Synaptic diversity is crucial for neuronal function. The heterogeneity of individual synaptic proteomes in a neuron underlies its input integration, compartmentalization of function, and neuronal plasticity during learning. The canonical classification of synapses based on neurotransmitter systems (e.g. the generic excitatory or inhibitory synapses) has become increasingly inadequate to explain the diverse synaptic responses in electrophysiology and plasticity. Without quantifying the variability of molecular combinations in synapses, our basic understanding of synapses is incomplete. Recently single-molecule localization microscopy has created an opportunity to interrogate individual synapses across a whole neuron. DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) is ideally suited for localizing and quantifying protein copies of interest compared to conventional stochastic optical reconstruction microscopy (STORM) because of its well-defined blinking kinetics and absence of photobleaching issues. This proposal aims to quantify the proteomic heterogeneity of synapses in individual neurons using DNA-PAINT and to interrogate the relationship between plasticity and synaptic diversification using multiple approaches. It will address the following:
1. The differential localization, variability, and stoichiometry of five representative synaptic proteins in the synapses of a neuron
2. The correlation between synapse activity and their proteomes
3. A potential change in synaptic diversity after single-spine plasticity
4. The effects of global homeostatic scaling on synaptic diversity
5. The remodeling of synapse diversity by newly synthesized proteins