Optogenetic Stimulation of Cortical Neurons

Postnatal-dissociated cortical neuron cultures were prepared as previously described (31 , 36 (link)), from newborn B6 mice. Neurons were plated into 35 mm glass bottom Petri dishes at a 1 million/ml density in culture medium consisting of Basal Medium Eagle supplemented with 10% bovine calf serum and 1% penicillin–streptomycin. On day in vitro 2 (DIV2), culture medium was changed to Neurobasal A medium, supplemented with B27-plus reagent (Invitrogen), Glutamax, and 1% penicillin–streptomycin. Neurons were transfected with the CryBAR optogenetic system (6 μg plasmid/plate) on DIV5 using Lipofectamine LTX reagent. On DIV7, culture medium was removed and neurons were placed in imaging solution (Mg-free HEPES-buffered artificial cerebrospinal fluid (37 (link))) Live-cell imaging was performed before and after illumination using a Leica DMi8 Live Cell Imaging System. Animal use protocols were approved by East Carolina University Institutional Animal Care and Use Committee.