Teorías que corroboran una relación entre la primera variable sobre

The M2 muscarinic receptor antagonist, methoctramine, decreased the variance of burst frequency and increased burst duration indicating that these receptors are involved in setting up the regularity of the drug-induced locomotor rhythm. Experiments with M2 blockers in ACh-induced bursts of activity in rat spinal cord preparations showed that methoctramine increased the frequency of locomotor events (Jordan et al., 2014). Also in the neonatal rat spinal cord, experiments stimulating sacral dorsal roots while simultaneously increasing the levels of ACh in the sacral region with ACh-esterase inhibitors, decreased the frequency of bursts of activity recorded from

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lumbar ventral roots, an effect that was blocked by methoctramine (Anglister et al., 2017). The current observations from ventral root recordings in the presence of NMDA, DA and 5-HT with methoctramine in mice do not show a statistically significant difference in burst frequency between control and drug but report an increase in burst duration by methoctramine which could be indicative of a slowing of the rhythm. The differences between these results and the observations from other authors (Jordan et al., 2014; Anglister et al., 2017) could indicate that (1) there are functional differences on M2 muscarinic receptor modulation of the frequency of spinal motor output between mouse and rat or (2) these modulations are different between NMDA, DA and 5-HT induced rhythmogenesis and locomotor activity generated in the presence of ACh- esterase inhibitors, which will greatly increase the levels of ACh present. As mentioned before, there are indeed differences regarding cholinergic-induced locomotion between mouse and rat, with perfusion of muscarine eliciting regular bursts of activity in the rat spinal cord but not in mice (Kiehn et al., 1996; Jiang et al., 1999). Another important factor is that in the present work rhythmic bursting evoked with the locomotor cocktail is more regular and exhibits phase alternation between right-left and extensor-flexor output, contrary to the experiments performed in the rat spinal cord (Jordan et al., 2014). In addition, the concentration of released ACh during alternating fictive locomotion might be more physiological than when in the presence of high concentrations of ACh elicited by ACh-esterase inhibitors or general cholinergic agonists, as illustrated by experiments in the mudpuppy spinal cord in which carbacol (ACh analog) and physostigmine disrupted the fictive walking rhythm induced by NMDA (Fok and Stein, 2002). These differences could underlie changes in M2 receptor activation and/or network excitability that might explain the variations in the results on

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burst frequency with methoctramine when comparing the current results with previous work (Jordan et al., 2014; Anglister et al., 2017). Despite these incongruities in the experimental setup, recordings performed in the rat lumbar spinal cord also indicated

that M2 receptor antagonism reduced burst amplitude (Jordan et al., 2014; Anglister et

al., 2017) which mirrors the decrease in ventral root amplitude elicited by

methoctramine that is reported in this work. This suggests that there is a level of conservation regarding M2 muscarinic receptor effects on motor output amplitude in mouse and rat, even with the different approaches used, indicating that activation of these receptors increases the strength of locomotor output.

Groups of cholinergic INs in the spinal cord could be responsible for the

reported muscarinic effects on the locomotor network. However, apart from Pitx2+ INs,

there remain a lack of genetic markers for subtypes of spinal cholinergic INs. Researchers proposed that cholinergic neurons from sacral segments that project to the

lumbar region could directly modulate CPG networks and MN output (Etlin et al., 2014;

Finkel et al., 2014; Anglister et al., 2017). These sacral projecting cholinergic neurons could include a variety of different populations of premotor ACh-releasing INs that are known to project intersegmentally (Stepien et al., 2010). Most of the cholinergic INs that could be involved in modulation of CPG-mediated rhythmogenesis are located in the ventromedial area of the spinal cord and they comprise Pitx2+ INs, partition cells and contralaterally projecting INs that form synapses with MNs and other spinal INs (Sherriff and Henderson, 1994; Huang et al., 2000; Zagoraiou et al., 2009; Bertrand and

Cazalets, 2011). Pitx2+ _{INs are suggested to directly modulate MN output through M2}

muscarinic receptors (Miles et al., 2007; Zagoraiou et al., 2009) thus, they are likely to be responsible for the decrease in burst amplitude and MN firing caused by

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methoctramine. The potential roles of these INs in drug-induced locomotion will be addressed in later sections discussing the results from DREADD experiments in which Pitx2+ INs were selectively activated and inhibited (section 5.2).

Phase alternation between antagonistic muscles and right-left sides is crucial for

adequate spinal locomotor activity (Brown, 1911). This can be replicated during in vitro

drug-induced locomotor-bursts of activity in the neonatal mouse spinal cord allowing to study effects of modulators that may affect these phase relationships (Jiang et al., 1999). This study did not systematically measure any eventual variations in extensor-flexor or right-left alternation, however a clear breakdown of these relationships upon blockade of muscarinic receptors was not observed. The data obtained during fictive locomotion reports changes in ventral root burst duration, frequency and variance in the presence of the antagonists used, which could reflect a modulation of different types of CPG INs in the mouse spinal cord. In V1 knockout mice, the duration of the bursts during stepping behaviour was decreased (Gosgnach et al., 2006). V1 INs do not express cholinergic markers however they receive some cholinergic innervation from primary afferents (Alvarez et al., 2005), which could suggest that M2 muscarinic receptors that are expressed in dorsal INs and control presynaptic release from these afferents (Stewart

and Maxwell, 2003; Wang et al., 2006; H. M. Zhang, Chen, et al., 2007; H. M. Zhang,

Zhou, et al., 2007; H. Zhang et al., 2007) could be acting directly or having an indirect network effect on this population of CPG INs that could be responsible for observed increases in burst duration. V2a INs that control right-left alternation in mice (Crone et al., 2008) have sparse cholinergic innervation (Zagoraiou et al., 2009). Furthermore, V2a genetic ablation did not change cycle period, amplitude or duration of bursts (Crone et al., 2008). Thus, it is unlikely that cholinergic modulation of the locomotor

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CPG involves this IN subpopulation. The dI6 INs are located around the ventromedial area of the spinal cord, are active during locomotion and seem to be important for the formation of rhythmic patterns (Lanuza et al., 2004; Dyck et al., 2012). The clustering of cholinergic INs in ventromedial regions (Bertrand and Cazalets, 2011) might facilitate local cholinergic modulation of INs near laminas X and VII involved in stabilizing the locomotor rhythm, which could include populations such as dI6 INs. Due to their sparse distribution in the spinal cord (Wilson et al., 2004), it is plausible that the effects of M2 muscarinic receptors on the drug-induced rhythm are a reflection of a modulation on several types of INs that form the locomotor CPG rather than an action on one particular type of IN.

5.1.2.

Modulation of spinal locomotor output by M3 muscarinic