The Mechanism of M2 and M3 Receptors on the Spinal Locomotor Circuits

I have read social media articles about the importance of acetylcholine (ACh) for muscles and memory. Since cholinergic drugs can inhibit, enhance, or mimic the action of the neurotransmitter acetylcholine, I wanted to find a study that showed the different effects of drugs on acetylcholine receptors.  A recent study hypothesized that acetylcholine modulates spinal locomotor control circuitry via M2 and M3 muscarinic receptors. The goal of the study was to prove that M2 and M3 receptors, which can be found in the heart, have opposing cellular mechanisms of action on the spinal locomotor circuits. This is important because the spinal cord executes the rhythmical and sequential activations of muscles in locomotion. 

For methodology, isolated in vitro spinal cord preparations were obtained from neonatal mice. Single cell recordings were used to measure the electrophysiological responses of single neurons using a microelectrode system. The in vitro isolated spinal cords were pinned in a recording chamber, which was continuously superfused with recording an artificial cerebrospinal fluid (CSF). NMDA, 5-HT, DA were added to the aCSF solution to trigger the fictive locomotion, meaning locomotion while the muscles are removed or cut. For data analysis, the single cell recordings were analyzed with Clampfit software and Mini-Analysis software for PSCs, postsynaptic currents. Two drugs were used. The methoctramine drug (MTT) is an M2 receptor antagonist and was applied at a concentration of 20 microM. The 4-DAMP drug (DP) is an M3 receptor antagonist and was applied at a concentration of 2 microM. The experiment used less amount of DP than MTT because it is within the range that has previously been used to block M3 receptor- related locomotor activity in the spinal cord. All drugs were dissolved in water except for DP which was dissolved in DMSO. 

The first result showed that the blockage of M2 muscarinic receptors, using MTT, increased the duration of the ventral root bursts, decreased burst variability and frequency, and reduced the amplitude of drug induced locomotor output (Fig 1c-e). However, the blockage of M3 muscarinic receptors, using DP, did not significantly affect burst frequency of the receptors, firing frequency, or had any effect on burst amplitude (Fig 2d,e). Second, M2 receptor activation led to an outward current, the upward deflection of the current trace. The M3 receptor activation led to an inward current in motoneurons, downward deflection of a current trace (Fig 3 a-c). These data demonstrate that ACh acts on motoneurons via M2 and M3 receptors and these two receptors have opposing actions. Third, the study concluded that M2 and M3 muscarinic receptors affect the motoneuron rheobase, the smallest current amplitude in a membrane potential, differently. When MTT was co-applied with muscarine, an increase in motor neuron rheobase was noted but no change was observed in the maximum firing rates of motoneurons. However, when DP was coapplied with muscarine, a decrease in motor neuron rheobase was noted but no change was noted in the maximum firing rates. These data reveal the opposing modulatory actions of M2 and M3 receptors with respect to regulation of the amount of stimulation required to induce repetitive firing (Fig 4e,f). Fourth, MTT had no effect on the amplitude of medium after-hyperpolarizations (mAHP) in motoneurons but increased action potential half-width, the duration of action potential halfway between threshold and the action potential peak. In contrast, DP reduced the mAHP amplitude and decreased action potential half-width (Fig5d,e). These results demonstrate opposing modulatory actions of M2 and M3 receptors. The study provides further evidence that ACh acting via M2 receptors regulate CPG neurons involved in rhythm generation while M3 receptors generate adequate frequencies of respiratory patterns. 


Nascimento, F., et al. “Balanced cholinergic modulation of spinal locomotor circuits via M2 and M3 muscarinic receptors.” Nature News, Nature Publishing Group, 01 October 2019.