In-vivo Whole-cell Recordings with Two-photon Guidance

 Two-photon guided in-vivo whole-cell recordings were performed as described in Polack et al. (2013) (link). Long-tapered micropipettes made of borosilicate glass (1.5 mm outer diameter, 0.86 mm inner diameter, Sutter Instrument) were pulled on Sutter Instruments P-97 pipette puller to a resistance of 3–7 MΩ, and filled with an internal solution containing 115 mM potassium gluconate, 20 mM KCl, 10 mM HEPES, 10 mM phosphocreatine, 14 mM ATP-Mg, 0.3 mM GTP, and 0.01–0.05 mM Alexa-594. Whole-cell current-clamp recordings were performed using the bridge mode of an Axoclamp 2A amplifier (Molecular Devices), further amplified and low-pass filtered at 5 kHz using a Warner Instruments amplifier (LPF 202A). Series of current pulses of small intensity (typically −100 pA) were used to balance the bridge and compensate the pipette capacitance. The membrane potential was not corrected for liquid junction potentials (estimated to be about 10 mV).