2.5. Electrophysiology
This protocol is extracted from research article:
Sex differences in the delayed impact of acute stress on the amygdala
Neurobiol Stress, Jan 7, 2021; DOI: 10.1016/j.ynstr.2020.100292

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.

Recording pipettes filled with an internal solution containing (in mM): 135 cesium methanesulfonate, 10 HEPES, 0.2 EGTA, 5 QX314, 5 NaCl, 2 MgATP, 0.3 NaGTP, 5 phosphocreatine; pH 7.3; ~295 mOsm were used to patch onto BLA principal neurons. The cells were held at −70 mV (VHold) and miniature excitatory post-synaptic currents (mEPSCs) were isolated using gabazine (10 μm, HelloBio, UK) and tetrodotoxin (TTX, 1 μm, HelloBio, UK) in the perfusate. Once the whole-cell configuration had been achieved, the cells were allowed to stabilize for 5 min followed by data acquisition. A total of 5 min of mEPSC recordings were analyzed for each cell. Mini Analysis Program (Synaptosoft Inc., USA) was used for the analysis and the threshold amplitude for each event was set at 5 pA. All cells included in the frequency and amplitude bar graph plots were included in the inter-event interval (IEI) cumulative plots. The frequency graph plotted at the cell level while the IEI plots were constructed using individual IEI values from the entire 5 min recording from all cells.

Neurons were patched and held at −70 mV (VHold) using pipettes filled with a high chloride internal solution containing (in mM): 135 cesium chloride, 10 HEPES, 10 EGTA, 5 QX314, 2 MgCl2, 4 MgATP, 0.3 NaGTP; pH 7.3; ~295 mOsm. Miniature inhibitory post-synaptic currents (mIPSCs) were isolated using TTX (1 μm), D-(−)-2-amino-5-phosphonopentanoic acid (D-AP5, 50 μm, HelloBio, UK) and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 μm, HelloBio, UK) in the perfusate. Similar criteria as described above for mEPSCs were also adopted for mIPSC recording and analysis. The threshold amplitude for each event was set at 10 pA.

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