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Voltage-clamp recordings performed in MNs from the neonatal mouse spinal cord have demonstrated opposing effects associated with the activation of M2 and M3 muscarinic receptors. Regarding synaptic inputs, M2 receptor activation decreased PSC frequency, whereas M3 receptor activation caused an increase in synaptic drive to MNs. M2 muscarinic receptors were responsible for a outward current and a decrease in input resistance while M3 receptors depolarized MNs and increased input resistance. Differences in the functional roles of M2 and M3 muscarinic receptors in spinal physiology have been highlighted previously. Stimulation of the saphenous nerve can evoke lumbar ventral root potentials that are potentiated by methoctramine and

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supressed by 4-DAMP (Kurihara et al., 1993). In the dorsal horn, M3 receptors are present at glycinergic INs and potentiate glycine release, while M2 receptors counteract the effects on synaptic glycine release to dorsal INs (Wang et al., 2006). In M3 receptor knockout mice, oxotremorine decreased the frequency of inhibitory PSCs whereas in M2 receptor knockout animals it increased the frequency of these inhibitory inputs to lamina II INs (H. M. Zhang, Zhou, et al., 2007). The opposing effects of M2 and M3 muscarine receptors have not only been reported in the spinal cord but also in other central synapses. In the cerebellum-projecting medial vestibular nucleus M2 muscarinic receptors inhibit glutamate release from vestibular afferents whereas M3 muscarinic receptors increase excitability of neurons projecting from this nucleus to the cerebellum

(Zhu et al., 2016). M2 muscarinic receptors were responsible for an inhibitory response

of ACh in parafascicular neurons whereas an excitatory component was mediated by

M3 receptors (Ye et al., 2009). Formation of heterodimers has often been implicated in

functional interactions between G-protein coupled receptors (Maggio et al., 1999). For

example, D2 dopamine receptors and A2A adenosine receptors can form heterodimers

that are involved in neuroplasticity in the basal ganglia and in neurogenerative diseases such as Parkinson’s disease (K. Fuxe et al., 2003; Kjell Fuxe et al., 2005). The A1

(inhibitory) and A2A (excitatory) adenosine receptors can also form dimeric structures

that may be responsible for functional crosstalk at several synapses prompting a balance between an adenosinergic status of excitation and inhibition (Sheth et al., 2014), which

can be dysfunctional in diseases involving spinal MNs such as ALS (Nascimento et al.,

2014, 2015). M2 and M3 muscarinic receptors can also cross-interact with each other and form heterodimeric receptors (Maggio et al., 1999; Novi et al., 2005; Goin and Nathanson, 2006; Clovis et al., 2016). However, immunoreactivity for M3 and M2

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muscarinic receptors in the spinal cord suggests that there might not be an overlap that could be indicative of heterodimerization, at least on the soma of MNs. M2 muscarinic receptors are preferentially clustered at the C-bouton synapse whereas the M3 receptor subtype was not present at this synapse but in fine cytoplasmic puncta in MNs (Wilson

et al., 2004). Some INs in laminas X, VII and VIII exhibited M3 receptor

immunoreactivity (Wilson et al., 2004) whereas M2 muscarinic receptors were mostly

found in laminas II-III, IV-VI and lamina X (Stewart and Maxwell, 2003). This suggests that there is a likelihood of both receptors being expressed in synapses around the ventromedial area of the spinal cord where some CPG INs responsible for the generation of locomotor patterns are located. Whether M2/M3 heterodimers or functional crosstalk is involved in the modulation of spinal neurons, remains a possibility that could be addressed in the future. The possibility of protein-protein interactions between M2 and M3 receptors in the spinal cord could be explored using bioluminescence resonance energy transfer to study the molecular details and functional role of such possible oligomeric assembly (Pfleger et al., 2006).

An interesting feature from the results obtained is the loss of muscarine-induced increase in MN maximum firing in the presence of 4-DAMP or methoctramine. Several studies suggested that some muscarinic receptor-dependent actions result from synergistic or a balanced activation of different receptors (Novi et al., 2005; González et al., 2011; Matsuyama et al., 2013). For example, in recordings from lamina II INs, both M2 and M3 muscarinic receptors were shown to regulate glutamate release with M2 receptor modulating release from primary afferents while the M3 subtype regulated transmission in a group of glutamatergic INs in the spinal cord (H. Zhang et al., 2007). In a different study, intrathecal administration of methoctramine or 4-DAMP in the rat

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spinal cord decreased the animal’s sexual behaviour, suggesting that blockade of any of the two receptors disrupts muscarinic receptor actions (Gómez-Martínez and Cueva- Rolón, 2009). Considering the contrasting role of M2 and M3 muscarinic receptors in the regulation of currents such as Ih (Chevallier et al., 2006; Zhao et al., 2016; Zhu et

al., 2016) and leak K+ channels (Jones and Baughman, 1992; Zhu and Uhlrich, 1998; Chevallier et al., 2006) and their different distribution on the surface of MNs but also on diverse spinal INs (Hellström et al., 2003; Stewart and Maxwell, 2003; Wilson et al., 2004), it could be assumed that the muscarine-induced increase in MN output is not a direct outcome of the activation of one type of muscarinic receptor but instead a result of a balance between M2 and M3 receptor actions.

Blocking M2 muscarinic receptors during NMDA, DA and 5-HT-induced locomotion decreased the variance of burst frequency and increased the duration of locomotor-related bursts. In contrast, M3 receptor antagonists disrupted the rhythmicity of the evoked bursts while also decreasing burst duration. These results indicate that M2 and M3 muscarinic receptors have contrasting effects on the CPG network and therefore suggest that ACh, which is released during locomotion, facilitates a balance between M2/M3 actions to ensure effectiveness of locomotor-related network output. M3 muscarinic receptors seem to be important in setting up adequate inter-burst intervals and thus avoiding formation of clusters of erratic bursts. The change between disorganized patterns of activity (e.g. multirhythm, diverse pattern) and continuously rhythmic activity relies on appropriate network excitability. Neuromodulators such as dopamine or 5-HT can shift disorganized bursts that were elicited at low doses into regular bursting when perfused at high concentrations (Sharples and Whelan, 2017). Regarding dopamine modulation, concentration-dependent sequential activation of

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different receptor subtypes seemed to be responsible for this transition (Sharples et al., 2015). Neuromodulation is largely influenced by network state, which could indicate that perhaps M3 muscarinic receptors are important in stabilizing the drug-induced rhythm by raising network excitability possibly through inactivation of the M-current in different CPG INs. On the other hand, M2 muscarinic receptors seem to be important in maintaining some degree of irregularity in the locomotor rhythm. By allowing burst events to be shorter (decreased duration) it could grant some degree of flexibility to the spinal network by allowing a modulator or a supraspinal stimulus to elicit a faster locomotor pace. Differences in M2 and M3 muscarinic receptor modulation of rhythmic outputs have not only been evidenced in the rat spinal cord (Jordan et al., 2014) but also in peripheral synapses where M2 and M3 receptors differentially regulate the intestinal motor activity with the M2 subtype being responsible for the generation of rhythmic motor activity while M3 receptors control periodicity of the rhythm (Tanahashi et al., 2013). In addition, synergistic responses to activation of both receptors is involved in the cholinergic excitation of intestinal motor activity (Matsuyama et al., 2013). Based on the above discussion one can assume that a balanced M2/M3 muscarinic receptor activation adequately regulates episodic bursting during generated by spinal locomotor networks.