Nature Neuroscience: doi: /nn Supplementary Figure 1

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1 Supplementary Figure 1 Subcellular segregation of VGluT2-IR and TH-IR within the same VGluT2-TH axon (wild type rats). (a-e) Serial sections of a dual VGluT2-TH labeled axon. This axon (blue outline) has two distinguishable microdomains: an axon terminal (AT) with VGluT2 signal (scattered dark material) and an adjacent area containing TH signal (gold particles, arrowheads). Both compartments establish synapses with postsynaptic structures (orange outlines). The VGluT2 AT establishes an asymmetric synapse (green arrows) with the head of a dendritic spine (sp1), which contains a spine apparatus (sa). The segment of the axon containing TH establishes a symmetric synapse (blue arrows) with a dendritic spine (sp2). Scale bars represent 200 nm.

2 Supplementary Figure 2 Specificity evaluation of anti-vmat2 antibodies by western blot analysis. (a) Graphic map of paav-ef1a-dio-vmat2(myc)-wpre-pa. A Myc epitope was inserted in-frame with VMAT2 at the N-terminus for immunodetection of transgenic VMAT2. (b) VMAT2 immunodetection by different anti-vmat2 antibodies. Western blots in lanes 1-8 from a gel loaded with the same amount of total protein from rat nacc homogenate, and tested with eight different anti-vmat2 antibodies as follows: (1) ab from Abcam; (2) H-V008 from Phoenix Pharmaceuticals, Inc; (3) ab81855 from Abcam; (4) VMAT2- Rb-Af720 from Frontier Institute Co. ltd; (5) from Synaptic Systems; (6) AB1598P from EMD Millipore; (7) SAB from Sigma-Aldrich; (8) EB06558 from Everest Biotech. Seven out of the 8 tested anti-vmat2 antibodies recognize multiple bands with exception of the EB06558 that recognizes two bands with a molecular weight 55 kda (arrow), expected for VMAT2. Lanes 9 and 10 correspond to western blot prepared from total protein from transfected cell homogenates expressing VMAT2 with Myc tag, and probed with anti-vmat2 antibody (EB06558) or anti-myc antibody from EMD Millipore (05-724). Each anti-vmat2 and anti-myc antibodies recognize a single band with similar molecular weight ( 55 kda), as detected in the nacc homogenate.

3 Supplementary Figure 3 VGluT2 and VMAT2 localize to distinct subpopulations of synaptic vesicles (wild type rats). (a) Full length western blots of proteins from isolated vesicles prior to immunoprecipitation, IP (T), and after IP with antibodies against VGluT2 (IP:VGluT2; B1) or VMAT2 (IP:VMAT2; B2). Western blots were immunolabeled (IB) with antibodies against VGluT2, VMAT2 or the vesicular marker synaptophysin. The vesicular nature of each fraction was confirmed by the detection of synaptophysin. VGluT2 and VMAT2 are present in the total pool of vesicles (T). In contrast, VGluT2 is detected only in the sample IP with anti-vglut2 antibody (IP:VGluT2), and VMAT2 is detected only in the sample IP with anti-vmat2 antibody (IP:VMAT2). (b) A model showing accumulation of dopamine by VMAT2 in a vesicle different from the one that accumulates glutamate by VGluT2.

4 Supplementary Figure 4 TH neurons expressing mcherry under the regulation of the TH promoter (TH::Cre mice). (a) Schematic representation of AAV5-DIO-ChR2-mCherry injection into the VTA of TH::Cre mice. (b) Low magnification of a coronal section, which was incubated with an anti-mcherry antibody and hybridized with a TH antisense radioactive riboprobe. Section is seen under bright field (for visualization of mcherry-ir), and under epiluminescence microscope (for visualization of silver grains indicating expression of TH mrna). Bregma mm. Rectangular area in b is shown at higher magnification in (c-e) to display two neurons coexpressing TH-mRNA and mcherry-ir. (f) The bars indicate the frequency (mean + s.e.m.) of neurons containing mcherry-ir or TH mrna from a total of 576 mcherry-ir neurons. Out of these neurons, ± 0.83% have mcherry-ir and TH mrna (t(2) = 17.07, P = , n = 3). Scale bars represent 100 µm (b) and 10 µm (e).

5 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 microdissected: 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).

6 Supplementary Figure 6 Mouse mesoaccumbens neurons establish VGluT2-asymmetric synapses or TH-symmetric synapses (TH::Cre mice). (a) Schematic representation of AAV5-DIO-ChR2-mCherry injection into the VTA of TH::Cre mice. (b) Electron micrograph of a nacc AT from a VTA neuron co-expressing mcherry-ir (scattered dark material) and VGluT2-IR (gold particles, green arrowheads) making an asymmetric synapse (green arrow) with a dendrite (De). (c) Electron micrograph of an AT from a VTA neuron co-expressing mcherry-ir (scattered dark material) and TH-IR (gold particles, blue arrowhead) making a symmetric synapse (blue arrow) on a dendrite (De). (d) Schematic representation of nacc inputs from VTA neurons expressing mcherry-ir under the regulation of TH promoter (TH-ChR2-mCherry mice). (e-g) A single AT from a VTA-VGluT2 neuron synapses on several postsynaptic structures. Serial sections of a single AT derived from a VTA neuron expressing mcherry-ir (scattered dark material) and containing VGluT2-IR (gold particles, green arrowheads) establishes asymmetric synapses (green arrows) with several postsynaptic structures, two dendritic spines (sp1 and sp2) and one dendrite (De). Scale bars represent 200 nm (b,c,e,f,g).

7 Supplementary Figure 7 TH immunoreactivity (TH-IR) is distributed in the entire axon and axon terminal lacking VGluT2 (TH::Cre mice). (a) Schematic representation of nacc inputs from VTA neurons expressing mcherry immunoreactivity (mcherry-ir) under the regulation of the TH promoter (TH-ChR2-mCherry mice). (b-c) Immunofluorescence detection of mcherry (red), TH (blue), and VGluT2 (green) in the nacc. Note mcherry detection throughout the axon (red in b), and VGluT2-IR restricted to a neighboring terminal-like structure lacking mcherry-ir (arrowheads in b). Under 2-D view, TH-IR appears to be present in segments within the mcherry-ir axon and terminal (arrow in b). However, the TH distribution throughout the axon and axon terminal is better seen after 3-D reconstruction from Z-stack of confocal microscopy images of triple labeled nacc (c). (d,e) Electron micrographs of serial sections of a TH-only axon containing mcherry-ir (scattered dark material) and TH-IR (gold particles, arrowheads). One axon terminal (AT) from this axon made a symmetric synapse (arrow) on a dendrite (De, orange outline). Scale bars represent 2 µm (b) and 200 nm (d,e).

8 Supplementary Figure 8 Compartmentalization of the storage and release of glutamate to axon terminals and the storage and release of dopamine to the adjacent axonal segment. Mesoaccumbens synapses established by 3 different classes of mesocorticolimbic neurons (TH-only, VGluT2-only and VGluT2-TH neurons), and subcellular segregation for dopaminergic and glutamatergic signaling. Axon terminals containing dopamine vesicles (blue circles) and establishing symmetric synapses on the side of dendritic spines (sp1) are originated from either TH-only or VGluT2-TH neurons. In contrast, axon terminals containing glutamate vesicles (green circles) derived from VGluT2-only or VGluT2-TH neurons make asymmetric synapses on the head of dendritic spines (or dendrites, no representation in this diagram). A single axon terminal from these glutamatergic terminals can establish asymmetric synapses with several postsynaptic targets (sp2 and sp3). A dual glutamatergic-dopaminergic axon from a VGluT2-TH neuron illustrates the compartmentalization of the storage and release of glutamate to axon terminals and the storage and release of dopamine to the adjacent axonal segment.