Figure 2.

RV-mediated retrograde tracing of projections from the VTA and SN to the IP. Inputs to the IP were retrogradely labeled with RV as described in Figure 1 (Case r1), producing a nuclear GFP label in presynaptic neurons. Serial 25-μm sections were examined at 100-μm intervals through the VTA and SN, at levels from bregma −2.8 to bregma −3.6 in a standard atlas (Paxinos and Franklin, 2001), and imaged as Z-stacks. DA neurons were labeled by immunostaining for TH, and all cells were counterstained for nucleic acids with DAPI. TH-positive neurons were counted if the TH immunostaining formed a cytoplasmic circle or semicircle around a nuclear hole, indicating that the labeled neuron was in the plane of section. Areas of interest were drawn manually around the area containing the VTA+SN (outlines), and all of the nGFP and TH-immunoreactive cells in the left hemisphere of these structures were counted. A, RV nGFP and TH labeling in the rostral VTA/SN. In the region of interest outlined, 9/1606 DAPI-labeled cells were labeled with GFP (0.56%). B, C, RV nGFP and TH labeling in the caudal VTA/SN. In the region of interest outlined, 83/2595 DAPI-labeled cells were labeled with GFP (3.2%). Co-localization of nGFP and TH was very rarely observed. In C, the arrow indicates an example of an nGFP-labeled nucleus which may be in a TH-labeled neuron. The arrowhead indicates an nGFP nucleus which overlies TH-labeling but does not appear to be the nucleus of the TH-expressing cell. D, Count of nGFP labeled, TH-positive, and dual-labeled neurons in the left VTA/SN in sections at the designated coordinates in Case r1. IP, interpeduncular nucleus; ml, medial lemniscus; MM, medial mammillary nucleus; PN, paranigral nucleus; SNC, substantia nigra, pars compacta; SNR, substantia nigra, pars reticulata; VTA, ventral tegmental area. Scale bar: 200 μm (A).