Locomotion is controlled by neuronal networks of the spinal cord, referred to as central pattern generators (CPGs), which can generate and sustain rhythmic patterns of muscle activity (Arber, 2012; Gosgnach et al., 2017; Goulding, 2009). The CPG for locomotion is located within the spinal cord and is formed by genetically distinct classes of interneurons that drive motoneuron activity in coordinated sequences that cause precise muscle activation necessary for behaviors such as walking or swimming. These spinal neurons are functionally diverse, as evidenced by the variety of different inhibitory, excitatory and modulatory transmitters they release to shape the pattern and frequency of motoneuron firing. Knowledge of the varying synaptic mechanisms by which interneuron subtypes participate in spinal circuits is crucial to better understand the flexibility and adaptability of spinal circuits in health and disease (Goulding, 2009; Miles and Sillar, 2011).

There are four broad, genetically distinct classes of spinal interneurons born in the ventral horn that are involved in motor function: V0, V1, V2 and V3 interneurons. V0 interneurons are derived from progenitors that express the developing brain homeobox protein 1 (Dbx-1) and can be further subdivided into V0_V (ventral) or V0_D (dorsal) interneurons based on the expression or absence, respectively, of the postmitotic determinant homeodomain protein even-skipped homeobox 1 (Evx1). V0 interneurons play an important role in left-right coordination (Moran-Rivard et al., 2001; Lanuza et al., 2004; Pierani et al., 2001). Less than 10% of the V0 neurons constitute a subpopulation of interneurons identified by expression of the Paired-like homeodomain 2 (Pitx2) transcription factor. Pitx2⁺ interneurons are preferentially clustered around the central canal and can be further subdivided into cholinergic (V0c) and glutamatergic (V0g) subtypes (Zagoraiou et al., 2009). V0c interneurons, receive excitatory inputs from spinal pathways involved in locomotion and innervate spinal motoneurons, forming 80–100 large synapses (Zagoraiou et al., 2009), termed C-boutons, on each motoneuron (Li et al., 1995). Recently, Rozani et al demonstrated that C-boutons of brainstem motoneurons also derive from Pitx2⁺ interneurons (Rozani et al., 2019).

Unlike other V0-derived interneurons, V0c interneurons are not involved in left-right or extensor-flexor coordination. Instead, they seem to be important for modulating the amplitude of motor output. In wild-type mice, the activation of specific hindlimb muscles is increased when animals switch from walking to swimming. However, when cholinergic transmission at C-boutons is genetically perturbed, this task-dependent increase in muscle activation is significantly reduced (Zagoraiou et al., 2009). These findings support that V0c interneurons and their C-bouton inputs to motoneurons provide task-dependent modulation of motor output by enhancing motoneuron firing when greater muscle activation is required.

A range of postsynaptic proteins, including M2 muscarinic receptors, Kv2.1 channels and SK channels, are clustered at C-bouton synapses (Wilson et al., 2004; Deardorff et al., 2014). Previous work indicated that activation of M2 receptors in spinal cord slices increases motor output through M2 receptor-mediated inhibition of SK channels, which reduces the medium afterhyperpolarization (mAHP) allowing increased motoneuron firing rates (Miles et al., 2007). Recent work utilizing Kv2.1 channel blockers has demonstrated that Kv2.1 currents also regulate the repetitive firing of motoneurons (Romer et al., 2019). However, the exact mechanisms by which V0c-derived C-boutons modulate motor output remain unknown (Deardorff et al., 2014; Witts et al., 2014). Given the important role of V0c interneurons in motor control, and data implicating C-boutons as therapeutic targets for Amyotrophic Lateral Sclerosis and peripheral nerve injury (Salvany et al., 2019; Herron and Miles, 2012; Landoni et al., 2019), we sought to identify the cellular mechanisms through which this distinct class of cholinergic neurons modulates motor output.

In this study, we employed a combination of genetic, pharmacological and electrophysiological approaches to determine the synaptic mechanisms by which V0c interneurons and their C-boutons modulate motor output. Using Ca²⁺ imaging, we show that populations of lumbar Pitx2⁺ interneurons are rhythmically active during fictive locomotion. Chemogenetic inhibition of Pitx2⁺ interneurons during fictive locomotion, using designer receptors exclusively activated by designer drugs (DREADDs), decreased the strength of motor output in an M2 receptor-dependent manner. Genetic ablation of V0c interneurons confirmed that C-boutons increase the amplitude, but do not affect rhythmicity, of locomotor output. Chemogenetic excitation of Pitx2⁺ interneurons showed that activation of M2 receptors and subsequent regulation of Kv2.1 channels at C-boutons increases motor output by decreasing motoneuron spike half-width and increasing the inter-spike AHP. Together, our findings provide the first direct evidence of the synaptic and cellular mechanisms involved in C-bouton-mediated modulation of spinal locomotor circuitry. Furthermore, we reveal a high degree of specificity within the modulatory control of spinal motor circuits, with discrete modulatory units likely to be devoted to the modulation of specific network output parameters.

In this study, we interrogated the roles and cellular mechanisms of cholinergic modulation within motor circuits by focusing on a uniquely discrete population of genetically identifiable cholinergic spinal interneurons marked by expression of the transcription factor Pitx2. By combining Ca²⁺ imaging, chemogenetic manipulation and conditional ablation of cholinergic Pitx2⁺ interneurons (V0c), we demonstrate that recruitment of V0c interneurons and their C-bouton synapses leads to activation of postsynaptic M2 receptors, which in turn regulate Kv2.1 channel function to facilitate increased and sustained motoneuron firing. Thus, activation of the V0c system results in augmented motor output that will translate into stronger muscle activity. Such intrinsic modulation will enable spinal motor output, and muscular contraction, to be matched to variable behavioral demands.

Previous single cell recordings from Pitx2⁺ interneurons during pharmacologically induced locomotor-related activity demonstrated that the majority of Pitx2⁺ cells exhibit bursts of activity in phase with segmentally aligned motoneuron targets (Zagoraiou et al., 2009). We have now extended these analyses to a population level by imaging Ca²⁺ fluctuations in groups of lumbar Pitx2⁺ interneurons during locomotor network activity. We find that a high proportion of Pitx2⁺ interneurons exhibit rhythmic activity that is phase locked to bursts of locomotor-related activity recorded from ventral roots. Similar to previous work (Zagoraiou et al., 2009), we found that the strength of the relationship between Pitx2⁺ cell activity and locomotor output was more robust in upper compared with lower lumbar regions. This may reflect rostro-caudal differences in the density of V0c and V0g interneurons in the lumbar spinal cord (Zagoraiou et al., 2009; Enjin et al., 2010).

Following the demonstration that Pitx2⁺ interneurons are rhythmically active during locomotor network activity, we next investigated their modulatory actions on locomotor-related output by using Cre-dependent expression of DREADDs to manipulate their activity. We first verified the effectiveness and specificity of our DREADD-based approach by showing that single-cell properties and baseline network output did not differ between DREADD and control mice prior to the application of CNO (Tables 1, 2 and 3, Supplementary file 1). Both high doses of CNO (10 µM) and the CNO derivative clozapine, which is only likely to be produced at significant levels when administered in vivo (MacLaren et al., 2016; Pirmohamed et al., 1995), can exhibit off-target effects on endogenous receptors, including 5-HT receptors known to contribute to the control of locomotor networks (Armbruster et al., 2007; Gomez et al., 2017). We therefore also verified that CNO (1 µM) had no detectable effects on ventral root output, single motoneuron firing, or subthreshold motoneuron properties when applied to preparations obtained from animals that do not express DREADD receptors (Figure 3—figure supplement 1 and Figure 7—figure supplement 1). Having verified our DREADD approach, we first assessed the effects of inhibiting Pitx2⁺ interneurons on ongoing locomotor-related activity. DREADD-mediated inhibition revealed that Pitx2⁺ interneurons modulate the amplitude of locomotor-related output in an M2 muscarinic receptor-dependent manner. This finding is consistent with previous reports of a reduction in the amplitude of locomotor-related bursts when applying antagonists of M2-type muscarinic receptors (Miles et al., 2007; Nascimento et al., 2019). We next addressed whether cholinergic V0c interneurons, and their C-bouton inputs to motoneurons, were solely responsible for the modulation of motor output we observed. In preparations from animals in which V0c cells had been genetically ablated, blockade of M2 receptors no longer affected the amplitude of locomotor-related output. Thus, Pitx2⁺ interneuron-dependent modulation of the intensity of motor output originates from cholinergic V0c interneurons and their C-bouton synapses on motoneurons. Despite previous evidence of a low density of V0c-derived synapses in the intermediate zone of the spinal cord (Zagoraiou et al., 2009), our findings demonstrate a primary role for V0c interneurons in the control of motoneuron output with little or no impact on locomotor CPG interneurons. Furthermore, although glutamatergic V0g interneurons are also known to project to the intermediate zone as well as the dorsal horn of the spinal cord (Zagoraiou et al., 2009), the fact that locomotor-related activity was unaltered when Pitx2⁺ cell activity was manipulated in the presence of muscarinic receptor blockers indicates that V0g interneurons do not contribute to the generation or modulation of locomotor network output and hence their functional roles remain elusive.

Recent studies have reported a diversity of muscarinic receptor-mediated effects on spinal locomotor circuits, as revealed by modulation of the frequency, duration and amplitude of locomotor-related output (Nascimento et al., 2019; Jordan et al., 2014; Finkel et al., 2014). In keeping with these observations, when V0c interneurons were genetically ablated, pharmacological blockade of M2 receptors revealed V0c-independent muscarinic modulation of the frequency and duration of locomotor bursts. Given that M2 receptors are not only juxtaposed to C-boutons but are also widely distributed throughout motoneuron somata (Wilson et al., 2004; Miles et al., 2007; Hellström et al., 2003; Welton et al., 1999) and on spinal interneurons (Höglund and Baghdoyan, 1997), it is likely that additional, as yet genetically undefined, neuronal sources of intraspinal acetylcholine (Bertrand and Cazalets, 2011; Gillberg et al., 1988; Gillberg et al., 1990; Sherriff and Henderson, 1994) are responsible for these M2 receptor-dependent changes in the rhythm and pattern of locomotor output. Given the specific nature of the effects of V0c interneurons on spinal motor circuits, it will be interesting to investigate whether similarly discrete subsets of spinal cells fulfil the other modulatory roles uncovered by blockade of endogenous cholinergic signalling. Recent work suggests that such separate modulatory pathways may act in concert, via different muscarinic receptor subtypes, to ensure balanced motor circuit output (Nascimento et al., 2019).

Inhibition of Pitx2⁺ interneurons during locomotor network activity was associated with both a reduction in population level motoneuron output, measured via ventral root recordings, and a reduction in motoneuron firing rates, evidenced by patch-clamp recordings from individual cells. We next took advantage of our genetic access to this specific population of interneurons, and our successful DREADD-based manipulation of their activity, to directly identify the synaptic and cellular mechanisms underlying the discrete modulatory pathway mediated by V0c interneurons.

DREADD-mediated excitation of V0c interneurons was associated with an M2 receptor-dependent increase in the excitability of motoneurons. This increased excitability was characterized by a decrease in rheobase due to hyperpolarization of the action potential threshold, greater resistance to depolarizing block, which allowed motoneurons to fire in response to greater amplitude stimuli, and an increase in maximum firing rates. Potential mediators of these effects include postsynaptic Kv2.1 potassium channels and SK-type calcium-dependent potassium channels, which are both clustered at C-boutons synapses (Wilson et al., 2004; Hellström et al., 2003; Muennich and Fyffe, 2004). Blockade of Kv2.1 channels during activation of V0c interneurons prevented the increased resistance to depolarizing block and the rise in maximum firing frequency, but not hyperpolarization of the action potential threshold. Given that recent modeling experiments indicate that blockade of SK channels depolarizes the action potential threshold in midbrain dopamine neurons (Iyer et al., 2017), SK channels at C-bouton synapses may also play a role in controlling the spike threshold of motoneurons. This is supported further by previous work indicating that motoneurons that express the SK3 channel isoform might have reduced rheobase when compared to motoneurons that do not express this isoform (Deardorff, 2013). In contrast, the other mechanisms leading to increased motoneuron excitability following activation of V0c interneurons appear to involve modulation of Kv2.1 channels, which are known to facilitate repetitive firing in a wide range of neuronal types (Liu and Bean, 2014; Malin and Nerbonne, 2002; Guan et al., 2013) and to play an important role in the repolarization phase of action potentials in motoneurons (Romer et al., 2019; Gao and Ziskind-Conhaim, 1998).

Our data, which support a predominant role for Kv2.1 channels in C-bouton-mediated modulation of motoneuron excitability, contrast previous work, which suggested modulation of SK channels and a reduction in the mAHP were responsible for increased motoneuron output upon C-bouton activation (Miles et al., 2007). However, this previous work relied on broad pharmacological manipulation of muscarinic receptors in spinal cord slices, prior to the discover of the C-bouton source cells, and therefore likely activated a wide population of receptors beyond those localized at C-bouton synapses. Given that activation of muscarinic receptors has a number of different effects on motoneurons (Nascimento et al., 2019), it is plausible that M2 receptor-dependent reductions in mAHP amplitude involve receptors outside the territory of C-bouton synapses. SK channel clusters preferentially appose large cholinergic C-bouton terminals (Deardorff, 2013). However, SK channels are also distributed throughout laminae VII and IX, and could therefore be present on more distal motoneuron dendrites where they might be subject to modulation by as yet uncharacterized, Pitx2-negative, populations of cholinergic spinal interneurons (Bertrand and Cazalets, 2011; Gillberg et al., 1988; Gillberg et al., 1990; Sherriff and Henderson, 1994; Deardorff, 2013). Another possible explanation for the mechanistic differences reported in the present study is that there may be differential, state-dependent levels of involvement of specific components of the C-bouton synapse, depending on the activity levels of motoneurons and V0c interneurons (Deardorff et al., 2014). For example, Kv2.1 channel-mediated modulation might dominate at the level of V0c activity induced by our DREADD-based stimulation, but calcium-dependent potassium channels might contribute at different rates of V0c interneuron firing. Testing this hypothesis will require the development of methods that enable finer control of V0c interneuron activity than were possible using the current DREADD-based approach.

Interestingly, in the current study, we found that activation of V0c interneurons was associated with a small increase in both the mAHP measured after single spikes, and the inter-spike AHP measured during repetitive firing. Given that both effects were blocked by guangxitoxin-1E, we propose that this reflects greater recruitment of Kv2.1 channels. This mechanism contrasts previous reports of a substantial reduction in the mAHP due to modulation of calcium-dependent potassium channels (Miles et al., 2007). However, as discussed above, these previous data were obtained using less direct, global activation of M2 receptors and reflected measurements of mAHPs evoked by single action potentials only. Furthermore, it remains to be determined whether these effects reflect activation of M2 receptors at C-bouton synapses. If these effects are C bouton-mediated, perhaps under certain conditions large changes in the mAHP, due to SK channel modulation, reduce the refractory period to allow greater firing frequencies. Meanwhile, in the absence of such large reductions in the mAHP, smaller Kv2.1-mediated increases in the amplitude of the inter-spike AHP may facilitate the removal of inactivation of sodium channels, enabling sustained, higher frequency firing in response to depolarizing stimuli, as has been shown in other neuron types (Liu and Bean, 2014; Johnston et al., 2008). This Kv2.1 channel-dependent mechanism is also supported by recent work in rat lumbar motoneurons, which has demonstrated that Kv2 channels permit greater motoneuron firing frequencies by maintaining hyperpolarized inter-spike membrane potentials (Romer et al., 2019). Another, likely linked, observation in our study was a Kv2.1 channel-dependent narrowing of action potentials following C-bouton activation. This is again consistent with recent reports of increased action potential half-width and a slowing of motoneuron firing when Kv2 channels are blocked (Romer et al., 2019; Fletcher et al., 2017).

While our findings are consistent with previous work demonstrating that Kv2.1 channels help maintain appropriate levels of repetitive firing in motoneurons (Romer et al., 2019; Fletcher et al., 2017), the intracellular mechanisms by which M2 receptor activation at C bouton synapses leads to changes in Kv2.1 channel function and motoneuron firing remain unclear. One potential pathway involves the calcineurin-meditated dephosphorylation of Kv2.1 channels. It has been proposed that inhibition of N-type Ca²⁺ channels, known to be located at C-bouton synapses (Wilson et al., 2004), by G_i/0-dependent pathways following M2 receptor activation could lower the levels of local intracellular Ca²⁺, thus reducing calcineurin-related dephosphorylation of Kv2.1 channels (Deardorff et al., 2014; Romer et al., 2019). Kv2.1 dephosphorylation in response to prolonged excitatory drive is thought to cause channel delustering in motoneurons, whilst reduced activity can increase Kv2.1 channel clustering (Romer et al., 2019; Romer et al., 2016; Romer et al., 2014). Interestingly, in motoneurons, declustering occurs in response to increased glutamatergic, but not cholinergic, input (Romer et al., 2019). Thus, activation of C-boutons should not lead to declustering of Kv2.1 channels and has in fact been proposed to maintain Kv2.1 clusters and promote repetitive firing at physiological activity levels (Romer et al., 2019). In addition to effecting channel clustering, the phosphorylation state of Kv2.1 channels has been shown to influence their gating parameters. Although data are lacking for motoneurons, dephosphorylation of Kv2.1 channels leads to a hyperpolarizing shift in channel activation and inactivation properties in hippocampal neurons, which is thought to suppress action potential firing frequency (Mohapatra and Trimmer, 2006; Mohapatra et al., 2009; Misonou et al., 2005; Park et al., 2006). Thus, it seems plausible that C-bouton activation helps maintain the phosphorylation state of Kv2.1 channels required to support motoneuron firing. It will therefore be interesting to directly test this, and other possible intracellular mechanisms, in future work. Regardless of the exact mechanisms, C-boutons appear well placed to ensure that motoneuron excitability and output is maintained or increased in a task or state-dependent manner, even in the face of elevated glutamatergic drive that would otherwise reduce motoneuron output via delustering of Kv2.1 channels.

An important consideration when interpreting our results is that our experiments were performed on neonatal tissue, because whole spinal cord preparations are not viable when obtained from older animals. Although our in vitro approach allows us to access, isolate and interrogate specific pathways, which are unlikely to be accessible or separable using in vivo approaches, an important caveat is the possibility of developmental changes in C-bouton signaling. The overall pre- and post-synaptic components of C-bouton synapses, including M2 receptors and Kv2.1 channels, appear to remain consistent from early neonatal stages into adulthood (Zagoraiou et al., 2009; Wilson et al., 2004). Rather than changes in the major components, postnatal maturation of C-boutons involves increased clustering of Kv2.1 channels and M2 receptors along with an increase in the size of C-boutons until motor networks become functionally mature (~P15) (Wilson et al., 2004; Phelps et al., 1984; Wetts and Vaughn, 2001). Given that maturation of spinal networks is sometimes accompanied by changes in the effects of modulators (e.g. 5-HT and acetylcholine [Jordan et al., 2014; Schmidt and Jordan, 2000]), we cannot exclude the possibility that the exact mechanisms of action at fully mature C-boutons differs from those revealed at neonatal stages. However, the fact that genetic perturbation of C-boutons in adult mice impairs the ability of motoneurons to increase muscle activation in response to behavioral demands (Zagoraiou et al., 2009), supports a consistency in the cellular effects of the C-bouton system into adulthood. Recordings from adult motoneurons within in vivo preparations, and associated modelling studies, have shown state-dependent changes in motoneuron properties including a decrease in AHP amplitude, hyperpolarization of action potential threshold, and a small increase in action potential half-width (Power et al., 2010; MacDonell et al., 2015; Krawitz et al., 2001; Brownstone et al., 1992; Dai et al., 2002). However, it is difficult to reconcile these results with those of the current study, since the in vivo findings reflect the net effect of multiple parallel modulatory processes and focusses on differences between quiescent states and baseline activity during locomotion or scratching, behavioral states during which the C-bouton system is unlikely to be strongly engaged (Zagoraiou et al., 2009). It will therefore be important in future studies to establish methods for interrogating the specific actions and mechanisms of C-bouton-mediated modulation of adult motoneurons during motor tasks of varying intensities.

Cholinergic pathways within the nervous system process a variety of inputs which enable them to fulfil roles in attentional modulation, enhancing responses to stimuli in a state-dependent manner (Ye et al., 2009; Mena-Segovia, 2016; Wikman et al., 2019; Herrero et al., 2008). The premotor, intraspinal cholinergic system, involving V0c interneurons and C-boutons, appears to parallel these supraspinal cholinergic systems. V0c interneurons receive synaptic inputs from a range of sources, including sensory pathways, supraspinal structures and CPG networks (Figure 9; Zagoraiou et al., 2009; Stepien et al., 2010; Murray et al., 2018), which ‘mirror’ inputs received by motoneurons and are therefore relevant to desired motor output. The C-bouton system seems to be less active during regular motor tasks such as walking but is particularly important during locomotor behaviors that require increased muscle activation such as swimming (Zagoraiou et al., 2009). Thus, V0c interneurons and their C-boutons are thought to provide task-dependent modulation of motor output (Zagoraiou et al., 2009). Here, we report novel findings regarding both the organization and mechanisms of action of intraspinal cholinergic modulatory systems. We provide evidence in support of a modular architecture for spinal cholinergic systems, in line with that recently proposed for the locus coerleus (Chandler et al., 2019; Totah et al., 2018), where V0c interneurons represent one functionally distinct neuromodulatory module devoted to task-dependent control of the intensity of motor output. Meanwhile, we provide the first demonstration of the synaptic and cellular mode of action of C-bouton inputs to motoneurons, demonstrating a role in shaping the action potential and therefore firing patterns via modulation of Kv2.1 channels. Given similarities in the roles of supraspinal and spinal cholinergic modulatory systems in task or state-dependent modulation, the insight gained from our study is likely to provide new insight into cholinergic modulatory systems throughout the mammalian nervous system.

Figure 9

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All of the data presented in this study are included in the manuscript and supporting files.

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Author details

1. Filipe Nascimento

   School of Psychology and Neuroscience, University of St Andrews, St Andrews, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9426-2807

2. Matthew James Broadhead

   School of Psychology and Neuroscience, University of St Andrews, St Andrews, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4078-5581

3. Efstathia Tetringa

   Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

   Contribution

   Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Eirini Tsape

   Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

   Contribution

   Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Laskaro Zagoraiou

   Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

6. Gareth Brian Miles

   School of Psychology and Neuroscience, University of St Andrews, St Andrews, United Kingdom

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   gbm4@st-andrews.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8624-4625

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