Brain slice electrophysiology

Scn1a^Stop/+mice were crossed with GAD67-GFP knock-in mice²². After genotyping, Scn1a^+/+; and Scn1a^Stop+/-;GAD67-GFP⁺ were tail-vein injected with PHP-e.B-Ctrl or PHP-e.B-Cre viruses. At P45-54, mice were sacrificed after deep isoflurane anesthesia, and brains were extracted. 350-μm-thick coronal hippocampal sections were cut using a Leica VT 1200 vibratome. After the cut, the slices were allowed to recover for 30 min at 32 °C in modified artificial cerebrospinal fluid (ACSF) containing 92 mM sucrose, 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃, 25 mM glucose, and 10 mM MgSO₄ aerated with 95% O₂ and 5% CO₂ (pH 7.4); slices were then allowed to recover at room temperature for at least 45 min before recording.

Current-clamp recordings were performed using a MultiClamp 700B amplifier (Molecular Devices) with pCLAMP 10 software. Signals were low-pass-filtered at 10 kHz and sampled at 50–100 kHz; the signal was digitized using a Digidata 1550 D/A converter (Molecular Devices).

Cells were held at 30 °C–32 °C. The extracellular solution contained 125 mM NaCl, 25 mM NaHCO₃, 2 mM CaCl₂, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 1 mM MgSO₄, and 10 mM D-glucose aerated with 95% O₂ and 5% CO₂ (pH 7.4). For current-clamp recordings, the internal solution contained the patch pipette contained 124 mM KH₂PO₄, 5 mM KCl, 2 mM MgCl₂, 10 mM NaCl, 10 mM HEPES, 0.5 mM EGTA, 2 mM Na-ATP, and 0.2 mM Na-GTP (pH 7.25, adjusted with KOH). Neurons with unstable resting potential (or more than −50 mV) and/or holding current of more than 200 pA at −70 mV were discarded. Bridge balance compensation was applied.

CA1 Gad67GFP⁺ hippocampal neurons were classified into fast spiking and not fast spiking interneurons according to their firing profile (AP frequency, >100 Hz at 400 pA current step, fast AHP).) Current step protocols were used to evoke APs, injecting 500 ms-long depolarizing current steps of increasing amplitude (Δ 10 pA). Passive properties were calculated from the hyperpolarizing steps of the current-clamp step protocol. Input resistance is an average of three steps (2 negative and 1 positive) and is defined as the as ΔV/I. Capacitance was determined in the current-clamp hyperpolarizing step as follows. First, the resistance was determined as voltage derivative (dV)/DI (voltage/current), and then the cell time constant (tau) was obtained, fitting the voltage changing between baseline and hyperpolarizing plateau. Capacitance was calculated as tau/resistance. AP amplitude was calculated from the AP threshold, defined as the voltage at which the first derivative (dV/dt) of the AP waveform reached 10 mV/ms, to the absolute value of the AP peak for the first spike obtained at the current threshold (defined as the minimal current injection able to elicit neuronal firing, determined through 10 pA current steps). Maximal rise and decay slope were defined, respectively, as the maximal and minimal value of the first derivative of the AP waveform.

For voltage-clamp recording the internal solution composition was the following: 125 mM CH₃O₃SCs, 10 mM NaCl, 2 mM MgCl₂, 10 mM HEPES, 2 mM Mg-ATP, and 0.2 mM Na-GTP (pH 7.25, adjusted with CsOH). sIPSCs were recorded from CA1 pyramidal neurons of Gad67GFP mice in voltage-clamp; cells were held at +10 mV for sIPSCs. Pipette capacitance and resistance were always compensated.

Post-synaptic currents (sIPSCs) were analyzed off-line using MiniAnalysis (Synaptosoft). 120 s recording were used to quantify IPSCs frequency and amplitude. First traces were lowpass filtered (8-pole Butterworth) at 800 Hz. PSCs threshold for detection was set to 5 times the baseline noise (1.5/2 pA), usually around 8 to 10 pA; area threshold was set to 3 pA*ms⁻¹; baseline was determined as the mean of the 2 ms preceding the IPSC. Individual currents were inspected to reject spurious events. Since IPSCs amplitude does not follow normal distribution, cell amplitude has been expressed as median value and then mean of the median of each cell was plotted. For each cell, 1000 events were considered for IPSCs distribution amplitude distribution analysis.