Loss of inhibitory synapses causes locomotor network dysfunction of the rat spinal cord during prolonged maintenance...

The isolated spinal cord of the neonatal rat is widely employed to clarify the basic mechanisms of network development or the early phase of degeneration after injury. Nevertheless, this preparation survives in Krebs solution up to 24 h only, making it desirable to explore approaches to extend its survival for longitudinal studies. The present report shows that culturing the spinal cord in oxygenated enriched Basal Medium Eagle (BME) provided excellent preservation of neurons (including motoneurons), glia and primary afferents (including dorsal root ganglia) for up to 72 h. Using DMEM medium was unsuccessful. Novel characteristics of spinal networks emerged with strong spontaneous activity, and deficit in fictive locomotion patterns with stereotypically slow cycles. Staining with markers for synaptic proteins synapsin 1 and synaptophysin showed thoroughly weaker signal after 3 days in vitro. Immunohistochemical staining of markers for glutamatergic and glycinergic neurons indicated significant reduction of the latter. Likewise, there was lower expression of the GABA-synthesizing enzyme GAD65. Thus, malfunction of locomotor networks appeared related to loss of inhibitory synapses. This phenomenon did not occur in analogous opossum preparations of the spinal cord kept in vitro. In conclusion, despite histological data suggesting that cultured spinal cords were undamaged (except for inhibitory biomarkers), electrophysiological data revealed important functional impairment. Thus, the downregulation of inhibitory synapses may account for the progressive hyperexcitability of rat spinal networks despite apparently normal histological appearance. Our observations may help to understand the basis of certain delayed effects of spinal injury like chronic pain and spasticity.