Figure 1.
Comparative localization of α7 nAChRs (green), glutamatergic axon terminals (A-D, red; E, VGluT1, red, VGluT2, blue), and dopaminergic neurons (dendrites and cell bodies, blue) in aldehyde-fixed sections through the adult rat VTA. A-D, α7 nAChRs were labeled with Alexa Fluor 488-conjugated αBgt; glutamatergic axon terminals were labeled with anti-VGluT1 or -VGluT2, followed by Alexa Fluor 546-conjugated secondary antibody; dopaminergic neurons were labeled with anti-tyrosine hydroxylase, followed by Alexa Fluor 633-conjugated secondary antibody. E, VGluT1 and VGluT2 terminals were labeled with rabbit anti-VGluT1 and guinea pig anti-VGluT2, respectively, followed by Alexa Fluor 546-conjugated anti-rabbit IgG and Alexa Fluor 633-conjugated anti-guinea pig IgG. A, B, Low-magnification images of VGluT1 (A) and VGluT2 (B) immunoreactivites in the VTA and surrounding nuclei. VGluT1 labeling is particularly strong in the interpeduncular nucleus (ipn), with moderate levels in the VTA (A). In contrast, VGluT2 immunoreactivity is stronger in the VTA but apparently absent from the ipn (B). Note that no αBgt signal was detected at this magnification. C, Higher-magnification image of the VTA labeled for both VGluT1 and VGluT2. Three populations of αBgt binding sites are revealed, associated with VGluT-immunoreactive axon terminals (yellow arrows), dopaminergic neurons (blue arrows), and nondopaminergic cells (green arrows). Note that not all glutamatergic axon terminals express detectable levels of α7 nAChRs (red arrows). D, Competition control, in which preincubation with an excess of unconjugated αBgt obliterated fluorescent αBgt labeling. Note the lack of green and yellow fluorescent spots. E, Dual labeling for VGluT1 (rabbit anti-VGluT1, red) and VGluT2 (guinea pig anti-VGluT2, blue) immunoreactivity in the VTA; no detectable colocalization of the two VGluT isoforms within the same structures was observed. snc, Substantia nigra pars compacta. Scale bars: A, B, 200 μm; C-E, 20 μm.