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

TL;DR

This study used super-resolution microscopy to analyze the distribution of VAChT and VGLUT3 in striatal cholinergic synaptic vesicles, revealing complex colocalization and segregation patterns that suggest diverse neurotransmitter release mechanisms.

Contribution

It provides the first detailed super-resolution analysis of VAChT and VGLUT3 distribution in single synaptic vesicles, uncovering their potential for independent or simultaneous neurotransmitter storage.

Findings

34% of vesicles contain both VAChT and VGLUT3

40% of vesicles express only VAChT

26% of vesicles contain only VGLUT3

Abstract

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.