Electrophysiological recordings

Mice (vehicle, n = 86; scPCP, n = 97) were deeply anesthetized with isoflurane and euthanized by decapitation 7 days after the last PCP injection (5 weeks old at the time of euthanasia). The brains were removed from the skull, and 300-μm-thick coronal brain slices of the mPFC (coordinates from bregma, +1.845 to +1.145 mm) were cut using a vibro-slicer (Leica VT-1200) in ice-cold artificial cerebrospinal fluid (ACSF) containing 125 mM NaCl, 25 mM NaHCO₃, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 25 mM glucose, 2 mM CaCl₂, 1 mM MgCl₂, and 3 mM kynurenic acid, saturated with 95% O₂ and 5% CO₂ to pH 7.4. Slices were then stored for ~20 min at 35°C and allowed to recover at room temperature (22° to 24°C) for at least 30 min in the same solution.

All electrophysiological measurements were performed using an Axopatch 200B amplifier. Signals were filtered at 2 kHz and sampled at 5 kHz for voltage-clamp recordings and filtered at 10 kHz and sampled at 20 kHz for current-clamp recordings. Data were acquired using pClamp9 software (Axon Instruments) running on a personal computer. Data and statistics were analyzed using software written in Matlab (MathWorks). Recordings were performed at 31° to 32°C in the presence of 3 mM kynurenic acid to block fast glutamatergic synaptic currents. Neurons were visualized using an upright microscope (Scientifica) with oblique illumination, a 60× water-immersion objective (Olympus) and a digital camera (DVC). Pyramidal cells were visually identified according to their location, size, and shape. Pipettes were pulled from thick-walled borosilicate glass (1.5-mm outer diameter; Sutter Instruments) using a horizontal puller (Sutter Instruments, P-97) and were filled with a KCl-based internal solution consisting of 148 mM KCl, 6 mM NaCl, 2 mM MgATP, 0.2 mM Na₃GTP, 0.1 mM EGTA, and 10 mM Hepes (pH 7.3 with KOH). Pipette resistances in the working solutions ranged from 4 to 6 megohms yielding series resistances of 20 to 30 megohms for whole-cell recordings. Resting membrane potential was measured in whole-cell configuration immediately after breaking into the membrane. Input resistance was calculated from the peak voltage responses to hyperpolarizing current injections (−200 to −40 pA, 40-pA steps). Spontaneous inhibitory postsynaptic currents (IPSCs) were recorded at −85 mV over a 3-min period. The total number of IPSCs in each recording was counted using the Mini Analysis Program 6 (Synaptosoft). For evaluation of IPSC amplitudes, the events identified using the Mini Analysis software were analyzed using custom-made code written in Matlab. Spontaneous excitatory postsynaptic current (EPSC) recordings were also 3 min long and were performed using a potassium-methylsulfate internal solution (146 mM K-methylsulfate, 8 mM NaCl, 2 mM MgATP, 0.2 mM Na₃GTP, 0.1 mM EGTA, and 10 mM Hepes, pH 7.3 with KOH) at −65 mV and analyzed as reported for IPSCs. The external solution for EPSCs recordings contained 50 μM picrotoxin and no kynurenic acid.

Giga-seals (1.5 to 8.5 gigohms) were obtained using 6- to 8-megohm pipettes filled with modified ACSF (NaHCO₃ was substituted with 10 mM Hepes and titrated to pH 7.4 with NaOH). We counted the number of spikes elicited by electrical stimulation (in layer 1; 0.15 to 0.35 mA, 20 Hz for 1 s) within a 10-s segment, in the absence and in the presence of kynurenic acid in the bath. Each neuron was only stimulated ≤2 times, and these recordings were limited to ≤5 min to prevent spontaneous break-in. If we observed any change in the leak current or if the cell became intrinsically active, then the neuron was discarded as these signs suggest partial breaking of the cellular membrane.

Our protocol was adapted from Heigele et al. (47). Fresh gramicidin (Sigma-Aldrich) stock solution [20 mg/ml in dimethyl sulfoxide (DMSO)] was prepared daily. Stock solution (2.5 μl) was added to 1 ml of warm (35°C) KCl internal (final concentration, 50 μg/ml). The solution was then vortexed for 1 min, sonicated for 15 min, and, lastly, filtered using a 0.2-μm filter. The solution was used within 2 hours. Recording pipettes (4 to 6 megohms) were tip filled with gramicidin-free KCl internal solution and then backfilled with the gramicidin solution. When approaching cell, small positive pressure was applied until the cell was patched. Seal properties were monitored measuring the current responses to square voltage steps (−5 mV, 150 ms; 0.1 Hz). Eighty- to 150-megohm access resistances could be attained within 10 to 15 min. Lucifer yellow (0.1%) was included in the internal solution to visually confirm in real time the integrity of the membrane. If any lucifer yellow staining of the cell body was observed during or after completion of the recordings, then data were discarded. All perforated patch recordings were performed at 27° to 30°C in the presence of 3 mM kynurenic acid. GABAergic synaptic currents were elicited by extracellular electrical stimulation (0.2 to 1.0 mA, 0.2 ms) using a bipolar electrode positioned in layer 1 of the mPFC, 100 to 300 μm apart from the recorded cell. Current/voltage (I/V) curves were obtained measuring the current at different holding voltages (−100 to −20 mV, 20-mV steps). For each voltage, 5 to 10 traces were averaged. If unclamped action potentials were recorded during any sweep, then that sweep was excluded from the analysis. The current peaks were plotted versus the holding potentials, and the I/V curves were obtained by fitting the data points using second-degree polynomial functions (using Clampfit 10, Axon Instruments).