Submerged type recording chamber

Slices were placed in submerged-type recording chamber (Warner Instruments, USA). A continuous perfusion of aCSF aerated with 95% O₂ and 5% CO₂ was maintained at 2.5 ml/min and 30 °C. The LA anatomy and principal neurons therein were visualized using infrared-differential interference contrast camera (Dage MTI, USA) attached to an upright widefield microscope (BX51WI, Olympus, USA). Patch-clamp recording electrodes of 2.5–3.5 MΩ tip resistance were pulled using P1000 micropipette puller (Sutter Instruments, USA). Patchmaster software (HEKA Elektronik, Germany) was used to deliver all stimuli and acquire the data. Data acquisition was done using an EPC-9 amplifier (HEKA Elektronik, Germany). A built-in Bessel filter was used to filter the data at 2.9 kHz and digitization was done at 20 kHz. Throughout all the recordings, access resistance was monitored once every minute, by application of brief 5 mV depolarizing step stimulus. Monitoring the access resistance at this frequency was done to assess whether all of the five 1-min epochs met the criterion for inclusion in the analysis. Testing once every minute helped ensure there was no deviation (beyond 20%) during the course of a 5 min recording. Only the cells where the series resistance (Rs) was always ≦ 25 MΩ and did not vary by more than 20% throughout recording were analyzed.