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.