Age-Related Changes in Pre- and Postsynaptic Partners of the Cholinergic C-Boutons in Wild-Type and SOD1G93A Lumbar Motoneurons

Abstract

Large cholinergic synaptic terminals known as C-boutons densely innervate the soma and proximal dendrites of motoneurons that are prone to neurodegeneration in amyotrophic lateral sclerosis (ALS). Studies using the Cu/Zn-superoxide dismutase (SOD1) mouse model of ALS have generated conflicting data regarding C-bouton alterations exhibited during ALS pathogenesis. In the present work, a longitudinal study combining immunohistochemistry, biochemical approaches and extra- and intra-cellular electrophysiological recordings revealed that the whole spinal cholinergic system is modified in the SOD1 mouse model of ALS compared to wild type (WT) mice as early as the second postnatal week. In WT motoneurons, both C-bouton terminals and associated M2 postsynaptic receptors presented a complex age-related dynamic that appeared completely disrupted in SOD1 motoneurons. Indeed, parallel to C-bouton morphological alterations, analysis of confocal images revealed a clustering process of M2 receptors during WT motoneuron development and maturation that was absent in SOD1 motoneurons. Our data demonstrated for the first time that the lamina X cholinergic interneurons, the neuronal source of C-boutons, are over-abundant in high lumbar segments in SOD1 mice and are subject to neurodegeneration in the SOD1 animal model. Finally, we showed that early C-bouton system alterations have no physiological impact on the cholinergic neuromodulation of newborn motoneurons. Altogether, these data suggest a complete reconfiguration of the spinal cholinergic system in SOD1 spinal networks that could be part of the compensatory mechanisms established during spinal development.

Experimental Design and Data Analysis Issues Contribute to Inconsistent Results of C-Bouton Changes in Amyotrophic Lateral Sclerosis

“…In sum, our data could not replicate reported differences in the G93A line (Pullen and Athanasiou, 2009; Herron and Miles, 2012; Saxena et al, 2013; Milan et al, 2015) by showing no change in C-bouton size, and also showed a decrease in C-bouton number and density only at end stage.…”

“…This was surprising, as bouton enlargement has been reported in the literature (Pullen and Athanasiou, 2009; Herron and Miles, 2012; Saxena et al, 2013; Milan et al, 2015) and has been interpreted as an example of an ALS disease change that influences motoneuron excitability. It is important to mention that our investigation, as opposed to most studies on this topic (Table 1), was based on large samples of WT and G93A motoneurons supported by statistical power analysis and was conducted with blinding to avoid potential bias.…”

“…Recently, a large number of studies have examined potential changes in C-bouton size during ALS disease progression in male mice of the G93A line (summarized in Table 1). However, the reported results have been conflicting: various studies report enlargement (Pullen and Athanasiou, 2009; Herron and Miles, 2012; Saxena et al, 2013), no change (Pullen and Athanasiou, 2009; Herron and Miles, 2012; Milan et al, 2015), or shrinkage (Milan et al, 2015) of C-boutons at various disease stages. Because C-bouton changes could potentially contribute to excitability dysregulation of motoneurons in ALS, reconciliation of these findings is a critical issue.…”

“…We performed histological analysis to test whether changes in C-bouton size took place, using standard published techniques (Pullen and Athanasiou, 2009; Herron and Miles, 2012; Saxena et al, 2013; Milan et al, 2015). We then performed additional examinations of C-boutons while varying methods of experimental design and data analysis to identify potential sources of research design variability and bias that might impact the results.…”

Neuregulin 1-ErbB module in C-bouton synapses on somatic motor neurons: molecular compartmentation and response to peripheral nerve injury

“…We have also demonstrated that ErbB2 and ErbB4 receptors are present in the presynaptic compartment, suggesting that NRG1 acts as a retrograde signalling molecule in C-type synapses. C-boutons are scarcely investigated structures19, which have been reported to suffer plastic changes in sick MNs after spinal cord injury2021, peripheral nerve lesion22, and amyotrophic lateral sclerosis (ALS)1823242526272829. In addition, mutations in S1R are considered risk factors for familial ALS, and abnormal accumulations of this protein in C-terminals have also been observed in patients with sporadic ALS303132.…”