Supplementary Figure 5: Mesoaccumbens axons from VGluT2 neurons expressing mCherry under the regulation of the VGluT2 promoter (VGluT2::Cre mice).

(a) Schematic representation of AAV5-DIO-ChR2-mCherry injection into the VTA of VGluT2::Cre mice. (b) Double labeled VTA cryosections for the detection of mCherry- IR or TH-IR. From this preparation, three different classes of neurons were laser micro-dissected: cells expressing only mCherry-IR (mCherry-IR⁺/TH-IR^– (arrows)), cells co-expressing mCherry-IR and TH-IR (mCherry-IR ⁺/TH-IR⁺ (arrowheads)) and cells expressing only TH-IR (mCherry-IR^–/TH-IR⁺ (stars)). (c) Percentage of neurons containing detectable levels of VGluT2 mRNA or TH mRNA in each of the three classes of laser micro-dissected neurons: mCherry-IR⁺/TH-IR^– (n = 30 neurons), mCherry-IR⁺/TH-IR⁺ (n = 35 neurons), and mCherry-IR^–/TH-IR⁺ (n = 40 neurons). Each mCherry-IR⁺/TH-IR^– neuron was confirmed to have VGluT2 mRNA and lack TH mRNA. Each mCherry-IR^–/TH-IR⁺ neuron was confirmed to have TH mRNA and lack VGluT2 mRNA. The majority of dissected dual mCherry-IR⁺/TH-IR⁺ neurons found in the medial VTA have both VGluT2 mRNA and TH mRNA (97%, 34 out of 35 neurons). (d-g) Subcellular Segregation of TH and VGluT2 in the nAcc of VGluT2-ChR2-mCherry mice. Detection of mCherry (red) under the VGluT2 promoter, VGluT2-IR (green), and TH-IR (blue) in the VTA and nAcc. (d) Low magnification of the VTA showing cells with mCherry within the midline aspects of this structure. (e) High magnification of the area delimited with white box in (d) showing a dual labeled mCherry/TH neuron (1), and single labeled mCherry (2) and single labeled TH (3) neurons. (f) Within the nAcc, mCherry-IR under the VGluT2 promoter is observed along an axon (arrows) and within terminal-like structures (arrowheads), which contain VGluT2-IR. TH-IR is adjacent to the VGluT2-IR axon terminal. (g) Segregation among the VGluT2 terminal-like structures and the TH-axon segment within the same nAcc axon from a VGluT2-TH neuron is better seen in this 3-D reconstruction from Z-stack confocal microscopy images of triple labeled nAcc. Scale bars represent 10 µm (b), 100 µm (d), 5 µm (e) and 2 µm (f).