Electrophysiological Recordings

Patch-clamp recordings were collected using an Axopatch 200B amplifier (Molecular Device, CA, USA). Data were digitized at 5 kHz and filtered at 1 kHz with a Digidata 1322 acquisition system. The acquisition software used was Clampex 9.2 (Axon Instruments, Inc., CA, USA). Patch pipettes (GB150F-8P with filament, Science Products) were pulled from hard borosilicate glass on a Brown-Flaming P-87 puller (Sutter Instruments, Novato, CA, USA) and fire-polished to a final electrical resistance of 5–7 MΩ for whole-cell recordings and 8–15 MΩ for cell-attached recordings. All the experiments were performed at room temperature on dissociated hippocampal pyramidal neurons maintained in a solution containing (in mM) 138 NaCl, 4 KCl, 2 CaCl₂, 1.2 MgCl₂, 10 HEPES, and 10 d-glucose, adjusted to pH 7.4 with NaOH. For experiments in whole-cell configuration, series resistance was monitored and, if it was > 20 MΩ, the recording was discarded. Data analysis was performed with Clampfit 10.3 (Molecular Devices, CA, USA).

GABA_AR-mediated tonic currents were recorded in whole-cell voltage-clamp mode at a holding potential (Vh) of − 70 mV in the presence of kynurenic acid (3 mM) to block glutamatergic transmission and GABA (0.1 μM) to increase the amplitude of tonic current and reduce variability among cultures. Pipettes were filled with a Cs-based solution containing (in mM) 140 CsCl, 1 MgCl₂, 1 CaCl₂, 10 EGTA, 10 HEPES, and 0.1 GTP-Na⁺ (adjusted to pH 7.4 with CsOH) supplemented with the Na⁺ channel blocker QX 314 bromide (4 mM). The amplitude of GABA_AR-mediated tonic current was calculated by the outward shift in the baseline current induced by the GABA_AR antagonist bicuculline (40 μM) [16, 28, 36, 37]. After obtaining a stable recording for at least 1 min (untreated control condition), bicuculline was applied by using a gravity-driven perfusion system (RSC-200, BioLogic, France) through a micropipette positioned close to the soma of the recorded cell. All-points histograms were generated from 60-s epochs relative to untreated and bicuculline conditions. Tonic currents were defined by fitting Gaussian curves to these histograms. The peak of the Gaussian distribution (μ) represents the mean holding current, while the standard deviation of the curve (σ) represents the root mean square (RMS) of the variance over the 60-s interval. Tonic current was calculated both as the difference between the μ values relative to untreated and bicuculline epochs (current shift, I_mean) and difference of RMS noise between the two conditions [28]. Changes in the RMS noise values before and during bicuculline application were also used to predict the single-channel conductance (γ) of tonic receptors. First, the squared standard deviation of the curve (σ²) was plotted against the I_mean to extrapolate the single-channel current (i), based on the parabolic relationship described by the following formula: σ²(I_mean) = i(1 − P_O)I_mean, where P_O is the channel open probability. In our experimental conditions, the P_O of the GABA_AR mediating tonic current is expected to be small, as the exogenous GABA concentration is low [36]. Therefore, the relationship between σ² and I_mean can be well approximated by the following equation: i = σ²/I_mean [16, 36]. Next, single-channel conductance was calculated using the equation γ = i/(Vh − ECl), where Vh is the holding potential and ECl is the chloride electrochemical equilibrium potential (or reversal potential), corresponding to approximately 0 mV in our experimental conditions.

GABA_AR single-channel currents were recorded in voltage-clamp mode, cell-attached configuration. Patch pipettes were filled with the extracellular solution (composition described above) supplemented with 100 μM 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS) to block ClC type Cl⁻ channel activity, 1 mM 4-aminopyridine (4-AP), and 5 mM tetraethylammonium chloride (TEA-Cl) to block K⁺ channel activity, and 100 μM GABA to evoke GABA_AR activity. Following the formation of a giga seal (> 10 GΩ), the output gain was set to 100 mV/pA and the headstage switched to the capacitive feedback mode. At the end of each single-channel recording, the patch membrane was ruptured to measure the resting membrane potential (RMP) of the cell. Although the intrapipette solution was not suitable for whole-cell experiments, RMP values, measured immediately after the rupture of the membrane, were analogous to those obtained in whole-cell experiments using a K-gluconate-based internal solution (values in Fig. ​Fig.5).5). GABA_AR single-channel currents were elicited by voltage steps ranging from − 60 to +100 mV (corresponding to a patch pipette voltage (Vpip) ranging from − 100 to + 60 mV) in 20-mV increments and 800-ms duration. For each patch, at each voltage step, all the sweeps where the channel opened were concatenated and analyzed. All-points histograms were generated from single-channel openings at each potential and fitted with the Gaussian equation. The peak of the Gaussian distribution represents the mean single-channel current, which was plotted against voltage to obtain the I/V relationship. Voltages were previously adjusted according to the measured RMP for each cell. The point at which the I/V curve crosses the voltage axis represents the reversal potential of the GABA_AR-mediated current, which corresponds to the chloride reversal potential (ECl). Single-channel slope conductance for an individual cell was calculated from the slope of the linear regression obtained from the I/V plots. For those cells in which slope conductance could not be obtained, the chord conductance (γ_chord) at single potential was calculated according to the equation γ_chord = i/(Vh − ECl) where i is the observed single-channel current, Vh the holding potential, and ECl the chloride reversal potential. Open and close times were analyzed at Vpip = + 60 mV, corresponding to a Vh approximately 60 mV below the ECl, which is the same condition used to measure whole-cell tonic current.

Duration histograms were fitted with a mixture of exponential distributions defined by

and characterized by a time constant τ and a relative area a. Since concatenated traces were analyzed, we reported only the short close times that likely characterize intra-cluster shutting events, and thus likely define the kinetic of a single channel.

The intrinsic excitability of neurons was evaluated by whole-cell recordings. To elicit action potentials (APs), neurons were held at − 60 mV and stimulated with depolarizing current injections of 4 s of duration and amplitude ranging from 0 to + 80 pA (current steps, ΔI = 10 pA). Patch pipettes were filled with a solution containing (in mM) 126 K-gluconate, 4 NaCl, 0.05 CaCl₂, 0.1 EGTA, 10 HEPES, 10 d-glucose, 1 MgCl₂, 3 ATP-Mg²⁺, and 0.1 GTP-Na⁺, adjusted to pH 7.2 with KOH. Bath application of bicuculline (40 μM) was used to block GABA_AR currents. To assess individual action potential (AP) waveforms, the first spike evoked by the minimum amount of current injected (rheobase) was analyzed. Spike width was measured at 50% of the peak amplitude, which was calculated from the AP voltage threshold to the peak of APs. To extrapolate the AP voltage threshold values, phase-plane plots were constructed from the first time derivative of voltage (dV/dt) plotted against the membrane voltage [38]. AP threshold was defined as the voltage value at which dV/dt was 4% of its maximal value (dV/dt_max) [39, 40]. Rheobase was calculated by measuring the minimum amount of current injection able to induce a spike from Vh = − 60 mV. Input resistance (R input) was calculated as the slope of the V/I relationship obtained by plotting the steady-state membrane depolarization elicited by a series of subthreshold current steps of 1 pA increment and 4 s of duration.