Context Striatal cholinergic interneurons (CINs) express vesicular transporters for acetylcholine (VAChT) and glutamate (VGLUT3) and consequently regulate the striatal network with both acetylcholine and glutamate. The synaptic vesicles of CINs have the potential to store and release simultaneously or independently acetylcholine and/or glutamate to regulate striatal activities. Yet, the molecular mechanisms through which striatal cholinergic interneuron releases these two classical neurotransmitters are still largely unknown. In the present proposal, we hypothesize that VAChT and VGLUT3 interact directly or indirectly through partner proteins to regulate acetylcholine/glutamate cotransmission. To address this hypothesis, it is critical to identify the synaptic partners of VAChT and VGLUT3, to determine their localization in synaptic vesicles in relation to VAChT and VGLUT3 and to analyze whether they are involved in acetylcholine and glutamate release. Objectives The central objective of this proposal is to understand the molecular, morphological and functional complexity of acetylcholine/glutamate co-transmission from CINs involving partner proteins of VAChT and VGLUT3. Using a multidisciplinary approach combining computational analyses, super-resolution microscopy, development of new pharmacological tools targeting vesicular glutamate transporters and optogenetics stimulation of CINs coupled to measures of acetylcholine and glutamate release, our ALLEGRO proposal will be organized along three aims: Aim 1. Computational analysis of molecular partners of VAChT and VGLUT3 Using different computational approaches, we will : 1. identify the protein partners of VAChT and VGLUT3 by a cutting-edge large-scale screening of pairs of human protein sequences based on deep learning and by a coevolution analysis addressing the identification of interacting residues between potential partners for VAChT and VGLUT3. 2. reconstruct the protein complexes with recent deep learning approaches for 3D reconstruction. 3. predict the binding sites of the interacting proteins and the modelling through molecular docking of the physical interaction of the VAChT and VGLUT3 domains with identified protein partners. Aim 2. Distribution of the selected partners in VAChT and VGLUT3-expressing synaptic vesicles in CINs and analysis of interactions of partners with VAChT or VGLUT3 Using innovative microscopic approaches, we will : 1. identify which of the partner proteins identified in Aim 1 are present in VAChT and VGLUT3-immunopositive synaptic vesicles by super resolution STED microscopy. 2. measure interactions between partner protein and VAChT or VGLUT3 in synaptic vesicles of cholinergic interneurons by Fluorescence lifetime imaging microscopy combined with Förster resonance energy transfer (FLIM-FRET). Aim 3. Functional role of molecular partners in acetylcholine and glutamate striatal release We will test the causal role of these partners by combining silencing strategies with the monitoring of acetylcholine and glutamate release from striatal cholinergic interneurons. We will : 1. Evaluate the protein partner role on acetylcholine and glutamate uptake on purified synaptic vesicles. 2. Develop glutamate sensors and validate acetylcholine biosensor (m4-based genetically encoded). 3. Characterize acetylcholine and glutamate release from striatal cholinergic interneurons. 4. Determine the effect of silencing of molecular partners on acetylcholine and glutamate release using shRNA. Conclusion This project will thus unravel the molecular actors of neuronal acetylcholine/glutamate cotransmission through the study of acetylcholine/glutamate release from striatal cholinergic interneurons. Beyond its fundamental dimension, the ALLEGRO project should also have profound impact on our understanding of the striatal network whose dysfunction is associated with substance use or eating disorders and possibly accelerates biomarker and therapeutic discovery.