Additional file 1:

Figure S1. MBH rhythmicity is independent of serum in culture medium. PMT recording of PER2::LUC expression in an MBH culture prepared in serum free, B27 containing medium. Addition of forskolin (10 μM) potently restarts rhythms in damped tissue, whereas medium change to control serum containing medium does not revive damped rhythms.

Figure S2. Photograph of electrode positioning on the DMHc and ArcD for electrophysiological recordings. Broken lines outline the glass suction electrodes, while component areas of the MBH are delineated in the lower panel as in Figure 1.

Figure S3. The phase of peak PER2::LUC expression in Arc and DMH is reset by cull. Rayleigh vector plots showing the phase of peak PER2::LUC expression in vitro, calculated as the time of first peak in bioluminescence after cull of animal, in the whole ArcD, ArcL and DMH regions for 12 cultures prepared at different times throughout the day/night. In all areas, the phase was significantly correlated with time of cull. Filled circles indicate the phase of peak bioluminescence for whole delineated ArcD, ArcL and DMH. The direction of the arrow indicates the mean phase vector and its length shows the significance of phase clustering, with the surrounding box indicating the variance of phase. The inner broken line indicates the significance threshold at p = 0.05.

Figure S4. Raster plots of circadian PER2::LUC bioluminescence expression in individual cells in the ArcD (A), ArcL (B) and DMHc (C) from a single MBH slice. Cells are stacked vertically, one cell per line; red indicates peak PER2::LUC emission, and green indicates minimal emission. Initially neuronal rhythms in PER2::LUC are synchronized then gradually drift out of phase and damp (shown by decreasing brightness on plot). 10 μM forskolin, added at 186 h, re-synchronizes circadian rhythms and increases the amplitude of oscillations of individual cells.

Figure S5. Frequency histograms showing the periods of PER2::LUC expression of individual cells resolved in the SCN, ArcD, ArcL and DMHc. SCN cells expressed the tightest range of periods, with most cells expressing a ~24 h period. Though cells of the ArcD, ArcL and DMHc expressed similar wide ranges of periods, the frequency distribution of cells in the ArcD peaked sharply at ~24 h whereas cells of the ArcL and DMHc expressed a more even distribution of periods across the range.

Figure S6. Extracellularly detected electrical signals in MBH nuclei are dependent on sodium channel activity. Multiunit (MUA; upper traces) and single unit activities (SUA; lower traces) detected in extracellular recordings from ArcD (A), DMHc (B) and VMH (C) are reversibly blocked by brief (5 min) applications of the Na⁺ channel blocker tetrodotoxin (TTX; 0.5 μM).

Figure S7: PMT recording of PER2::LUC expression in an MBH slice in the presence of 0.5 μM TTX. Despite the presence of TTX, forskolin revived damped PER2::LUC rhythms.

Figure S8: KCl stimulation restarts damped rhythms circadian rhythms. PMT recordings of PER2::LUC expression in representative Arc/ME/PT (A) and MBH (B) slice cultures from food deprived animals. Addition of 10 μM KCl to the culture medium consistently restarted damped rhythms in all previously rhythmic MBH regions.

Format: PDF Size: 348KB Download file

This file can be viewed with: Adobe Acrobat Reader

Additional file 2:

Movie 1. EM-CCD recording over 326 hours showing bioluminescence emission from an MBH slice culture prepared from a PER2::LUC mouse; one frame every 30 min. 10 μM forskolin was added to the slice at 186 h. Note the damping of cellular and tissue rhythms over the first 186 hours, which are revived by forskolin stimulation.

Format: MP4 Size: 1.3MB Download file

Playing the movie within this page requires QuickTime 6 or later and JavaScript. Read more

Additional file 3:

Movie 2. EM-CCD recording over 214 hours showing bioluminescence emission from an MBH slice culture prepared from a PER2::LUC mouse; one frame every hour. 10 μM forskolin was added to the slice at 164 h. Note the revival of individual cellular rhythms particularly in the DMHc.

Format: MP4 Size: 832KB Download file

Playing the movie within this page requires QuickTime 6 or later and JavaScript. Read more