  Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and
glutamate (Glut) to regulate the striatal network since they express vesicular
transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut
are released simultaneously and/or independently from cholinergic varicosities
is an open question. The answer to that question requires the multichannel
detection 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 in
CINs SVs. Nearest-neighbor distances analysis between VAChT and
VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT
and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs
contain only VGLUT3. These results suggest that SVs from CINs have the
potential to store simultaneously or independently ACh and/or Glut. Overall,
these morphological findings support the notion that CINs varicosities can
signal with either ACh or Glut or both with an unexpected level of complexity.
