Summary Mammalian motor programs are controlled by networks of spinal interneurons that set the rhythm and intensity of motor neuron firing. Motor neurons have long been known to receive prominent ‘C-bouton’ cholinergic inputs from spinal interneurons, but the source and function of these synaptic inputs have remained obscure. We show here that the transcription factor Pitx2 marks a small cluster of spinal cholinergic interneurons, V0C neurons, that represents the sole source of C-bouton inputs to motor neurons. The activity of these cholinergic interneurons is tightly phase-locked with motor neuron bursting during fictive locomotor activity, suggesting a role in the modulation of motor neuron firing frequency. Genetic inactivation of the output of these neurons impairs a locomotor task-dependent increase in motor neuron firing and muscle activation. Thus V0C interneurons represent a defined class of spinal cholinergic interneurons with an intrinsic neuromodulatory role in the control of locomotor behavior.

To effect movement, motoneurons must respond appropriately to motor commands. Their responsiveness to these inputs, or excitability, is regulated by neuromodulators. Possible sources of modulation include the abundant cholinergic ''C boutons'' that surround motoneuron somata. In the present study, recordings from motoneurons in spinal cord slices demonstrated that cholinergic activation of m 2-type muscarinic receptors increases excitability by reducing the action potential afterhyperpolarization. Analyses of isolated spinal cord preparations in which fictive locomotion was elicited demonstrated that endogenous cholinergic inputs increase motoneuron excitability during locomotion. Anatomical data indicate that C boutons originate from a discrete group of interneurons lateral to the central canal, the medial partition neurons. These results highlight a unique component of spinal motor networks that is critical in ensuring that sufficient output is generated by motoneurons to drive motor behavior.T o generate movement, it is necessary for motoneurons (MNs) to integrate the inputs (motor commands) they receive and produce an output sufficient to effect muscular contraction. The relationship of input to output is determined by neuronal excitability, which in the case of MNs is known to be regulated by identified descending modulatory systems (1). Given that these descending systems are disrupted after spinal cord injury, strategies aimed at restoring movement need to address not only premotor circuits that provide motor commands but also any modulatory systems that ensure MNs are sufficiently excitable to respond to these commands. Spinal premotor circuits for locomotion can be activated after spinal transection (2) and provide one clear target for treatments designed to produce functional recovery. Should an intrinsic spinal modulatory system exist, this would be an important additional target for such strategies.The somata and proximal dendrites of MNs are contacted by large cholinergic varicosities named ''C boutons'' (3-11). It has been known since 1972 (12) that C boutons originate from spinal cord neurons, but the location of these cells remains unknown (10). Although the C bouton synapse has been anatomically characterized and shown to be associated with postsynaptic type 2 muscarinic (m 2 ) receptors (8-10), neither the physiological effects of m 2 receptor activation on MNs nor the roles of C boutons in motor activity are known. In the absence of motor behavior, exogenous application of cholinergic agonists affects MN excitability via undefined mechanisms (13-17). We therefore studied the possibility that the intrinsic spinal neurons that give rise to the C boutons regulate MN excitability via activation of m 2 receptors, and that this system is used during motor behavior. ResultsThe Effects of Muscarinic Receptor Activation on Spinal MNs. Because C boutons are closely associated with postsynaptic m 2 receptors by the second postnatal week (8-10), we investigated the effects of muscarinic receptor activatio...

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease for which a greater understanding of early disease mechanisms is needed to reveal novel therapeutic targets. We report the use of human induced pluripotent stem cell (iPSC)-derived motoneurons (MNs) to study the pathophysiology of ALS. We demonstrate that MNs derived from iPSCs obtained from healthy individuals or patients harbouring TARDBP or C9ORF72 ALS-causing mutations are able to develop appropriate physiological properties. However, patient iPSC-derived MNs, independent of genotype, display an initial hyperexcitability followed by progressive loss of action potential output and synaptic activity. This loss of functional output reflects a progressive decrease in voltage-activated Na+ and K+ currents, which occurs in the absence of overt changes in cell viability. These data implicate early dysfunction or loss of ion channels as a convergent point that may contribute to the initiation of downstream degenerative pathways that ultimately lead to MN loss in ALS.

Mutations in C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS). Here, through a combination of RNA-Seq and electrophysiological studies on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs), we show that increased expression of GluA1 AMPA receptor (AMPAR) subunit occurs in MNs with C9ORF72 mutations that leads to increased Ca2+-permeable AMPAR expression and results in enhanced selective MN vulnerability to excitotoxicity. These deficits are not found in iPSC-derived cortical neurons and are abolished by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs. We also demonstrate that MN-specific dysregulation of AMPAR expression is also present in C9ORF72 patient post-mortem material. We therefore present multiple lines of evidence for the specific upregulation of GluA1 subunits in human mutant C9ORF72 MNs that could lead to a potential pathogenic excitotoxic mechanism in ALS.