Whole cell patch clamp recordings were made at 22–25 °C. Slices were superfused at a rate of 3 ml min−1. Patch-clamp recordings were made using a MultiClamp 700A amplifier with Clampex 10.3 software. 3–5 MΩ borosilicate glass recording electrodes were filled with internal solution containing 120 mM potassium gluconate, 11 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 1 mM EGTA, pH adjusted to 7.4 with KOH (290 mOsm). To determine if EPSCs recorded in voltage clamp are glutamate dependent, the glutamate receptor blocker kynurenic acid (1 mM) was added to the bath solution following at minimum 5 min of baseline recording. Evoked responses were produced by extracellular monopolar tungsten electrode placed 100–300 μm from the whole cell recording. At the end of the recording, biocytin was added to the internal solution, and the slice was fixed and immunostained (Streptavidin, Alexa Fluor 555 conjugate, Life Technologies) to visualize neuronal morphology. All electrophysiological experiments were performed between days 90 and 130 of in vitro differentiation.
