Vs120 epifluorescence microscope

For labelling D1 and D2 dopamine receptor-expressing medium spiny neurons (MSNs), adult Adora2a-Cre (GENSAT 036158-UCD, for labelling D2-MSNs) and Drd1a-Cre (GENSAT 017264-UCD, for D1-MSNs) congenic mice on a C57BL6/J background were obtained from GENSAT and backcrossed with wild type C57BL mice (Jackson Laboratories) for several generations. Surgeries were performed between 10–12 weeks of age. Subjects were anaesthetized with 2% isoflurane in oxygen. Buprenorphine SR (1 mg kg⁻¹) was administered at the beginning of the surgery as an analgesic. Glass micropipettes (10–30 µm diameter tip) filled with AAV-DIO-EGFP (AAVDJ-hSyn-DIO-EGFP-WPRE-bGH, B.K.L. laboratory) were lowered into the target region and delivered a localized injection by pressure (50–150 nl volume) at a rate of 100 nl min⁻¹. After 3 weeks, animals were deeply anaesthetized and perfused. Tissue sections were stained with DAPI and imaged on an Olympus VS120 epifluorescence microscope with a 10× objective lens. All procedures to maintain and use mice were approved by the Institutional Animal Care and Use Committee at the University of California, San Diego.

For cell-type-specific anterograde tracing of Cbln2+ and Pitx2+ SC neurons, AAV-DIO-EGFP (200 nl) was stereotaxically injected into the SC of Cbln2-IRES-Cre and Pitx2-Cre mice, respectively. The mice were then maintained in a cage individually. Three weeks after viral injection, mice were perfused with saline followed by 4% PFA in PBS. After 8 hr of post-fixation in 4% PFA, coronal or sagittal brain sections at 40 μm in thickness were prepared using a cryostat (Leica CM1900). All coronal sections were collected and stained with primary antibody against EGFP and DAPI. The coronal brain sections were imaged with an Olympus VS120 epifluorescence microscope (10× objective lens).

Animal subjects were deeply anaesthetized with an overdose bolus of sodium pentobarbital (Euthasol, 2 mg kg⁻¹, intraperitoneal injection), and cardiac perfused with normal saline followed by 4% boric acid-buffered paraformaldehyde. Brains were post-fixed overnight, embedded in 4% agarose, and sectioned on a vibratome at 50 μm thickness (50–150 μm for rabies-labelled tissues), collected in a 1-in-4 manner into 4 equivalent series, and stored in cryoprotectant at −20 °C until staining. Tissue series were stained with rabbit polyclonal anti-PHAL antibody (Vector Labs AS-2300) at 1:5,000 and donkey anti-rabbit AlexaFluor 647 (Jackson ImmunoResearch, 711-605-152). Nissl substance was stained with NeuroTrace 435/455 (ThermoFisher, N21479) at 1:500 to reveal cytoarchitecture. Sections were scanned on an Olympus VS120 epifluorescence microscope running VS-Desktop software with a 10× lens (Plan Apochromat) to capture the Nissl, FG, GFP, RFP and far red tracers in multichannel photomicrographs; these images were processed for the striatofugal network analysis. High-resolution images of some tissue samples (including the rabies-labelled tissue from Figs. 1e, 3b) were captured with an Andor Dragonfly spinning disk confocal microscope running Fusion software with a 60× lens with a z step of 1 μm.