4.2. Whole-Cell Patch Clamp Recordings

Registration of miniature excitatory postsynaptic currents (mEPSC) from pyramidal neurons in primary neuroglial culture of hippocampus was carried out by means of whole-cell patch clamp. Pyramidal neurons were identified based on the soma morphology using a SliceScope (Scientifica, UK) equipped with a CCD camera. Recordings were done with a PC505B amplifier (Warner Instruments, Hamden, CT, USA) in voltage clamp mode. Signals were filtered at 2 kHz with the amplifier, then acquired and digitized at 10 kHz sampling frequency with ADC/DAC Digidata 1440A (Molecular Devices, USA), and the pClamp 10 software package for data acquisition and analysis (Molecular Devices, USA). Recording electrodes of 4–5 mOhm resistance were pulled from borosilicate glass capillaries (Harvard Apparatus, USA) using a PC10 vertical puller (Narishige, Japan). The composition of the intracellular solution used for electrode filling was as follows (in mM): 115 K-gluconate, 10 KCl, 2 Na₂ATP, 10 Na₂phosphocreatine, 1 MgCl₂, 0.5 EGTA, 10 HEPES, pH 7.2, and osmolality of 280 ± 5 mOsm. Resistance of the electrodes was 4–5 MOhm. Hank’s solution used as an extracellular solution and contained (in mM): 139 NaCl, 4.17 NaHCO3, 0.4 NaH2PO4, 2.1 KCl, 0.44 KH2PO4, 2 CaCl₂, 1.25 MgSO4, 5 HEPES, 8 d-glucose, pH 7.4, and osmolality 305 ± 2 mOsm. In addition, for mEPSC isolation from other synaptic events, 20 µM of bicuculine, in order to block GABAergic synaptic transmission, and 1µM of TTX to block action potential driven synaptic activity were added to the extracellular solution. All experiments were conducted at 27–28 °C. Access resistance was monitored throughout the recording and was typically <30 MΩ. Neurons which had an access resistance change of more than 15–20% during recording were excluded from the analysis.