An Organotypic Spinal Cord‐Dorsal Root Ganglion‐Skeletal Muscle Coculture of Embryonic Rat. II. Functional Evidence for the Formation of Spinal Reflex Arcs In Vitro

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Abstract
Electrical properties of motoneurons, muscle fibres and dorsal root ganglion (DRG) cells were studied in an organotypic coculture of embryonic rat spinal cord, dorsal root ganglia and skeletal muscle. The motoneurons were identified by their morphology and position in culture. Their size and input conductance were significantly larger than those of spinal interneurons. Intracellular current injection evoked action potentials in all motoneurons, but only evoked stable repetitive firing patterns in some. Excitability was correlated to somatic size and the rate of spontaneous excitatory input. It is suggested that the somatic growth and the increase in excitability is regulated by the excitatory afferents. The motoneurons showed spontaneous excitatory and inhibitory postsynaptic potentials and action potentials which disappeared with the application of various agents known to inhibit excitability or excitatory synaptic transmission. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs respectively) were distinguished by their shape, reversal potential and pharmacology. IPSPs could be depolarizing or hyperpolarizing in different cells. A higher percentage of cells with hyperpolarizing IPSPs was found in older cultures and in the presence of skeletal muscle, suggesting a reversal of the polarity of IPSPs with development. The spontaneous muscle contractions observed in the cultures could be due either to innervation, spontaneous oscillations of the membrane potential, or electrical coupling between neighbouring fibres. A small percentage of DRG cells showed spontaneous action potentials, all of which were found in cultures with spontaneous muscle contractions. The electrical stimulation of DRG afferents evoked mono- and polysynaptic EPSPs in motoneurons, endplate potentials and muscle contractions. The stimulation of the ventral horns evoked endplate potentials and muscle contractions via mono- or polysynaptic pathways. Together these results indicate that appropriate and functional contacts were established in the culture between myotubes and DRG cells, between DRG cells and motoneurons, and between motoneurons and muscle fibres.