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Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) andglutamate (Glut) to regulate the striatal network since they express vesiculartransporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glutare released simultaneously and/or independently from cholinergic varicositiesis an open question. The answer to that question requires the multichanneldetection of vesicular transporters at the level of single synaptic vesicle(SV). Here, we used super-resolution STimulated Emission Depletion microscopy(STED) to characterize and quantify the distribution of VAChT and VGLUT3 inCINs SVs. Nearest-neighbor distances analysis between VAChT andVGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChTand VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVscontain only VGLUT3. These results suggest that SVs from CINs have thepotential to store simultaneously or independently ACh and/or Glut. Overall,these morphological findings support the notion that CINs varicosities cansignal with either ACh or Glut or both with an unexpected level of complexity.

frontiers in molecular neuroscience

Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic  varicosities suggests colocalization and segregation of the two transporters  in synaptic vesicles