Whole-Cell Patch Clamp Recordings

Male rats were anesthetized, and using previously described techniques (Faria and Prince 2010), brains were removed and coronal neocortical slices cut and maintained for in vitro slice recordings. Neocortical slices (~300 μm) were cut with a vibratome in cold (4 ± 1 °C) “slicing” ACSF containing (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 1 CaCl₂, 2 MgSO₄, 26 NaHCO₃, and 10 glucose; pH 7.4, when saturated with 95% O₂–5% CO₂. After 1 h of incubation in standard ACSF containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 1 MgSO₄, 26 NaHCO₃, and 10 glucose (32 ± 1 °C), single slices were transferred to a recording chamber where they were minimally submerged (32 ± 1 °C) and perfused at the rate of 2.5–3 mL/min with standard ACSF. Patch electrodes were pulled from borosilicate glass tubing (1.5 mm OD) and had impedances of 4–6 MΩ when filled with intracellular solution containing (in mM): 60 K-gluconate, 67 KCl, 0.1 CaCl₂, 10 HEPES, 1.1 EGTA, 4 ATP-Mg, and 6 phosphocreatin-tris and 0.2% biocytin. The osmolarity of the pipette solution was adjusted to 285–295 mOsm and the pH to 7.35–7.4 with KOH.

Whole-cell voltage clamp recordings were made from visually identified layer Va Pyr cells using infrared video microscopy and differential interference contrast optics (Zeiss Axioskop2) and a Multiclamp 700A amplifier (Axon Instruments). The estimated chloride equilibrium potential (E_Cl) was −18 mV based on the Nernst equation. Access resistance (Ra) was measured in voltage clamp mode from responses to 2 mV hyperpolarizing voltage pulses. Data from recordings in which Ra varied by more than 15% or was >20 MΩ were rejected. Pyramidal neurons were identified as cells with large somata and a single emerging apical dendrite extending toward the pial surface. In addition, in some slices, intracellular labeling with biocytin was used to confirm cell type and position. Pharmacologically isolated (monosynaptic) IPSCs were reliably evoked with monopolar tungsten-stimulating electrodes, placed in layer V, ~80 μm below the recorded Pyr soma to obtain steep input/output slopes (Salin and Prince 1996). 2-Amino-5-phosphonovaleric acid (D-AP5; 50 μM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX; 20 μM) (Ascent Scientific) in ACSF were continuously applied via bath perfusion. ω-Conotoxin GVIA (1 μM, Ascent Scientific) and ω-agatoxin IVa (0.2 μM, Bachem Bioscience) were bath applied to assess the effects of N channel and P/Q channel blockade on eIPSCs, respectively.

To obtain the threshold (T) for evoking IPSCs, the stimulus duration was initially set at 100 μs and stimulus current increased until a stable evoked (e)IPSC with a failure rate of ≤ 50% was evoked. The pulse duration was then increased to 150 μs (1.5 T) for determination of peak amplitude. In some experiments, bath perfusion of gabazine (10 μM) was used to block the evoked events and verify that they were mediated by GABA_A receptors (not shown). Responses in which spontaneous (s) IPSCs were superimposed on evoked IPSCs were not included in the data analysis.

Baseline values for eIPSC peak amplitude were obtained in UC rats during the first 5 min of stable whole-cell recording in standard ACSF. This was followed by a single 10-min local perfusion of ω-agatoxin IVa and/or ω-conotoxin GVIA. The effects of calcium channel blockade on eIPSCs were routinely assessed for 5 min, beginning after the end of toxin perfusion. Mean values for peak amplitude were calculated by averaging eIPSCs over 5 min prior to, and after the 10-min toxin treatment in UC. The prolonged actions of the toxins did not allow washout of the effects. Statistical significance was determined with two-tailed Student’s t-test (P < 0.05). Data are expressed as mean ± SEM and “n” corresponds to the number of neurons, unless otherwise noted. One neuron was recorded per slice and no more than 3 slices were used per rat. “Baseline” values below are those for UCs before toxin exposure.