Coronal brain slices (300 μm) were prepared including the ACC region by a vibratome (7000smz-2, Campden, Loughborough, Leics, England)12 (link). Brain slices were put in a chamber for storing slices filled with oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 25 NaHCO3, 1 NaH2PO4, and 10 glucose at room temperature for about 1 h. All experiments were performed in a recording chamber on the stage of a BX51WI microscope (Olympus, Center Valley, PA, USA) with infrared differential interference contrast optics for neuron visualization. Whole-cell patch clamp recordings were performed from layer II/III pyramidal cells in the ACC with an amplifier (IPA, Sutter Instrument, Novato, CA, USA) at room temperature. In the voltage-clamp and current-clamp modes, tip microelectrodes were filled with internal liquid constituted of (in mM) 120 K-gluconate, 5 NaCl, 1 MgCl2, 0.5 EGTA, 2 Mg-ATP, 0.1 Na3GTP, and 10 HEPES; pH 7.2, 280–300 mosmol were used for spontaneous excitatory postsynaptic currents (sEPSCs), resting membrane potentials (RMPs) and action potentials (APs). For recording sEPSCs, the holding membrane was kept at − 70 mV in voltage-clamp mode with a GABAA receptors blocker, picrotoxin (100 μM) in ACSF continuously. APs were recorded by adjusting the membrane potentials about − 70 mV by changing the holding currents in the current-clamp mode. When spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded, tip microelectrodes were filled with internal liquid constituted of (in mM) 120 Cs-gluconate, 5 NaCl, 1 MgCl2, 0.5 EGTA, 2 Mg-ATP, 0.1 Na3GTP, and 10 HEPES; pH 7.2, 280–300 mosmol. To record sIPSCs, holding membrane potential was kept at 0 mV in voltage-clamp mode. The APs, sEPSCs and sIPSCs were analyzed by Mini Analysis Software, and membrane potentials were analyzed by Clampfit 10.7. The rise and decay time of APs, sEPSCs and sIPSCs were monitored between 10 and 90% of their peak and amplitude.
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