Using molecular proximity to refine synapse quantification in neuronal cultures.

Synapses are specialized connections between neuronal cells that determine the wiringpatterns, which are essential for memory and cognition. Synaptic dysfunction is a commonpathological hallmark of neurodevelopmental and - degenerative conditions. Thus, accurateand reliable quantification of synaptic state and number in neuronal networks is crucial. Inprevious work, we have shown that primary neuronal cultures from rodents preserve manymorphological and functional properties of in vivo neuronal networks and can be used toevaluate the impact of chemo-genetic perturbations on synaptic state. However, the largenumber of synapses on the one hand, and the variable specificity of existing microscopytechniques on the other hand, makes synapse quantification in these cultures a balancing actbetween accuracy and throughput. To improve both, we propose to make use of a novelmethod, termed Proximity Ligation Assay (PLA), that can directly visualize molecularinteractions using a standard fluorescence microscope. To do so, we will first identify anoptimal set of trans-synaptic protein interactors. Then, we will validate the PLA technique anduse it to measure synapse density after application of targeted perturbations. By applying PLAto trans-synaptic proteins, we aim at detecting true synapses with superior specificity. Thisshould enhance the sensitivity with which we can detect changes in synapse density, andtherefore it has the potential to accelerate the identification of synaptic modulators in ourongoing screening efforts.

Keywords:PROTEIN-PROTEIN INTERACTIONS, SYNAPS, NEURONAL CULTURES, NEURODEGENERATIVE DISORDERS

Disciplines:Neurophysiology, In vitro testing