Ex vivo electrophysiological recordings

Mice (7–11 weeks) were deeply anesthetized with pentobarbital (200 mg/kg i.p.; Virbac) and perfused intracardially with 10 ml ice-cold sucrose-based artificial cerebrospinal fluid (ACSF) containing (in mM): 75 sucrose; 87 NaCl, 2.5 KCl, 7 MgCl₂, 0.5 CaCl₂, 1.25 NaH₂PO₄, 25 NaHCO₃ and continuously bubbled with carbogen (95% O₂, 5% CO₂). Brains were extracted and 200 μm coronal slices were cut in sucrose-ACSF using a Leica Vibratome (vt1200). Slices were transferred to a perfusion chamber containing ACSF at 31°C (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl₂, 2.4 CaCl₂, 1.4 NaH₂PO₄, 25 NaHCO₃, 11 glucose, continuously bubbled with carbogen. After at least 45 min recovery, slices were transferred to a recording chamber continuously perfused with ACSF (2–3 ml/min). Patch pipettes (3.5–5.5 MΩ) were pulled from borosilicate glass (King Precision Glass) and for voltage-clamp recordings filled with internal recording solution containing (in mM): 120 CsCH₃SO₃, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 TEA, 2.5 Mg-ATP, 0.25 Na-GTP, at pH 7.25 and 285 ± 5 mOsm. For cell-attached and current-clamp recordings of ChR2- and eNpHR3.0-expressing STN neurons, a potassium-based recording solution was used (in mM): 123 KCH₃SO₃, 10 HEPES, 0.2 EGTA, 8 NaCl, 2.5 Mg-ATP, 0.25 Na-GTP, at pH 7.25 and 280 ± 5 mOsm.

Fluorescent STN neurons and terminals were visualized by epifluorescence and visually-guided patch recordings were made using infrared-differential interference contrast (IR-DIC) illumination (Axiocam MRm, Examiner.A1, Zeiss). ChR2 was activated by flashing blue light through the light path of the microscope using a light-emitting diode (LED460, Prizmatix) under computer control. Excitatory postsynaptic currents (EPSCs) were recorded in whole-cell voltage clamp or action potentials were recorded in cell-attached mode (Multiclamp 700B amplifier, Molecular Devices), filtered at 2 KHz, digitized at 10 KHz (Axon Digidata 1550, Molecular Devices) and collected on-line using pClamp 10 software (Molecular Devices). Series resistance and capacitance were electronically compensated prior to whole-cell recordings. Estimated liquid-junction potential was 12 mV and left uncorrected. Series resistance was monitored during recordings and cells that showed >25% change during recordings were considered unstable and discarded from analyses. To assess the effects of ChR2 activation and Halo inhibition in the STN we used cell-attached or current-clamp and assessed responses to a single 50 ms pulse, or 10 10 ms pulses (40 Hz) of blue light (ChR2), or 1 s pulse of green light (Halo), delivered every 55 s and 3 responses were averaged. For post-synaptic firing rates, cell-attached recordings were averaged over the 5 s before, 5 s during photostimulation (40 Hz, 200 pulses, 5 ms pulse width) and 5 s after; 3 responses were averaged per neuron. Average effect is also shown as timeplot histograms where each bar (200 ms bin) is relative to the baseline (baseline has been calculated as the average of all the pre-photostimulation bins for each neuron separately). To assess EPSCs, neurons were held in voltage-clamp at −60 mV, a single pulse (5 ms) photostimulus was applied every 60 s and 10 photo-evoked EPSCs were averaged per neuron per condition. DMSO stock solution of DNQX (Sigma) was diluted 1000-fold in ACSF and bath applied at 10 μM. Current sizes were calculated by using peak amplitude from baseline.