In vivo whole-cell recordings.

We used in vivo patch-clamp techniques to record from layer 2/3 neurons in the mouse somatosensory cortex, similar to our previously published methods (46). Mice were anesthetized using urethane (1.5 g/kg, i.p.) (82) supplemented with isoflurane (~0.5%), if necessary. The depth of anesthesia was assured by continuous monitoring of the breathing rate, which was more than 90 breaths per minute. This was maintained by altering the isoflurane level. Additionally, to maintain a consistent depth of anesthesia, recordings were started when up states occurred at approximately 1 Hz frequency (83). Neuronal recordings were made approximately 2.0 mm from the center of the CCI lesion (Figure 1, A and B). Briefly, we used patch electrodes of 3 to 4 MΩ resistance that were filled with intracellular solution containing 135 mM K-gluconate, 8 mM NaCl, 5 mM EGTA, 10 mM HEPES, 0.3 mM GTP, 2 mM ATP, 7 mM phosphocreatine (pH 7.2), and an osmolality of approximately 295 mOsm. In current-clamp configuration, recordings were made at the cells’ RMP (–71.58 ± 1.08 mV; range: –63.78 to –75.84 mV). Voltage-clamp experiments were performed with a potassium gluconate internal solution at a clamped potential of –70 mV.

Signals were amplified, digitized at 10 kHz, and stored for analysis as previously described (46). If access resistance changed more than 20%, the recording was excluded from analysis. We examined the neuronal I-V curve, which was determined by measuring the frequency of AP firing in response to stepped current injections (–50 to +500 pA, 1000 ms square pulses). R_in was measured in voltage-clamp at –70 mV from the steady-state current in response to –20 mV voltage steps. Rheobase was the smallest current evoking an AP. Amplitude, width, AUC, and IEI were characterized using the first 50 events.