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.
Highlights
Synapses contain complex proteomes that organize into multiprotein signaling complexes (Husi et al, 2000; Collins et al, 2006)
Using HDBSCAN applied to the uniform manifold approximation and projection (UMAP) output, we identified six unique clusters of synaptic subtypes (Fig. 2B,C)
The results indicate that TTX treatment does not produce unique synaptic subtypes, but it does change the number of synapses present within each cluster, reducing the number of synapses in clusters defined by low protein expression
Summary
Synapses contain complex proteomes that organize into multiprotein signaling complexes (Husi et al, 2000; Collins et al, 2006). Synapses are ;2 mm in size; and while advances in mass spectrometry-based imaging have achieved 1mm resolution (Zavalin et al, 2015), the majority of commercial matrix-assisted laser desorption/ionization (MALDI) mass spectrometers are not sufficiently accurate for examination of individual synapses. Microscopy techniques, such as immunofluorescence, remains the optimal technique for the examination of individual synapses. Conventional fluorescence microscopy is generally limited to four channels as a result of the maximal spectral resolution of organic and biomolecular fluorophores This limitation presents a challenge to comprehensive analysis of synaptic architecture because of the large number of different protein species within each synapse
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