Brain slice preparation
- Decapitate 4-6 weeks-old C57BL/6J mice.
- Remove brains and cut transversal (350 μm thick) hippocampal slices in sucrose-based solution(in mM):87 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 26.2 NaHCO3, 1.25 NaH2PO4, 25 glucose, and 50 sucrose; saturated with 95% CO2/5% O2, ~300mOsm using Microm HM650V sectioning system.
- Collect the slices and maintain in oxygenated artificial cerebrospinal fluid (ACSF)(in mM):119 NaCl, 2.5 KCl, 1.3 MgSO4, 1 CaCl2, 26.2 NaHCO3, 1 NaH2PO4, and 11 glucose: for 30 min at 34°C and then at least 1 hr at 25°C before recording.
Whole-cell recording for synaptic and tonic GABAAR signaling
Transfer individual slices to a submersion recording chamber that is continuously perfused with the oxygenated ACSF at 34°C throughout the recording.
All recordings were made from CA1 pyramidal cells and str. radiatum interneurons visually identified with an infrared differential interference contrast microscope.
Whole-cell pipettes used in voltage-clamp recordings of tonic GABAA current and IPSCs contained (in mM): 130 CsCl, 8 NaCl, 10 Cs-HEPES, 2 EGTA, 0.2 MaCl2, 2 MgATP, 0.3 Na3GTP, and 5 QX314Br (pH 7.2, osmolarity 295 mOsm) in the presence of NBQX(25 µM), CGP52342(5 µM), and D-AP5(50 µM).
7.Whole-cell current-clamp recordings were performed to obtain input-output characteristics of the neurons with pipettes containing (in mM): 132.3 K-gluconate, 7.7 KCl, 4 NaCl, 0.5 CaCl2, 10 HEPES, 5 EGTA, 2 MgATP, and 0.5 Na3GTP.
Amplify electrical signals with a MultiClamp 700B amplifier (Molecular Devices), digitalized at 5 KHz by National Instruments converter board (BNC-2110, Molecular Devices), acquired and analyzed by WinWCP software (Univ of Strathclyde).
Analyze Series resistance (Rs), input resistance (Ri), and membrane capacitance (cm) were monitored throughout the recordings. These parameters were obtained in voltage-clamp mode from the current in response to hyperpolarizing voltage steps (Vstep=-5 mV).
where, Ipeak is the peak amplitude of the current transient immediately after the step is applied, τ is the decay time constant of the current, and Iss is the steady-state current. Analyze data only when the change in series resistance or input resistance is less than 20% during recording.
Gramicidin-perforated patch recording
8.Perforated patch recording were conducted with pipettes with a resistance 4-6 MΩ and back-filled with an solution containing (in mM) 145 KCl, 10 HEPES, 5 ATP-Mg, 0.2 GTP-Na, 2 QX314, and 2 EGTA, adjusted to pH7.2 with KOH. 20µg/ml gramicidin-D(Sigma)
9.The pipettes tips were first filled with the KCl-based solution described for whole-cell recordings to prevent leakage of the antibiotic while approaching the cell. The pipettes were then back-filled with the same solution containing 20 µg/ml gramicidin.
- Fresh gramicidin solution was made every 2 hour.
- After the pipette tip contacted the cell membrane, the Rs was monitored. Recordings were started when the Rs stabilised at 30 to 45 MΩ (usually within 15-20 min).
- To get reversal potential of tonic GABAA currents, the cells were voltage-clamped at -65 mV. Membrane I-V characteristics were then obtained with 2000-ms voltage steps delivered from 90 mV to 70 mV with 10 mV intervals and from 55 mV to 20 mV with 5-mV intervals.
- I-V characteristics for GABAA tonic current were obtained as the difference between membrane I-V characteristics in 5 µM GABA and in 100 µM picrotoxin, added sequentially.
Cell-attached experiments for detecting action currents
1.Pipettes were filled with superfusion solution and loosely attached to the cell membrane to measure interneuron firing without perturbing intracellular ionic concentrations.
Recordings were performed in the voltage-clamp mode. The command potential was set to the potential at which the holding current was 0 pA (0-current potential) to avoid direct cell stimulation by the electrode.
3.The evoked GABAA mediated postsynaptic conductance were triggered by 200-µs current pulses at 50 µA delivered by a tungsten monopolar electrode placed in the str. radiatum.
GABAA activation was also produced with a 100-µM GABA puff application (100 ms, 50 psi) using Pneumatic picopump.