Calcium imaging

For all calcium imaging experiments, Fluo-4AM (Invitrogen) was mixed with 10 μl Powerload (Invitrogen) and vortexed for 15 s. Fluo-4AM/Powerload mix was subsequently mixed with aCSF for a final concentration of 10 μM Fluo-4AM. Individual coverslips were removed from culture media and incubated in 600 μl of Fluo-4AM mixture for 1 h at 37 °C. Following incubation, coverslips were placed in a recording chamber on an Olympus BX51W microscope and rinsed for 15 min with continuous flow of aCSF before imaging. Cells were imaged at 470 nM (Cairn OptoLED). Areas of the coverslip with >10 healthy looking cells with neuronal morphology were chosen for imaging. Calcium signals were acquired using Turbo-SM software and an SM-CCD67 camera (RedShirtImaging, Decatur, GA, USA). For spontaneous activity, images were acquired for 6 min at 100 Hz. For plasticity experiments, images were acquired for 90 min (see plasticity protocol above) at 10 Hz. Following data acquisition, all experiments were randomized and coded and then analysed by a blinded experimenter. Cells with neuronal morphologies were chosen as regions of interest across all experiments. Traces were then blindly analysed offline in Clampfit (Axon Instruments). For 100 Hz experiments, transients were counted if they had rise times ≤15 s. For 10 Hz experiments, transients were counted if they had rise times ≤120 s.