Preparation of SCN Slices for Optogenetics

Removed brains were mounted and cut into coronal slices (300 μm) on a vibrating blade microtome (Leica) in cold HBSS supplemented with 100 U/ml penicillin/streptomycin, 10 mM HEPES, and 4.5 mM sodium bicarbonate. The SCN slices were dissected out and transferred onto a PTFE membrane insert (Millipore) in 35 mm culture dishes with 1.2 ml of DMEM (D5030, Sigma) supplemented with 3.5 g/L D-glucose, 2 mM Glutamax (Gibco), 10 mM HEPES, 25 U/ml penicillin/streptomycin, 2% B-27 Plus (Gibco), and 0.1 mM D-Luciferin sodium salt (Tocris). The SCN slice position was adjusted to the center of the dish and 1.5 μl AAV (pAAV1-Syn-ChrimsonR-tdT, Addgene) (Klapoetke et al., 2014 (link)) was placed directly onto the SCN slice. The culture dishes were then sealed with an optically clear PCR plate film (Bio-Rad) and maintained in a non-humidified incubator at 36.8 °C for about 10 days. The opsin expression was checked after about 10 days of viral transduction by imaging tdT fluorescence.