BRIDGING SYNAPTIC MOLECULAR ARCHITECTURE TO FUNCTION: A MULTIMODAL APPROACH TO STUDY HETEROGENEITY OF CONNECTIONS

Brain function relies on the precise organization of neuronal connections, which is disrupted in neurodevelopmental disorders such as autism spectrum disorders (ASD). These connections exhibit substantial heterogeneity in synapse size, strength and molecular architecture, yet are often studied primarily at the population level. Consequently, the determinants underlying connection diversity remain poorly understood. We hypothesize that synaptic heterogeneity reflects discrete connection states defined by coordinated functional, structural, and molecular features, and may respond differently to physiological, pathological, and therapeutic perturbations. Addressing this heterogeneity in tissue therefore requires an integrated, multimodal approach.

We developed a strategy combining patch-clamp recordings, single-cell transfection, live imaging, and expansion microscopy in CA3-CA3 connections in organotypic hippocampal slices to study connection states. This approach enables: 1/ genetic manipulation and imaging of pre- and postsynaptic elements at individual connections; 2/ simultaneous monitoring at structural, molecular, and functional levels; 3/ assessment of disease-related perturbation on state modulation. As proof of principle, we overexpressed Neuroligin-1, an ASD-associated cell-adhesion molecule affecting nanodomains, spine structure, and AMPAR function. Our results show pronounced functional heterogeneity across CA3-CA3 connections and distinct functional connection states by correlating multiple pre- and postsynaptic parameters via unsupervised clustering, with potential links to synapse morphology and RIM1/2-PSD95 nanocolumn number. Preliminary data indicate that neuroligin-1 overexpression biases synaptic states, increasing synaptic strength through nanocolumns rather than synapse number. In conclusion, this multimodal approach enables correlative analysis of synaptic function, structure, and molecular organization at single-connection resolution in intact tissue, providing a platform to study synaptic heterogeneity and dysfunction in disease.