On the following articles, the articles and reviews for my initial master work will be summarized.
1. Witts EC, Zagoraiou L and Miles GB. Anatomy and function of cholinergic C bouton inputs to motor neurons. J Anat. 2013:
Motor neurons receive many presynaptic inputs, which are integrated by them into the electric signals that finally act over the muscles, producing controlled, coordinated movement. The neuromodulatory systems that act over motor neurons are therefore important (maybe as important as the motor neuron itself) to understand how movement is regulated. Much work has been conducted over control systems that arise from the brainstem nuclei, but much less has studied spinal systems. C-boutons, which are discreet, spinally-derived modulatory inputs to motor neurons, have recently been proposed to have a role in movement modulation.
Anatomy of the C bouton synapse
C boutons are large (2-6 micrometers diameter) synaptic inputs in the soma and proximal dendrites of motor neurons. They are associated with highly-specialised postsynaptic structures, its sub-surface cisternae probably being the most disctinctive. The sub-surface cisternae are flattened membrane discs, just below the synaptic cleft, extending all along the synaptic surface. These particular synapses were given the name of "C-type synapsis", to distinguish them from other types. C-synapses have been reported in a wide range of mammals.
C boutons express choline acetyl transferase (ChAT), acetylcholinesterase (AChE) and vesicular acetylcholine transporter (vAChT). They are, therefore, cholinergic inputs.
There are specific sets of proteins clustered in the postsynaptic membrane and in the postsynaptic density of C boutons, presumably mediating transmission of C-boutons input. Clusters of metabotropic m2-type muscarinic receptors are present. Nicotinic ACh receptors have not been reported postsynaptically, but presynaptically instead. ATP receptors (P2X7) have also been reported presynaptically. Those presynaptic receptors are thought to contribute to feedback control at the synapsis. There is also postsynaptic clustering of ion channels, being Kv2.1 one of the most clearly clustered. Ca2+ dependent K+ channels are also present, and it seems that, at least in rodents, the type of Ca2+-mediated K+ receptor could depend in the motor neuron subtype that the C bouton is targeting, thus indicating a certain degreee of specificity in C bouton signalling. N-type calcium channels also show high levels of postsynaptic expression at C bouton synapses, perhaps due to Ca2+ requierements of the SK. Finally, sigma-1 receptors also form clusters at C bouton synapses, particularly on sub-surface cisternae. The enzime that converts tryptamine to an agonist of sigma-1 receptors, dimethyltryptamine, is also close to the postsynaptic membrane of C bouton synapses.
C boutons are not cells, but they are just the axonal terminal of a particular type of neuron, making a synapsis with a motor neuron. The neurons that produce C boutons are spinal cord cholinergic interneurons, which are a medially positioned population of partition cells (V0C interneurons). These interneurons and the C boutons:
V0Cs circuitry
C boutons selectively innervate the soma and proximal dendrites of motor neurons throughout the spinal cord, showing selectivity for alpha over gamma motor neurons. They also seem to preferentially innervate motor neurons innervating large proximal muscles, instead of those innervating small distal muscles, and also fast twitch muscles compared with slow twitch muscles. There is great divergence in a single V0C signal, since each of them makes around 1000 synaptic contacts with different motor neurons. Transynaptic viral experiments show that most C boutons project to ipsilateral motor neurons, althought up to a third V0Cs have contralateral projections. A work has suggested that some V0C interneurons may project bilaterally to functionally equivalent motor neurons in each side of the spinal cord, but the study was centered in a very specific population of interneurons, and they were not defined as Pitx2+, V0C interneurons. This work also raised the possibility that motor neurons received C bouton inputs from various V0C interneurons situated in the rostrocaudal axis of the spinal cord.
In addition to innervate motor neurons, V0C also send axons to Ia inhibitory interneurons in the spinal cord and to other interneurons within the intermediate zone of the spinal cord which have not been thoroughly characterised yet.
To determine the circuitry of C boutons, it is also important to determine the inputs V0C receive. Data are limited, but there is evidence of descending serotoninergic inputs and a lack of direct primary afferent input. V0C receive inputs from the spinal locomotor central pattern generation (CPG), but are not involved in the generation of the locomotor rythm (which is the function of CPG). These inputs are similar to those that motor neurons receive themselves, and therefore, it has been suggested that V0Cs may receive copies of the input that motor neurons are receiving, which may enable them to control the activity of the motor neurons to adapt it to the circumstances.
C bouton function
Given the clustering of m2 muscarinic receptors at C bouton synapses, pharmacological activation of these was used to determine the function of C boutons. These studies proved that m2 receptors activation reduced the action potential after hiperpolarization, therefore increasing motor neuron excitability. This effect was mediated by a blockade of Ca2+ dependent K+ channels, while voltage-dependent Ca2+ channels were left unaltered.
Surprisingly, despite their representativeness of C bouton synapse, the roles of Kv2.1 and sigma1 receptors are still unknown. It has been proposed that Kv2.1 could underlie m2-induced hyperpolarization, but it seems unlikely. Although, Kv2.1 are known to control neuronal excitability, perhaps by limiting neuron hyperexcitability. Interestingly, the position and function of Kv2.1 depends on their phosphorilation state: dephosphorilated channels are dispersed and show a leftward shift in their activation curve, while phosphorilated Kv2.1 produces a rightward shift in their activation curve. This makes plausible that activation of C boutons -leading to an activation of m2 receptors, which causes phosphorilation of Kv2.1-, may need a greater depolarization in order to be opened, thus providing another mechanism to increase motor neuron excitability -the first one was the one mediated by m2 receptors-. Kv2.1 also have non-conducting roles, which they conduct predominantly when clustered on the plasma membrane. In this state, they may instead act as cell surface insertion platforms for ion channel trafficking.
One possible function for sigma-1 receptor is the regulation of Ca2+ concentration at C-synapses. This possibility is supported by their localization at sub-surface cisternae, and their known association with IP3 receptors. Therefore, sigma-1 receptors could countrol the release of Ca2+ from sub-surface cisternae, or alternatively, they could modulate the function of ion channels at C-synapses to control motor neuron excitability: given their direct interaction with Kv2.1 channels, they may regulate their traffick between intracellular compartments and the plasma membrane.
In addition to modulate the intrinsic properties of motor neurons, C boutons may also modulate synaptic inputs to motor neurons. A recent study in rodents showed that the activation of projections from cholinergic neurons close to the central canal potentiated commissural glutamatergic inputs to motor neurons via muscarinic receptor-dependent mechanisms. Another recent study, though, showed that muscarinic receptor activation could inhibit synaptic currents mediated by postsynaptic AMPA receptors on motor neurons. Further research is required in this area.
Research into the function of C boutons has primarily focused on their contribution to the control of locomotion. Fictive locomotor activity induces c-fos expression in cholinergic interneurons near the central canal. It has also been demonstrated that V0Cs activity is tighly related with the phases in motor neuron activity during fictive locomotion. Another study proved that blockade of m2 receptors reduced the amplitude of locomotor-related bursts of motor neurons output, while cholinesterase inhibition had the opposite effects. Neither treatment affected the frequency or pattern of locomotor-related output, which points to a role of C boutons in the modulation of movement, but not in CPG functions.
In rodents, C boutons have proved to have an impact on animal behaviour. On a study, normal rodents and choline acetyltransferase KOs were subjected to walking and swimming tasks. Muscle activation was assessed through EMG measurements performed during the tasks. Greater activation of some hind limb muscles was expected during swimming, which is normal in rodents. However, KOs showed significantly diminished muscle activation enhancement. This suggests C boutons regulate the activation of the different muscles involved in a movement in order to match the biomechanical environment and needs for that movement. Possible mechanisms to regulate V0Cs are feedback inputs from sensory systems and feedforward inputs from higher motor control centres.
Many aspects of C boutons remain to be elucidated: the inputs they receive, or the synapsis the make with motor neurons (it seems there is a certain selectivity, beyond the motor neuron preferences that have been addressed before). But despite this, it seems clear that C bouton regulation of motor neuron activity is highly dynamic, and it allows V0Cs to finely tune movements.
Clinical significance of the C bouton system
Changes in the C bouton system have been described in a number of studies involving animal models of spinal cord injury. Rodent studies of contusion or complete spinal cord transection have shown a reduction in C boutons after injury, although C bouton loss was not observed in all motor neuron types. Following injury, Kv2.1 levels and membrane clusters reduced. Interestingly, post-injury training seems to trigger a recovery of C boutons in these animals. However, studies conducted in cats failed to show reductions in C boutons after injury, recovering function, and normal number and size of C boutons between 72 and 200 days post-injury. Overall, though, these studies seem to point to a correlation between motor function and C boutons, so these structures could be interesting therapeuthical targets to treat spinal cord injury.
There is also evidence of C boutons alteration in Amyotrophic Lateral Sclerosis (ALS). C boutons are perturbed in human ALS ans rodent models of the disease. Animal models show larger C boutons, and in rodent models and human studies, a reduction in the number of C boutons has been observed (it has been related to the last stages of the disease). It has been hypothesized that enlarged C boutons represent a compensatory effort to increase motor neuron output, but since they are present in the early development of the mutant mice, long before motor symptoms arise, these enlarged C boutons seem likely to have a role in the pathogenesis of ALS. Larger C boutons could lead to motor neuron death by excitotoxic mechanisms, but a major caveat to this hypothesis is the finding that C boutons are only altered in male ALS model mice. However, this observation suggests that, although C boutons may not be central to ALS pathology, they may explain the greater susceptibility of males to the disease (3:1 to females).
Another link between C boutons and ALS has come from the demonstration that sigma-1 receptors are mutated in some forms of familial ALS. In addition, an abnormal distribution and an overall reduction of sigma-1 receptors has been observed in human patients and in animal models of ALS. Finally, treatment of ALS model mice with a sigma-1 receptor agonist reduces motor neuron loss and reduces also deficits in motor function, improving locomotor performance and survival.
Conclusion
C boutons seem to be of great importance for the spinal control of movement, and this control seems to be produced in a dynamic way, allowing them to adjust muscle activation to the needs imposed by the environment or other factors. They also show alterations in pathological states, which have been suggested to be correlated with functional findings. Therefore, there is a great interest in elucidating the physiology of these structures in normal conditions, to understand motor control, and also in pathological states, because of their potential as therapeuthic targets for neurological diseases.
Anatomy of the C bouton synapse
C boutons are large (2-6 micrometers diameter) synaptic inputs in the soma and proximal dendrites of motor neurons. They are associated with highly-specialised postsynaptic structures, its sub-surface cisternae probably being the most disctinctive. The sub-surface cisternae are flattened membrane discs, just below the synaptic cleft, extending all along the synaptic surface. These particular synapses were given the name of "C-type synapsis", to distinguish them from other types. C-synapses have been reported in a wide range of mammals.
C boutons express choline acetyl transferase (ChAT), acetylcholinesterase (AChE) and vesicular acetylcholine transporter (vAChT). They are, therefore, cholinergic inputs.
There are specific sets of proteins clustered in the postsynaptic membrane and in the postsynaptic density of C boutons, presumably mediating transmission of C-boutons input. Clusters of metabotropic m2-type muscarinic receptors are present. Nicotinic ACh receptors have not been reported postsynaptically, but presynaptically instead. ATP receptors (P2X7) have also been reported presynaptically. Those presynaptic receptors are thought to contribute to feedback control at the synapsis. There is also postsynaptic clustering of ion channels, being Kv2.1 one of the most clearly clustered. Ca2+ dependent K+ channels are also present, and it seems that, at least in rodents, the type of Ca2+-mediated K+ receptor could depend in the motor neuron subtype that the C bouton is targeting, thus indicating a certain degreee of specificity in C bouton signalling. N-type calcium channels also show high levels of postsynaptic expression at C bouton synapses, perhaps due to Ca2+ requierements of the SK. Finally, sigma-1 receptors also form clusters at C bouton synapses, particularly on sub-surface cisternae. The enzime that converts tryptamine to an agonist of sigma-1 receptors, dimethyltryptamine, is also close to the postsynaptic membrane of C bouton synapses.
C boutons are not cells, but they are just the axonal terminal of a particular type of neuron, making a synapsis with a motor neuron. The neurons that produce C boutons are spinal cord cholinergic interneurons, which are a medially positioned population of partition cells (V0C interneurons). These interneurons and the C boutons:
| Expression of | Lack expression |
| Dbx-1 Pitx2 | nNOS |
V0Cs circuitry
C boutons selectively innervate the soma and proximal dendrites of motor neurons throughout the spinal cord, showing selectivity for alpha over gamma motor neurons. They also seem to preferentially innervate motor neurons innervating large proximal muscles, instead of those innervating small distal muscles, and also fast twitch muscles compared with slow twitch muscles. There is great divergence in a single V0C signal, since each of them makes around 1000 synaptic contacts with different motor neurons. Transynaptic viral experiments show that most C boutons project to ipsilateral motor neurons, althought up to a third V0Cs have contralateral projections. A work has suggested that some V0C interneurons may project bilaterally to functionally equivalent motor neurons in each side of the spinal cord, but the study was centered in a very specific population of interneurons, and they were not defined as Pitx2+, V0C interneurons. This work also raised the possibility that motor neurons received C bouton inputs from various V0C interneurons situated in the rostrocaudal axis of the spinal cord.
In addition to innervate motor neurons, V0C also send axons to Ia inhibitory interneurons in the spinal cord and to other interneurons within the intermediate zone of the spinal cord which have not been thoroughly characterised yet.
To determine the circuitry of C boutons, it is also important to determine the inputs V0C receive. Data are limited, but there is evidence of descending serotoninergic inputs and a lack of direct primary afferent input. V0C receive inputs from the spinal locomotor central pattern generation (CPG), but are not involved in the generation of the locomotor rythm (which is the function of CPG). These inputs are similar to those that motor neurons receive themselves, and therefore, it has been suggested that V0Cs may receive copies of the input that motor neurons are receiving, which may enable them to control the activity of the motor neurons to adapt it to the circumstances.
C bouton function
Given the clustering of m2 muscarinic receptors at C bouton synapses, pharmacological activation of these was used to determine the function of C boutons. These studies proved that m2 receptors activation reduced the action potential after hiperpolarization, therefore increasing motor neuron excitability. This effect was mediated by a blockade of Ca2+ dependent K+ channels, while voltage-dependent Ca2+ channels were left unaltered.
Surprisingly, despite their representativeness of C bouton synapse, the roles of Kv2.1 and sigma1 receptors are still unknown. It has been proposed that Kv2.1 could underlie m2-induced hyperpolarization, but it seems unlikely. Although, Kv2.1 are known to control neuronal excitability, perhaps by limiting neuron hyperexcitability. Interestingly, the position and function of Kv2.1 depends on their phosphorilation state: dephosphorilated channels are dispersed and show a leftward shift in their activation curve, while phosphorilated Kv2.1 produces a rightward shift in their activation curve. This makes plausible that activation of C boutons -leading to an activation of m2 receptors, which causes phosphorilation of Kv2.1-, may need a greater depolarization in order to be opened, thus providing another mechanism to increase motor neuron excitability -the first one was the one mediated by m2 receptors-. Kv2.1 also have non-conducting roles, which they conduct predominantly when clustered on the plasma membrane. In this state, they may instead act as cell surface insertion platforms for ion channel trafficking.
One possible function for sigma-1 receptor is the regulation of Ca2+ concentration at C-synapses. This possibility is supported by their localization at sub-surface cisternae, and their known association with IP3 receptors. Therefore, sigma-1 receptors could countrol the release of Ca2+ from sub-surface cisternae, or alternatively, they could modulate the function of ion channels at C-synapses to control motor neuron excitability: given their direct interaction with Kv2.1 channels, they may regulate their traffick between intracellular compartments and the plasma membrane.
In addition to modulate the intrinsic properties of motor neurons, C boutons may also modulate synaptic inputs to motor neurons. A recent study in rodents showed that the activation of projections from cholinergic neurons close to the central canal potentiated commissural glutamatergic inputs to motor neurons via muscarinic receptor-dependent mechanisms. Another recent study, though, showed that muscarinic receptor activation could inhibit synaptic currents mediated by postsynaptic AMPA receptors on motor neurons. Further research is required in this area.
Research into the function of C boutons has primarily focused on their contribution to the control of locomotion. Fictive locomotor activity induces c-fos expression in cholinergic interneurons near the central canal. It has also been demonstrated that V0Cs activity is tighly related with the phases in motor neuron activity during fictive locomotion. Another study proved that blockade of m2 receptors reduced the amplitude of locomotor-related bursts of motor neurons output, while cholinesterase inhibition had the opposite effects. Neither treatment affected the frequency or pattern of locomotor-related output, which points to a role of C boutons in the modulation of movement, but not in CPG functions.
In rodents, C boutons have proved to have an impact on animal behaviour. On a study, normal rodents and choline acetyltransferase KOs were subjected to walking and swimming tasks. Muscle activation was assessed through EMG measurements performed during the tasks. Greater activation of some hind limb muscles was expected during swimming, which is normal in rodents. However, KOs showed significantly diminished muscle activation enhancement. This suggests C boutons regulate the activation of the different muscles involved in a movement in order to match the biomechanical environment and needs for that movement. Possible mechanisms to regulate V0Cs are feedback inputs from sensory systems and feedforward inputs from higher motor control centres.
Many aspects of C boutons remain to be elucidated: the inputs they receive, or the synapsis the make with motor neurons (it seems there is a certain selectivity, beyond the motor neuron preferences that have been addressed before). But despite this, it seems clear that C bouton regulation of motor neuron activity is highly dynamic, and it allows V0Cs to finely tune movements.
Clinical significance of the C bouton system
Changes in the C bouton system have been described in a number of studies involving animal models of spinal cord injury. Rodent studies of contusion or complete spinal cord transection have shown a reduction in C boutons after injury, although C bouton loss was not observed in all motor neuron types. Following injury, Kv2.1 levels and membrane clusters reduced. Interestingly, post-injury training seems to trigger a recovery of C boutons in these animals. However, studies conducted in cats failed to show reductions in C boutons after injury, recovering function, and normal number and size of C boutons between 72 and 200 days post-injury. Overall, though, these studies seem to point to a correlation between motor function and C boutons, so these structures could be interesting therapeuthical targets to treat spinal cord injury.
There is also evidence of C boutons alteration in Amyotrophic Lateral Sclerosis (ALS). C boutons are perturbed in human ALS ans rodent models of the disease. Animal models show larger C boutons, and in rodent models and human studies, a reduction in the number of C boutons has been observed (it has been related to the last stages of the disease). It has been hypothesized that enlarged C boutons represent a compensatory effort to increase motor neuron output, but since they are present in the early development of the mutant mice, long before motor symptoms arise, these enlarged C boutons seem likely to have a role in the pathogenesis of ALS. Larger C boutons could lead to motor neuron death by excitotoxic mechanisms, but a major caveat to this hypothesis is the finding that C boutons are only altered in male ALS model mice. However, this observation suggests that, although C boutons may not be central to ALS pathology, they may explain the greater susceptibility of males to the disease (3:1 to females).
Another link between C boutons and ALS has come from the demonstration that sigma-1 receptors are mutated in some forms of familial ALS. In addition, an abnormal distribution and an overall reduction of sigma-1 receptors has been observed in human patients and in animal models of ALS. Finally, treatment of ALS model mice with a sigma-1 receptor agonist reduces motor neuron loss and reduces also deficits in motor function, improving locomotor performance and survival.
Conclusion
C boutons seem to be of great importance for the spinal control of movement, and this control seems to be produced in a dynamic way, allowing them to adjust muscle activation to the needs imposed by the environment or other factors. They also show alterations in pathological states, which have been suggested to be correlated with functional findings. Therefore, there is a great interest in elucidating the physiology of these structures in normal conditions, to understand motor control, and also in pathological states, because of their potential as therapeuthic targets for neurological diseases.
