GENETIC AND FUNCTIONAL CHARACTERIZATION OF DISSOCIATED SPINAL CORD NEURONS USING HIGH-DENSITY MICROELECTRODE ARRAYS

The spinal cord relays sensory and motor information between the brain and the periphery through well-defined circuits. Although dysfunction of these circuits underlies chronic pain and motor impairment, their analysis is complicated by the diversity of neuronal subtypes and complex connectivity. Dissociated cultures serve as a simplified experimental model for spinal cord networks. However, little is known about whether cell-type identity and functional diversity are preserved in vitro. Here, we investigated genetic and functional properties of dissociated rat spinal cord neurons using high-density microelectrode arrays (HD-MEAs) combined with single-cell transcriptomic analyses. Neurons isolated from the dorsal horn were cultured on HD-MEAs, allowing large-scale extracellular recording with high spatial resolution. Single-cell RNA sequencing showed that neuronal subtypes corresponding to those observed in vivo were preserved in the cultured neurons. Spontaneous synchronized bursting was observed approximately ten days after seeding, suggesting the formation of synaptic connections. Extracellular signals were separated into single-unit activities by spike sorting, allowing quantitative analysis of waveforms, firing patterns, and functional connectivity for individual neurons. Following electrical recording, neurons were fixed and subjected to spatial transcriptomic analysis, enabling direct correspondence between neuronal activity and gene expression profiles. Cell types were classified based on gene expression patterns, and their functional contributions to network dynamics were evaluated. Specific neuronal subtypes were found to be associated with the emergence of synchronized activity. These results suggest that HD-MEAs represent a useful approach for linking cell-type identity with functional properties in spinal cord cultures.