Neuronal Cell Culture, Western Blotting, and Ca2+ -Sensor Imaging

Primary cortical neuron / glia co-cultures were prepared from embryonic day 18 mouse pups as described (Kügler et al., 2001). Cultures were infected with AAV6 vectors on day 1 in vitro (DIV1) by diluting the proper amount of viruses in 10 μl of phosphate-buffered saline which was then directly added to cells grown in 24-well plates; 1 × 10⁷ vector genomes (vg)/250.000 cells of AAV6-ZFN1-WP(D) and AAV6-ZFN2-WP(D), AAV6-ZFN1-P(D) and AAV6-ZFN2-P(D), AAV6-ZFN1-NoP(D), and AAV6-ZFN2-NoP(D), along with AAV6-EGFP as transduction control were applied. To reduce CatD expression and secretion from glial cells, 1-β-D-arabinofuransylcytosine (ARA-C, 0.5 μM) was added to the cultures on DIV2 to transduction and medium was changed every 3rd day to minimize extracellular CatD. For western blots cells were harvested at day 22 post-transduction. For bicistronic expression of CatD-ZFNs, AAV6- ZFN1+2-P(S) was used at 1 × 10⁷ vg/250.000 cells and the cells were harvested after 7 or 22 days post-transduction. Blots were developed using the following primary antibodies: goat anti CatD mouse (R&D, AF1029); mouse anti Flag M2 (Sigma, F3165); mouse anti EGFP (Roche, 11814460001) and mouse anti β-tubulin (Sigma, T4026) followed by secondary anti-mouse and anti-rabbit HRP antibodies (Sigma-Aldrich). Tubulin served as loading control. Blots were imaged with ChemiDoc MP System with ImageLab 4.1 software (Bio-Rad), and quantified using ImageJ software 1.49.

Neuronal functionality was addressed through co-transduction of the eGFP based genetically encoded calcium indicator (GECI) GCaMP3.5 (Tian et al., 2009) and Ca²⁺ imaging was performed essentially as described (Mironov et al., 2009). Electrical stimulation was done using a SIU-102 (Warner Instruments) stimulus isolation unit and custom built function generator. Neurons were in all cases stimulated by emulating a train of 50 action potentials (AP; 10 Hz, 1 ms pulse-width, 100 mA current and 2 cm electrode separation) and data was evaluated using ImageJ 1.49 software.