Screening shRNA Effects on Neuronal Activity

Primary cortical neurons were AAV infected on DIV6 with the pathScreener vector expressing individual hit shRNAs or a non-targeting control (NTC). On DIV10, half of the cultures were silenced with 1 µM TTX, 100 µM D-APV (TTX cocktail) for 48 hrs. On DIV12, the remaining cultures were stimulated for 4 hours with 50 μM BIC, 100 μM 4-AP, 100 μM glycine, 1 μM strychnine (BIC cocktail). Subsequently, all cultures were lysed in passive lysis buffer (Promega). Lysate was transferred into white 96-well assay plates and measurement of luciferase bioluminescence was performed in a Mithras LB 940 Microplate Reader (Berthold Technologies) as described previously^{26 (link)}.