Smart-seq2 of spinal cord motoneurons in spinal cord injury model  after dual electrical stimulation with specific frequencies
Electrical stimulation can augment or modify neuronal function and can have therapeutic benefits for certain neurological disorders. There is evidence that enhancing spinal excitability with either epidural or transcutaneous stimulation can restore some volitional motor output after spinal cord injury (SCI). Lumbosacral epidural stimulation temporarily improves locomotor and autonomic function in both rodents and humans with SCI. When combined with overground locomotor training enabled by a weight-supporting device, epidural electrical stimulation (EES) promotes extensive reorganization of residual neural pathways that improves locomotion after stopping stimulation. However, the exact mechanism underlying the reconstruction of spinal cord neural circuits with electrical stimulation is not yet known. Thus, we developed a epidural electrical and muscle stimulation(EEMS) system at the interface of the spinal cord and muscle to mimic feedforward and feedback electrical signals in spinal sensorimotor circuits. Using methods of motor function evaluation, neural circuit tracing and neural signal recording, we discovered a unique stimulus frequency of 10-20 Hz under EEMS conditions that was required for structural and functional reconstruction of spinal sensorimotor circuits. Single-cell transcriptome analysis of EEMS activated motoneurons characterized molecular networks involved in spinal sensorimotor circuit reconstruction. This study provides insights into neural signal decoding during spinal sensorimotor circuit reconstruction, and indicates a technological approach for the clinical treatment of SCI. Overall design: Spinal cord motoneurons of mice in the sham group (n = 3 mice), untrained group (n = 3 mice), 10-20 Hz MS group (n = 3 mice),  10-20 Hz SCS group (n = 3 mice), 10-20 Hz DES group (n = 3 mice), 1-40 Hz DES group (n = 3 mice) were labeled with TRDA and sacrificed 4 weeks after SCI (DES was renamed EEMS). 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.