Smart-seq2 of spinal cord motor neurons reveals transcriptomic changes in mice with spinal cord injury after synergistic bidirectional electrical stimulation of specific frequencies

The characteristics of electrical signals-mediated neural circuits reconstruction remain poorly understood. We initially developed a spinal-muscle synergistic bidirectional electrical stimulation (SBES) protocol to mimic feedforward and feedback electrical signals in spinal sensorimotor circuits. The results indicate that sensorimotor circuits could be precisely reassembled in structure and function levels by 10-20 Hz SBES. To gain mechanistic insights into the reassembly of the spinal sensorimotor circuits with 10-20 Hz SBES, we performed gene expression profiling analysis in motoneurons. Gene ontology (GO) analysis showed that one group of GO terms accounted for a large fraction of the upregulated transcripts. This group includes axon growth-related cellular functions, such as cell adhesion, regulation of cell growth, and cell projection assembly. Statistical analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed sixteen signaling pathways were involved, two of which were related to the regulation of axonal regeneration (PI3K-Akt signaling pathway and cAMP signaling pathway). This study provides insights into neural signal decoding during spinal sensorimotor circuit reconstruction. Spinal cord motoneurons of mice in the sham group (n = 3 mice), untrained group (n = 3 mice), 10-20 Hz SBES group (n = 3 mice), were labeled with TRDA and sacrificed 4 weeks after SCI. The freshly isolated spinal cord was sliced on a Vibratome (Leica, Germany) into 200- to 300-μm-thick coronal sections. Fluorescence-labeled motoneurons were visualized under a fluorescence microscope (Zeiss) using a miniature operating system (Eppendorf, Germany), and the motoneurons were aspirated by glass electrodes and placed in preservation solution (Invitrogen, USA). TRDA-labeled neurons were individually homogenized in 1 ml TRIzol (Invitrogen), and RNA was extracted with a RNeasy Mini Kit (Qiagen) or equivalent. Total RNA (1 μg) was used to synthesize double-stranded cDNA.