Supplementary Figure 9: Expression of vesicular acetylcholine transporter (VAChT) in YFP-labeled axons.

Axons expressing YFP in the VTA from both PPN- and LDT-transduced animals were tested for the presence of vesicular transporters by immunofluorescence. VAChT immunofluorescence was detected in the majority of axonal varicosities expressing YFP (arrowheads), as has also been reported in the striatum (Dautan et al (2014) J Neurosci 34, 4509-4518). In contrast, the vesicular glutamate transporter 2 (VGluT2) was never observed to co-localize with YFP in axonal varicosities, even though we detected large numbers of VGluT2-positive varicosities within the VTA. Thus, in agreement with in situ hybridization data (Wang and Morales (2009) Eur J Neurosci 29,340-358) the axons of cholinergic neurons from the brainstem lack VGluT2.