Balanced cholinergic modulation of spinal locomotor circuits via M2 and M3 muscarinic receptors

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.

Figure 1

M2 muscarinic receptors modulate active CPG networks and motoneuron firing during drug-induced locomotion. (a) Schematics of ventral root and individual motoneuron recordings from intact spinal cords and (b) single cell recordings from isolated spinal cord slices. (c) Raw (top) and integrated/rectified (bottom) ventral root recordings with (d) averaged time course plots and (e) box-plots of pooled data illustrating the effects of methoctramine on the frequency, duration and amplitude of drug-induced locomotor output (n = 14). (f) Motoneuron firing (top) and simultaneous raw (middle) and integrated/rectified (bottom) ventral root recordings during drug-induced locomotor output. (g) Motoneuron firing frequencies plotted for all motoneurons tested showing the effects of methoctramine during drug induced locomotor activity (n = 17). *p < 0.05, ***p < 0.001.

Figure 2

M3 receptors modulate the stability of the locomotor rhythm during drug-induced activity. (a) Raw (top) and integrated/rectified (bottom) ventral root recordings with (b) averaged time course plots and (c) box-plots of pooled data showing the effects of 4-DAMP on the frequency, duration and amplitude of drug-induced locomotor output (n = 22). (d) Motoneuron firing (top), and simultaneous raw (middle) and integrated/rectified (bottom) ventral root recordings during drug-induced locomotor output. (e) Motoneuron firing frequencies plotted to illustrate the effects of 4-DAMP during drug induced locomotor activity (n = 8). *p < 0.05.

Figure 3

Muscarine preferentially induces a M2 receptor-mediated outward current in smaller motoneurons and a M3 receptor-dependent inward current in larger motoneurons. (a) Voltage-clamp recordings illustrating an inward current and (b) outward current elicited by muscarine with respective I-V plots depicting an increase (n = 9) or decrease (n = 6) in input resistance. (c) Plot of average capacitance values for motoneurons exhibiting inward versus outward currents in response to muscarine. (d) Illustrative trace of the inward current elicited by muscarine in the presence of methoctramine with respective I-V plot (n = 8). (e) Representative trace of the outward current induced by muscarine co-applied with 4-DAMP and I-V plots for motoneurons that showed an increase (left, n = 6) or decrease in input resistance (right, n = 9). (f) Muscarine in the presence of both M2 and M3 receptor antagonists did not affect membrane current (n = 10) or input resistance (n = 7). All recordings were performed at a holding voltage of −60 mV. ***p < 0.001.

Figure 4

M2 and M3 receptors differently affect rheobase and both modulate motoneuron firing rates. (a) Current-clamp recordings showing the effects of muscarine on motoneuron firing in response to current injection. (b) Graphs showing the effects of muscarine on rheobase (n = 14), an illustrative f-I relationship and maximum firing frequencies (n = 14). (c) Motoneuron firing in response to current injection illustrating the effects of muscarine in the presence of methoctramine. (d) Graphs showing the effects of muscarine, in the presence of methoctramine, on rheobase (n = 11), an illustrative f-I relationship and maximum firing (n = 10). (d) Motoneuron firing in response to current injection illustrating the effects of muscarine in the presence of 4-DAMP. (f) Graphs showing the effects of muscarine, in the presence of 4-DAMP, on rheobase (n = 11), an illustrative f-I plot and maximum firing (n = 15). *p < 0.05.

Figure 5

M2 receptor activation modulates the mAHP and decreases spike half-width while M3 receptor activation increases action potential duration. (a) Truncated single action potentials showing the effects of muscarine on the mAHP (n = 17). (b) Superimposed action potentials recorded from a motoneuron before and after muscarine with plots of average values of action potential half-width and rise-time in each condition (n = 9). (c) Truncated single action potentials illustrating the effect of muscarine co-applied with methoctramine on the mAHP (n = 10). (d) Representative traces of action potentials in the presence of methoctramine or muscarine and methoctramine, with plots of average half-width and rise rime (n = 10). (e) Truncated single action potentials illustrating the effect of muscarine co-applied with 4-DAMP on the mAHP (n = 10). (f) Representative traces of action potentials in the presence of 4-DAMP or muscarine and 4-DAMP, with plots of average half-width and rise-time (n = 13). *p < 0.05, **p < 0.01.

Figure 6

Muscarine has a biphasic effect on the synaptic drive to motoneurons. (a) Representative voltage-clamp traces of PSCs recorded before, during and after muscarine application. Box-plots showing the time-dependent effects of muscarine on PSC inter-event interval (b) and PSC amplitude (c) (n = 12). (d) Example traces showing the effects of muscarine on PSCs when applied in the presence of methoctramine. Box-plots showing the effects of muscarine co-applied with methoctramine on PSC inter-event interval (e) and PSC amplitude (f) (n = 12). (g) Example traces showing the effects of muscarine on PSCs when co-applied with 4-DAMP. Box-plots showing the effects of muscarine co-applied with 4-DAMP on PSC inter-event interval (h) and PSC amplitude (i) (n = 10). All recordings were performed at a holding potential of −60 mV. *p < 0.05.

Figure 7

Muscarine modulates both the amplitude and frequency of miniature postsynaptic currents through M2 and M3 receptors. (a) Voltage-clamp recordings of mPSCs (with 0.5 μM TTX) before, during and after application of muscarine. Box plots and cumulative frequency plots of mPSC inter-event interval (b) and mPSC amplitude (c) showing the effects of muscarine on spontaneous activity (n = 15). (d) Example traces of mPSCS recorded in the presence of muscarine and methoctramine. Box plots and cumulative frequency plots of mPSC inter-event interval (e) and mPSC amplitude (f) showing the effects of muscarine when co-applied with methoctramine (n = 16). (g) Representative traces of mPSCs recorded in the presence of 4-DAMP and muscarine. Box plots and cumulative frequency plots of mPSC inter-event interval (h) and mPSC amplitude (i) showing the effects of muscarine when co-applied with 4-DAMP (n=16). All recordings were performed at a holding potential of −60 mV. *p < 0.05.

Figure 8

Schematic showing how shifts in the balance between M2 and M3 receptor activation can affect motor output. During rhythmic locomotion acetylcholine (yellow) is released and modulates CPG neurons (orange) as well as motoneurons (green) through M2 (red) and M3 receptor (blue) activation. A fine balance between M2 and M3 receptor activation ensures adequate locomotor patterns, however a decrease in M3 receptor activation (red) disrupts the locomotor pattern whereas a reduction of M2 receptor activation (blue) slows the rhythm and decreases burst amplitude.