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. 2021 Dec 20;10:e70137. doi: 10.7554/eLife.70137

Figure 1. Long-term Optogenetic Stimulation System for Circadian Entrainment Ex Vivo.

(A) Representative PER2::LUC bioluminescence rhythms of adult SCN slices exposed to either red (top) or blue (bottom) 10 Hz light pulses (red or blue bars) for 12 hr. The black arrow indicates the timing of media change. (B) Fold change in the rhythm amplitude following sham, blue, or red light exposure (Student’s t-test, mean ± SEM, n = 3, ***p < 0.001). (C) Merged ChrimsonR-tdT fluorescence and the brightfield images of an SCN slice. Scale = 100 μm. (D) Diagrams showing a multi-channel luminometer integrated with an optogenetic stimulation apparatus.

Figure 1—source data 1. Source data for Figure 1B.

Figure 1.

Figure 1—figure supplement 1. Improved PER2::LUC rhythmicity in SCN slices explanted from young mice to culture medium containing stabilized glutamine.

Figure 1—figure supplement 1.

Representative PER2::LUC rhythms of SCN slice cultures from an adult (P60) and a young (P12) mouse. Young SCN slice cultures with stabilized glutamine (alanyl-glutamine) showed higher amplitude in PER2::LUC rhythms for a longer duration, compared to rhythms from adult SCN slices in culture medium containing regular glutamine.
Figure 1—figure supplement 2. Diagram of an integrated system for long-term luminescence recording and optogenetic stimulation.

Figure 1—figure supplement 2.

Custom-written Matlab code has access to a luminometer data collection software, a multifunction I/O device turning on/off the photomultiplier tubes (PMTs), and a signal generator controlling LEDs. Thus, it can schedule periodic stimulation and execute a series of events during optogenetic stimulation — pause PMT recording, target positioning, LED stimulation, and PMT recording resumption.