Figure 1. Experimental design for dopamine connectomics.

(A) Adeno-associated viruses (AAVs) expressing Cre-dependent Apex2 were bilaterally injected into the ventral tegmental area (VTA) of transgenic mice expressing Cre in dopamine transporter positive neurons (Slc6a3 (DAT)-CRE). (B) Approximately 4 weeks after AAV injections, vibratome sections (~300 µm thick) show strong Apex2 labeling in VTA and nucleus accumbens (NAc) after staining with 3’3’-diaminobenzidene (DAB) and hydrogen peroxide (H₂O₂) before electron microscopy (EM) processing (see Materials and methods). Black arrows point to an Apex2-positive region and black arrowhead points to an Apex2-negative region. Green rectangles highlight the VTA and medial shell of the NAc region dissected out and processed for EM. Red ovals highlight areal landmarks to ensure the same region was dissected across all animals. (C–D) Representative EM images of cytoplasmic (C, top row) and mitochondrial (D, bottom row) Apex2+ DA neurons. Left panel: Apex2 soma in the VTA (red arrows). Middle panel: Apex2 top panel shows a DA dendritic spine forming a synapse (purple arrow) with presynaptic bouton (orange) in the VTA, and the bottom panel shows a narrow DA dendrite expressing mitochondrial Apex. Right panel: Apex2 axon in the NAc with narrow (yellow arrow) and thick varicosities (yellow arrowhead). Cytosolic Apex2 does not obscure mitochondria (top panel, asterisk), and mitochondrial Apex2 (bottom) only fills up mitochondria. Scale bar = C,D, soma = 10 µm, dendrite and axon = 1 μm.