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All-optical voltage imaging-guided postsynaptic single-cell transcriptome profiling with Voltage-Seq
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DOI：10.1038/s41596-024-01005-y.png)
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Neuronal pathways recruit large postsynaptic populations and maintain connections via distinct postsynaptic response types (PRTs). Until recently, PRTs were accessible as a selection criterion for single-cell RNA sequencing only through probing by low-throughput whole-cell electrophysiology. To overcome these limitations and target neurons on the basis of specific PRTs for soma collection and subsequent single-cell RNA sequencing, we developed Voltage-Seq using the genetically encoded voltage indicator Voltron in acute brain slices from mice. We also created an onsite analysis tool, VoltView, to guide soma collection of specific PRTs using a classifier based on a previously acquired database of connectomes from multiple animals. Here we present our procedure for preparing the optical path, the imaging setup and detailing the imaging and analysis steps, as well as a complete procedure for sequencing library preparation. This enables researchers to conduct our high-throughput all-optical synaptic assay and to obtain single-cell transcriptomic data from selected postsynaptic neurons. This also allows researchers to resolve the connectivity ratio of a specific pathway and explore the diversity of PRTs within that connectome. Furthermore, combining high throughput with quick analysis gives unique access to find specific connections within a large postsynaptic connectome. Voltage-Seq also allows the investigation of correlations between connectivity and gene expression changes in a postsynaptic cell-type-specific manner for both excitatory and inhibitory connections. The Voltage-Seq workflow can be completed in similar to 6 weeks, including 4-5 weeks for viral expression of the Voltron sensor. The technique requires knowledge of basic laboratory techniques, micromanipulator handling skills and experience in molecular biology and bioinformatics.
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