Whole-Cell Patch-Clamp Electrophysiology

Methods used for whole-cell patch clamp in acute coronal slices were as described previously (Threlfell et al. 2012). Briefly, mice were anaesthetized with pentobarbital and transcardially perfused with ice-cold high Mg²⁺ aCSF containing in mM: 85 NaCl, 25 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 7 MgCl₂, 10 glucose, 65 sucrose. Coronal 300 μm slices were cut on a vibratome (Leica VT1200S) at coordinates between +1.5 mm and +0.5 mm from bregma (Paxinos and Franklin 2008). Slices recovered at 32°C for 30–40 min after dissection and were subsequently kept at room temperature. Slices were maintained and recorded from in aCSF containing in mM: 130 NaCl, 25 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 2 MgCl₂, 10 glucose. The aCSF was saturated with 95% O₂/5% CO₂; recordings were made at 32°C.

Glass electrodes for whole-cell patch clamp (5–9 MΩ) were filled with an intracellular solution containing in mM: 120 K-gluconate, 10 KCl, 10 HEPES, 4 MgATP, 0.3 NaGTP, 10 Na-phosphocreatine and 0.5% neurobiotin. ChIs in the striatum were identified by their distinctive morphological features, that is, large somas (>20 µm) and their characteristic electrophysiological properties, that is prominent Ih, after-hyperpolarization (AHP) and broad action potential. A minimum negative holding current (<40 pA) was applied to prevent spike activity during recordings of laser-induced excitatory post-synaptic potentials (EPSPs). Recordings were acquired using a Multiclamp 700B at 10–20 kHz. All data were analyzed offline with Clampfit (pClamp10), and custom-written Matlab (R2013b) scripts. ChIs were confirmed by post hoc labelling for choline acetyltransferase (ChAT) and neurobiotin, as previously (Threlfell et al. 2012).