AimStriatal cholinergic interneurons (CINs) use both acetylcholine (ACh) and glutamate (Glu) to regulate the striatal network. CINs express vesicular transporters for acetylcholine (VAChT) and Glu (VGLUT3). We recently demonstrated, using super resolution STimulated Emission Depletion microscopy (STED) microscopy, that part of cholinergic synaptic vesicles (SVs) contains both VAChT and VGLUT3, whereas others express only VAChT or only VGLUT3. CINs SVs are thus able to release either ACh or Glu or both. However, the link between neurotransmitters accumulation into SVs and their final exocytosis is unknown. Proteins of the SNARE complex, including vesicular associated membrane proteins (VAMPs), facilitates the SVs fusion with presynaptic terminals and the release of neurotransmitters. SNARE complex dysfunction might contribute to synaptic impairment. VAMP2 and VAMP7 were shown to facilitate ACh and Glu release. They might involve direct interaction with VAChT or VGLUT3. Our aim was thus to determine the distribution of VAMP2 or VAMP7 in VAChT or VGLUT3 expressing SVs. MethodsWe used super-resolution STED microscopy to characterize and quantify the distribution of VAMP2 and VAMP7 in VAChT and or VGLUT3 expressing SVs. ResultsFluorescent in situ hybridization demonstrated that VAMP2 and VAMP7 are expressed by CINS. We observed that the canonical VAMP2 is expressed in a large part of VAChT and VGLUT3 striatal SVs (63% and 83%). In nucleus accumbens, ≈25% of VAChT and VGLUT3-immunopositive SVs also express VAMP7-positive. ConclusionOur results suggest that VAMP2 and VAMP7 may be putative molecular partners of ACh-Glu cotransmission.