1,2^−/− and Cry1,2^−/−/Vipr2^−/− SCN.

(A and B) Representative circadian PER2::LUC rhythms rescued in the arrhythmic adult SCN of Cry1,2^−/− (A) and Cry1,2^−/−/Vipr2^−/− (B) mice by coculture of a neonatal WT SCN before and after treatment of AVP receptor (V1a and V1b) antagonists (blue and red) or vehicle (black). An arrow indicates the day of coculture, and a horizontal magenta line indicates the days under treatment. Coculture was done without exchanging culture medium, whereas the drug treatment was done by exchanging into a new medium containing drug or vehicle. The histogram shown on the right side demonstrates the mean ratios of circadian amplitude on the third day after drug treatment to that immediately before the treatment (Cry1,2^−/−, n = 6; Cry1,2^−/−/Vipr2^−/−, n = 4; each for vehicle and antagonists). **P < 0.01, Student’s t test, significant difference between antagonists and vehicle treatments. (C and D) Representative period maps and period distributions of the rescued circadian PER2::LUC rhythm (left), after AVP antagonist treatment (middle) and washout of the antagonists (right) in the adult SCN of Cry1,2^−/− (C) and Cry1,2^−/−/Vipr2^−/− (D) mice by coculture with a neonatal WT SCN on the pixel level. Superimposed curves in the period distribution indicate three Gaussian curves fitted to the data. Scale bars, 200 μm. (E) Schematic drawing of the coculture of the SCN slices and representative circadian PER2::LUC rhythm of the adult SCN slice of Cry1,2^−/−/Vipr2^−/− mice (orange) cocultured with the SCN from the heterozygote (top) and homozygote (bottom) of Avp knockout mice (green). Recipient adult SCN exhibits PER2::LUC bioluminescence (orange) and graft neonatal (P4 + 3 days culture) SCN exhibits Avp-ELuc bioluminescence (green), which were calculated after alternatively monitoring total and filtered bioluminescence in the same cocultured SCN slices. The abscissa indicates days in coculture. (F) χ² periodograms for the circadian Avp-ELuc rhythms of graft SCN (left) and PER2::LUC rhythms of recipient SCN (right) in culture. An oblique line in the periodogram indicates the level of significance (P < 0.01).