Key issues to consider include: 1) the AAV-GCaMP6f infection step needs to be optimized for human neuronal cultures and may require repeated AAV infections to maintain adequate GCaMP levels, 2) human neurons mature at a slower rate than rodent neurons and synapsin I expression is required for GCaMP6f expression, and 3) functional neural network formation may take longer in human cultures than in rodent cultures and therefore Ca2+ imaging time points may need to be adjusted accordingly. This evidence concerns the gene SYN1 and infection.