Modulation of the activity of the locomotor central pattern generator in the rat spinal cord in vitro

The present study has investigated the rhythmic properties of spinal networks in the neonatal rat spinal cord in vitro, by means of intracellular recordings from single motoneurons (MNs) and extracellular recordings from ventral and dorsal roots (VRs;DRs). Distinct subclasses of metabotropic glutamate receptors (mGluRs) on rat spinal neurons mediated complex facilitatory and inhibitory effects. The class I agonist DHPG evoked MN depolarization (via the mGluR1 subtype) mostly at network level and generated sustained, network-dependent oscillations (via the mGluR5 subtype). DHPG also decreased the amplitude of reflex responses induced by DR stimuli, an effect unrelated to depolarization but dependent on glycinergic transmission. Single reflex responses were insensitive to group I mGluRs antagonists, suggesting no phasic activation of group I receptors during this process. Finally, DHPG depressed the glycinergic recurrent IPSP, perhaps by impairing the cholinergic input to Renshaw cells. Thus, the cellular distribution of those mGluRs at strategic circuit connections may determine the functional outcome of the network in terms of excitation or inhibition. Activation of class II or III mGluRs had no direct action on MNs although it strongly blocked evoked synaptic transmission, presumably acting at presynaptic level. To extend our understanding of the network-based properties, which enable a neuronal circuit to produce sustained electrical oscillations, we explored the potential contribution of mGluRs to generate rhythmic discharges. During cumulative depolarization or fictive locomotion, spinal mGluRs were minimally activated by endogenous glutamate, although they could potently modulate these responses once activated by exogenously applied mGluR agonists. Disinhibited bursting was associated with the activation of mGluR1 receptors (facilitating network excitability) and of group II mGluRs (depressing it). We investigated if the K+ channel blocker 4-aminopyridine (4-AP) could facilitate spinal locomotor networks in addition to its well-known effect on motor nerve conduction. 4-AP produced synchronous VR oscillations, which did not develop into fictive locomotion. These oscillations had network origin, required intact glutamatergic transmission and were probably amplified via electrotonic coupling. 4-AP slightly increased input resistance of lumbar MNs, without affecting their action or resting potentials. DR evoked synaptic responses were enhanced by 4-AP without changes in axon conduction. 4-AP accelerated chemically or electrically induced fictive locomotion and facilitated the onset of fictive locomotion in the presence of subthreshold stimuli, that were previously insufficient to activate locomotor patterns. Thus, although 4-AP per se could not directly activate the locomotor network of the spinal cord, it could strongly facilitate the locomotor program initiated by neurochemicals or electrical stimuli. On DRs, 4-AP induced sustained synchronous oscillations smaller than electrically evoked synaptic potentials, persistent after sectioning off the ventral region and preserved in an isolated dorsal quadrant, indicating their dorsal horn origin. 4-AP oscillations were network mediated via glutamatergic, glycinergic and GABAergic transmission. Isolated ventral horn areas could not generate 4-AP oscillations, although their intrinsic, disinhibited bursting was accelerated by the substance. Activation of fictive locomotion by either application of neurochmicals or stimulus trains to a single DR reversibly suppressed DR oscillations induced by 4-AP. The present electrophysiological investigation also examined whether the broad spectrum potassium channel blocker tetraethylammonium (TEA) could generate locomotor-like patterns. Low concentrations of TEA induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations evoked alternating discharges between flexor and extensor motor pools, plus a large depolarization of MNs with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABAA and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I mGluRs. Furthermore, TEA also evoked irregular discharges on DRs. The rhythmic alternating patterns elicited by TEA on VRs were relatively stereotypic, had limited synergy with the fictive locomotion induced by DR stimuli, and were not accelerated by 4-AP. Horizontal section of the spinal cord preserved irregular VR discharges and DR discharges, demonstrating that the action of TEA on spinal networks was fundamentally different from that of 4-AP.