Slicing and electrophysiology

Brain slice experiments were performed on male and female adult mice of at least 6 weeks in age. Mice were anesthetized with isoflurane, decapitated, and brains rapidly extracted. Horizontal sections (electrophysiology, dLight, calcium imaging) or coronal sections (voltammetry) were cut at 330–400 µm thickness on a vibratome while immersed in warmed, modified, slicing ACSF containing (in mM) 198 glycerol, 2.5 KCl, 1.2 NaH₂PO₄, 20 HEPES, 25 NaHCO₃,10 glucose, 10 MgCl₂, 0.5 CaCl₂, 5 Na-ascorbate, 3 Na-pyruvate, and two thiourea. Cut sections were promptly removed from the slicing chamber and incubated for 30–60 min in a heated (34°C) chamber with holding solution containing (in mM) 92 NaCl, 30 NaHCO₃, 1.2 NaH₂PO₄, 2.5 KCl, 35 glucose, 20 HEPES, 2 MgCl₂, 2 CaCl₂, 5 Na-ascorbate, 3 Na-pyruvate, and 2 thiourea. Slices were then stored at room temperature and used 30 minutes to 6 hours later. Following incubation, slices were moved to a heated (33–35°C) recording chamber that was continuously perfused with recording ACSF (in mM): 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 3.5 KCl, 10 glucose, 1 MgCl₂, 2 CaCl₂. Whole-cell recordings were made using borosilicate pipettes (5–10 MΩ, axon; 2–3 MΩ, soma) filled with internal solution containing (in mM) 122 KMeSO₃, 9 NaCl, 1.8 MgCl₂, 4 Mg-ATP, 0.3 Na-GTP, 14 phosphocreatine, 9 HEPES, 0.45 EGTA, 0.09 CaCl₂, adjusted to a pH value of 7.35 with KOH. For high chloride experiments, KMeSO₃ was substituted with KCl.

Perforated-patch recordings were made using borosilicate pipettes (5–10 MΩ) filled with internal solution containing (in mM) 135 KCl, 10 NaCl, 2 MgCl₂, 10 HEPES, 0.5 EGTA, 0.1 CaCl₂, adjusted to a pH value of 7.43 with KOH, 278 mOsm. Pipette tips were back-filled first with ~1 µL of internal lacking gramicidin followed by internal containing either 4–8 (perforated-patch on the main axon) or 80–100 µg/mL (perforated-patch in the striatum) gramicidin. Patch integrity was monitored by the addition of Alexa-488 to the gramicidin-containing internal. Experiments were discarded upon visual evidence of membrane rupture (Alexa-488 entering the axon).

To enable post-hoc reconstruction, pipette solutions in some experiments included 0.1–0.3% w/v neurobiotin (Vector Labs), and 0.01 mM AlexaFluor 594 hydrazide or AlexaFluor 488 hydrazide. Current clamp recordings were manually bridge balanced. Liquid junction potential for KMeSO₃ based internal solutions was −8 mV and was corrected offline.

Electrical stimulation was evoked with tungsten bipolar electrodes (150 µm tip separation, MicroProbes). For experiments where the site of electrical stimulation is distal to the site of imaging or recording, electrodes were placed at the caudal end of horizontal brain slices, or at the medial end of coronal slices. Stimulations were evoked using an Isoflex (A.M.P.I.), amplitudes ranging from 0.1 to 75 V.

Pressure ejection was performed using a borosilicate micropipette pulled on a horizontal puller (pipette size ~2–4 MΩ). The pharmacological agent being tested, either GABA or muscimol, was added to a modified external solution containing (in mM): 125 NaCl, 25 NaHCO₃, 1.25 NaH₂PO₄, 3.5 KCl, 10 HEPES, 0.01 either Alexa 488 (for experiments in DAT-Cre x Ai9 animals) or Alexa 594 (for experiments in TH-GFP animals), final osmolarity 280–290 mOsm. This puffing solution was then spin filtered, loaded into the glass pipette, and lowered to within 30–50 µm of the axon using a micro-manipulator. The puffing solution was then applied onto the axon with a short pressure ejection (80–300 ms in duration) using a PV 820 Pneumatic PicoPump (WPI).