Molecular diversity of glutamatergic and GABAergic synapses from multiplexed fluorescence imaging

Abstract

Neuronal synapses contain hundreds of different protein species important for regulating signal transmission. Characterizing differential expression profiles of proteins within synapses in distinct regions of the brain has revealed a high degree of synaptic diversity defined by unique molecular organization. Multiplexed imaging of in vitro rat primary hippocampal culture models at single synapse resolution offers new opportunities for exploring synaptic reorganization in response to chemical and genetic perturbations. Here, we combine 12-color multiplexed fluorescence imaging with quantitative image analysis and machine learning to identify novel synaptic subtypes within excitatory and inhibitory synapses based on the expression profiles of major synaptic components. We characterize differences in the correlated expression of proteins within these subtypes and we examine how the distribution of these synapses is modified following induction of synaptic plasticity. Under chronic suppression of neuronal activity, phenotypic characterization revealed coordinated increases in both excitatory and inhibitory protein levels without changes in the distribution of synaptic subtypes, suggesting concerted events targeting glutamatergic and GABAergic synapses. Our results offer molecular insight into the mechanisms of synaptic plasticity.

Significance Statement An immense number of proteins are present at synapses to regulate synaptic function. Recent efforts characterizing synaptic protein expression patterns suggest their differential expression gives rise to diverse synapse sub-populations. In this work, we use multiplexed fluorescence imaging with advanced image analysis to detect and quantify protein levels using CellProfiler. We apply our technique to develop a robust approach for unbiased synaptic subtype identification based on protein expression profiles using UMAP dimensional reduction and HDBScan clustering. Finally, we apply this approach to examine synaptic diversity in cultured hippocampal neurons and examine the molecular events of 11 proteins at excitatory and inhibitory synapses following synaptic scaling.