Abstract
Neuromodulation ensures that neural circuits produce output that is flexible whilst remaining within an optimal operational range. The neuromodulator acetylcholine is released during locomotion to regulate spinal motor circuits. However, the range of receptors and downstream mechanisms by which acetylcholine acts have yet to be fully elucidated. We therefore investigated metabotropic acetylcholine receptor-mediated modulation by using isolated spinal cord preparations from neonatal mice in which locomotor-related output can be induced pharmacologically. We report that M2 receptor blockade decreases the frequency and amplitude of locomotor-related activity, whilst reducing its variability. In contrast, M3 receptor blockade destabilizes locomotor-related bursting. Motoneuron recordings from spinal cord slices revealed that activation of M2 receptors induces an outward current, decreases rheobase, reduces the medium afterhyperpolarization, shortens spike duration and decreases synaptic inputs. In contrast, M3 receptor activation elicits an inward current, increases rheobase, extends action potential duration and increases synaptic inputs. Analysis of miniature postsynaptic currents support that M2 and M3 receptors modulate synaptic transmission via different mechanisms. In summary, we demonstrate that M2 and M3 receptors have opposing modulatory actions on locomotor circuit output, likely reflecting contrasting cellular mechanisms of action. Thus, intraspinal cholinergic systems mediate balanced, multimodal control of spinal motor output.
Highlights
Locomotion is generated by a hierarchy of diverse motor control centres within the nervous system that together program, adapt and execute a plurality of actions
Distinct actions of M2 and M3 receptors were demonstrated by experiments performed on neonatal rat spinal cord preparations in which rhythmic locomotor-like activity was induced via administration of the acetylcholinesterase inhibitor edrophonium[26]
We first investigated the modulatory actions of M2 muscarinic receptors within spinal locomotor networks by applying the M2 receptor antagonist methoctramine while recording drug-evoked (NMDA, 5 μM; 5-HT, 10 μM; and DA, 50 μM), stable, fictive locomotion via suction electrodes attached to the lumbar ventral roots (Fig. 1c–e)
Summary
Locomotion is generated by a hierarchy of diverse motor control centres within the nervous system that together program, adapt and execute a plurality of actions. Www.nature.com/scientificreports affect motor output[27,28] with M2 and M3 muscarinic receptors later identified as the subtypes of muscarinic cholinergic receptors that play an active role in the modulation of CPG networks in the mammalian spinal cord[15,29]. Both subtypes of metabotropic receptors are widely distributed in the spinal cord but the role and identity of the spinal neurons expressing M2 and M3 receptors remains unclear[3,30,31]. Given the evidence provided by these previous studies that muscarinic receptors play a range of roles, via differing mechanisms, in the control of locomotor output, we aimed to decipher and directly compare the actions and cellular mechanisms of M2 and M3 receptor-mediated modulation within spinal motor circuits
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