Anatomy and function of cholinergic C bouton inputs to motor neurons

Abstract

Motor control circuitry of the central nervous system must be flexible so that motor behaviours can be adapted to suit the varying demands of different states, developmental stages, and environments. Flexibility in motor control is largely provided by neuromodulatory systems which can adjust the output of motor circuits by modulating the properties and connectivity of neurons within them. The spinal circuitry which controls locomotion is subject to a range of neuromodulatory influences, including some which are intrinsic to the spinal cord. One such intrinsic neuromodulatory system, for which a wealth of anatomical information has recently been combined with new physiological data, is the C bouton system. C boutons are large, cholinergic inputs to motor neurons which were first described over 40 years ago but whose source and function have until recently remained a mystery. In this review we discuss how the convergence of anatomical, molecular genetic and physiological data has recently led to significant advances in our understanding of this unique neuromodulatory system. We also highlight evidence that C boutons are involved in spinal cord injury and disease, revealing their potential as targets for novel therapeutic strategies.

Keywords: C terminal; motor control; neuromodulation; spinal cord.

Fig. 1

Anatomy of the C bouton synpase. (A) An electron micrograph (adapted from Conradi, 1969) showing a C bouton (C) contacting a motor neuron (MN). Note the elongated subsurface cisternae (arrowheads) located postsynaptically. (B) A single motor neuron, labelled via injection of CTB into the gastrocnemius muscle, with numerous C boutons (vAChT-immunolabelled terminals) contacting the soma and proximal dendrites. (C,D) Immunolabelling reveals postsynaptic clusters of Kv2.1 channels (C) and m2 (D) receptors aligned with vAChT-labelled C boutons. (E) Schematic representation of a C bouton synapse. Nicotonic acetylcholine (NACh) and ATP (P2X7) receptors are thought to be located presynaptically. Muscarinic acetylcholine receptors (m2), Ca²⁺-dependent K⁺ channels (SK), voltage-activated Ca²⁺ channels (N-type) and delayed rectifier K⁺ channels (Kv2.1) are clustered postsynaptically. Sigma-1 receptors (σ1R) appear to be located beneath the postsynaptic plasma membrane, on subsurface cisternae (SSC).

Fig. 2

Source and distribution of C boutons. (A,B) A Pitx2-Cre mouse line, which directs expression of Cre recombinase selectively in Pitx2⁺ neurons, was crossed with conditional promoter-stop-FP reporter lines (Zagoraiou et al., 2009). Fluorescent protein (FP) is expressed in a small population of neurons located close to the central canal (CC). Inset shows a higher magnification view of fluorescent Pitx2⁺ interneurons. (C) Coexpression of vAChT and FP in a Pitx2-Cre; promotor-stop-FP mouse, confirming Pitx2⁺ interneurons as the sole source of C boutons. (D) Schematic representation of C bouton source neurons (V0_C interneurons) and their axonal projections. The majority of V0_C neurons (∼70%) project solely to ipsilateral motor neurons (MN), whereas the remaining cells project contralaterally, or possibly bilaterally. The axons of V0_C interneurons may also project rostrocaudally to innervate motor neurons in different segments of the spinal cord.

Fig. 3

Function of the C bouton system. (A) C bouton source cells (V0_C interneurons) are well positioned to control motor neuron (MN) output in a task-dependent manner. They receive a range of inputs including: oligosynaptic sensory input; descending input likely from the brainstem; and rhythmic input from the locomotor central pattern generator (CPG). DRG, dorsal root ganglion. (B) Signalling at C bouton synapses is thought to involve release of acetylcholine (ACh), activation of postsynaptic m2-type muscarinic acetylcholine receptors (m2R), and the subsequent blockade of Ca²⁺-dependent K⁺ channels (SK-type) which underlie the action potential after hyperpolarisation (AHP). (C) Increases in C bouton activation should lead to reductions in the motor neuron AHP, increased motor neuron firing rates, greater activation of muscles, and ultimately more intense muscle contractions.

Fig. 4

Involvement of C boutons in spinal cord injury and disease. Compared to ‘healthy’ control conditions (A), C boutons appear to be reduced in number in spinal cord injury (B) and enlarged in amyotrophic lateral sclerosis (ALS; C). Decreased C bouton numbers in spinal cord injury should reduce motor neuron excitability and the frequency of motor neuron output (B, bottom trace), perhaps contributing to overall motor dysfunction. Conversely, enlarged C boutons in ALS should lead to greater motor neuron excitability, which might contribute to excitotoxic disease mechanisms.