Whole-cell patch clamp recording

Twenty-four hours after the last kindling stimulation (2–3 hours following the last LFS application), electrophysiological experiments were conducted. Hippocampal slices were prepared in vitro as follows. Rats were anesthetized with isoflurane and decapitated. Brains were rapidly extracted, and the right hemispheres were dissected. Tissue was placed in ice-cold cutting solution containing (in mM) 2.5 KCl, 0.5 CaCl₂, 2 MgCl₂, 1 NaH₂PO₄, 26.2 NaHCO₃, 238 sucrose and 11 D-glucose and bubbled with 95% O2- 5% CO2. Osmolarity was adjusted to 290–300 mOsm, and the pH was adjusted to 7.2–7.35 in the cutting solution. Transverse slices (400 μm) were cut using a vibratome (1000 Plus Sectioning System, Vibratome, MO, USA). Subsequently, the sliced right hippocampi were incubated for 1 hour at 32°C. For incubation, we used standard ACSF that was continuously bubbled with 95% O₂- 5% CO₂, and contained (in mM): 125 NaCl, 3 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 10 D-Glucose, 2 CaCl₂, 1.3 MgCl₂. Osmolality was in the range of 290–300 mOsm and pH was adjusted to 7.2–7.35 by NaOH 1 M. Then slices were individually transferred to a submerged recording chamber.

The recording chamber was mounted on a fixed-stage, upright microscope (Axioskop 2 FS MOT; Carl Zeiss, Germany) and was continuously perfused at 1.5–2 ml/minutes with standard ACSF at room temperature (24 ± 1°C). CA1 pyramidal neurons were visualized using an IR-CCD camera (IR-1000, MTI, USA) with a 40× water immersion objective lens. Neurons were selected for recording based on their relative pyramidal shape and smooth, low-contrast appearance. Whole-cell patch clamp recordings were made under voltage clamp mode. Recording microelectrodes (1.5 mm outer diameter, borosilicate glass, GC150-11; Harvard Apparatus, UK) were pulled with a horizontal puller (P-97, Sutter Instrument, USA).

For excitatory LTP (eLTP) induction in excitatory synapses and EPSC recording, glass microelectrodes were filled with intracellular solution containing (in mM): 135 K-gluconate, 20 KCl, 10 HEPES, 0.2 EGTA, 7 disodium-phosphocreatine, 2 MgATP, 0.3 NaGTP and 1 QX-314. For inhibitory LTP induction (iLTP) in inhibitory synapses and eIPSC recording, the intracellular solution contained (in mM): 140 CsCl, 1 CaCl2, 5 QX-314, 10 HEPES, 2 MgCl2, 2 Mg-ATP, 2Na-GTP and 0.5 EGTA. pH was set at 7.2–7.35 and osmolality was in the range of 290–300 mOsm. The electrode tip resistance in the bath was 5 to 7 MΩ, and series resistance ranged from 18 to 30 MΩ. Cells were rejected if the series resistance was changed more than 20% during the experiment. Capacitance compensation and bridge balance were carried out. Data were low-pass filtered at 3 kHz and acquired at 10 kHz with a Multiclamp 700B amplifier equipped with Digidata 1440 A/D converter (Molecular Devices, CA, USA). The signal was recorded on a PC for offline analysis using the Axon pClamp 10 acquisition software. After establishment of a gigaseal (more than 2 GΩ), the whole-cell configuration was achieved by applying a brief suction.

Synaptic responses were evoked by constant monophasic current pulses (0.1 ms, 50–200 μA) delivered through a stimulus isolation unit (WPI-A360, World Precision Instruments, Sarasota, FL, USA) to a stimulating electrode (bipolar teflon-coated, stainless steel electrodes; A-M Systems, Inc., WA, U.S.A.; 127 μm in diameter); placed in the Schaffer collateral pathway to record excitatory monosynaptic responses and placed in the striatum radiatum to record inhibitory monosynaptic responses. The distance between stimulating and recording electrodes was less than 50 μm for inhibitory synapses and 100–150 μm for excitatory synapses. Minimal stimulation protocol was used to determine the threshold intensity and 1.5×threshold intensity to record excitatory and inhibitory monosynaptic responses respectively.

Monosynaptic excitatory responses were recorded following the addition of bicuculline (GABA_A receptor antagonist, 20 μM; Tocris Bionscience, England) into ACSF. Monosynaptic inhibitory responses were pharmacologically isolated using 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPA receptor antagonist, 20 μM; Tocris Bionscience, England) and (±)-2-amino-5-phosphopentanoic acid (AP5;NMDA receptor antagonist, 50 μM; Tocris Bionscience, England) to block non-NMDA and NMDA receptors, respectively.

High frequency stimulation (HFS) was used for LTP induction both in excitatory synapses (2 trains of 100 pulses at 100 Hz for 1 s, 20-s interval) and inhibitory synapses (2 trains of 7 pulses at 100 Hz for 1 s, 20-s interval).