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

Milan L, Courtand G, Cardoit L, Masmejean F, Barrière G, Cazalets JR, Garret M, Bertrand SS - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: INCIA, Université de Bordeaux, CNRS UMR5287, Bordeaux, France.

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.

No MeSH data available.


Related in: MedlinePlus

M2 receptor expression in the postsynaptic membrane of C-boutons.(A) Representative intensity plot profiles of CholineAcetylTransferase (ChAT, red channel and trace) and M2 receptor (green channel and trace) immunolabeling computed from the contour cutting of wild type (WT, left panels) and SOD1 (right panels) motoneurons of postnatal (P) 10 (A1) and P100 (A2) mice. (B) The portion of postsynaptic membrane containing M2 receptors in front of C-boutons was computed as the ratio of the area of M2 receptor labeling to that of C-bouton labeling and expressed as a function of the postnatal (P) WT (black bars) and SOD1 (white bars) mouse age. (C) Histogram of the mean M2 receptor labeling intensity under C-boutons normalized by the mean M2 labeling computed in Mn membrane devoid of C-bouton. Comparisons between groups were made with two-way ANOVA with Sidak’s multiple comparison tests. Asterisks indicate positive significance (p<0.05). The number of animals used in each group is stated in Table 1. Note the significant decrease in the M2 receptor/C-bouton area ratio with age between P10 and P75 in WT motoneurons and the absence of difference in age matched SOD1 motoneurons.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549056&req=5

pone.0135525.g004: M2 receptor expression in the postsynaptic membrane of C-boutons.(A) Representative intensity plot profiles of CholineAcetylTransferase (ChAT, red channel and trace) and M2 receptor (green channel and trace) immunolabeling computed from the contour cutting of wild type (WT, left panels) and SOD1 (right panels) motoneurons of postnatal (P) 10 (A1) and P100 (A2) mice. (B) The portion of postsynaptic membrane containing M2 receptors in front of C-boutons was computed as the ratio of the area of M2 receptor labeling to that of C-bouton labeling and expressed as a function of the postnatal (P) WT (black bars) and SOD1 (white bars) mouse age. (C) Histogram of the mean M2 receptor labeling intensity under C-boutons normalized by the mean M2 labeling computed in Mn membrane devoid of C-bouton. Comparisons between groups were made with two-way ANOVA with Sidak’s multiple comparison tests. Asterisks indicate positive significance (p<0.05). The number of animals used in each group is stated in Table 1. Note the significant decrease in the M2 receptor/C-bouton area ratio with age between P10 and P75 in WT motoneurons and the absence of difference in age matched SOD1 motoneurons.

Mentions: To go one step further, analysis of M2 receptor accumulation under C-boutons during normal development and ALS pathogenesis was performed using ChAT and M2 receptor double immunofluorescence staining (Figs 1 and 4, Table 1). For this purpose, Mn contour cutting including both presynaptic C-boutons in ChAT labeling (red channel in Figs 1 and 4A) and postsynaptic M2 receptors (green channel in Figs 1 and 4A) were realized (for details see Material and Methods). Fig 4 shows the plots of ChAT and M2 labeling intensities of pixels along the Mn contour extracted from a P10 SOD1 Mn (Fig 4A1) and a P100 WT Mn (Fig 4A2) with red peaks highlighting the presence of C-boutons. To characterize M2 receptor expression in C-bouton PSM, two different parameters were computed: 1) the portion of the PSM containing M2 receptors in front of C-boutons expressed as the ratio of the area of M2 receptor labeling to that of C-bouton labeling (Fig 4B) and 2) the M2 receptor labeling intensity under C-boutons normalized by the mean M2 labeling computed in Mn membranes devoid of C-boutons (Fig 4C). The histogram in Fig 4B shows that in WT Mns, the PSM occupied by M2 receptors under C-boutons progressively decreased from P10 to P75 and then re-increased in P100 Mns. This evolution was mirrored in the intensity values of the M2 receptor staining (Fig 4C). Indeed, M2 intensity progressively increased from P10 to P75 and then decreased in P100 Mns. As previously mentioned, we found that the mean C-bouton area exhibits a progressive increase from P10 to P75 in WT Mns (Fig 2D). Our data, therefore, interestingly suggest that in this age range, while M2 receptor expression is enhanced under enlarging C-boutons, these receptors do not spread into the PSM but are sequestrated/clustered in specific areas. At P100, in contrast, we observed a re-increase in the number of C-boutons juxtaposed to lumbar WT Mns (Fig 2C) and a return to immature (P10-like) features with the dispersion of M2 receptors under these synaptic contacts.


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

Milan L, Courtand G, Cardoit L, Masmejean F, Barrière G, Cazalets JR, Garret M, Bertrand SS - PLoS ONE (2015)

M2 receptor expression in the postsynaptic membrane of C-boutons.(A) Representative intensity plot profiles of CholineAcetylTransferase (ChAT, red channel and trace) and M2 receptor (green channel and trace) immunolabeling computed from the contour cutting of wild type (WT, left panels) and SOD1 (right panels) motoneurons of postnatal (P) 10 (A1) and P100 (A2) mice. (B) The portion of postsynaptic membrane containing M2 receptors in front of C-boutons was computed as the ratio of the area of M2 receptor labeling to that of C-bouton labeling and expressed as a function of the postnatal (P) WT (black bars) and SOD1 (white bars) mouse age. (C) Histogram of the mean M2 receptor labeling intensity under C-boutons normalized by the mean M2 labeling computed in Mn membrane devoid of C-bouton. Comparisons between groups were made with two-way ANOVA with Sidak’s multiple comparison tests. Asterisks indicate positive significance (p<0.05). The number of animals used in each group is stated in Table 1. Note the significant decrease in the M2 receptor/C-bouton area ratio with age between P10 and P75 in WT motoneurons and the absence of difference in age matched SOD1 motoneurons.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549056&req=5

pone.0135525.g004: M2 receptor expression in the postsynaptic membrane of C-boutons.(A) Representative intensity plot profiles of CholineAcetylTransferase (ChAT, red channel and trace) and M2 receptor (green channel and trace) immunolabeling computed from the contour cutting of wild type (WT, left panels) and SOD1 (right panels) motoneurons of postnatal (P) 10 (A1) and P100 (A2) mice. (B) The portion of postsynaptic membrane containing M2 receptors in front of C-boutons was computed as the ratio of the area of M2 receptor labeling to that of C-bouton labeling and expressed as a function of the postnatal (P) WT (black bars) and SOD1 (white bars) mouse age. (C) Histogram of the mean M2 receptor labeling intensity under C-boutons normalized by the mean M2 labeling computed in Mn membrane devoid of C-bouton. Comparisons between groups were made with two-way ANOVA with Sidak’s multiple comparison tests. Asterisks indicate positive significance (p<0.05). The number of animals used in each group is stated in Table 1. Note the significant decrease in the M2 receptor/C-bouton area ratio with age between P10 and P75 in WT motoneurons and the absence of difference in age matched SOD1 motoneurons.
Mentions: To go one step further, analysis of M2 receptor accumulation under C-boutons during normal development and ALS pathogenesis was performed using ChAT and M2 receptor double immunofluorescence staining (Figs 1 and 4, Table 1). For this purpose, Mn contour cutting including both presynaptic C-boutons in ChAT labeling (red channel in Figs 1 and 4A) and postsynaptic M2 receptors (green channel in Figs 1 and 4A) were realized (for details see Material and Methods). Fig 4 shows the plots of ChAT and M2 labeling intensities of pixels along the Mn contour extracted from a P10 SOD1 Mn (Fig 4A1) and a P100 WT Mn (Fig 4A2) with red peaks highlighting the presence of C-boutons. To characterize M2 receptor expression in C-bouton PSM, two different parameters were computed: 1) the portion of the PSM containing M2 receptors in front of C-boutons expressed as the ratio of the area of M2 receptor labeling to that of C-bouton labeling (Fig 4B) and 2) the M2 receptor labeling intensity under C-boutons normalized by the mean M2 labeling computed in Mn membranes devoid of C-boutons (Fig 4C). The histogram in Fig 4B shows that in WT Mns, the PSM occupied by M2 receptors under C-boutons progressively decreased from P10 to P75 and then re-increased in P100 Mns. This evolution was mirrored in the intensity values of the M2 receptor staining (Fig 4C). Indeed, M2 intensity progressively increased from P10 to P75 and then decreased in P100 Mns. As previously mentioned, we found that the mean C-bouton area exhibits a progressive increase from P10 to P75 in WT Mns (Fig 2D). Our data, therefore, interestingly suggest that in this age range, while M2 receptor expression is enhanced under enlarging C-boutons, these receptors do not spread into the PSM but are sequestrated/clustered in specific areas. At P100, in contrast, we observed a re-increase in the number of C-boutons juxtaposed to lumbar WT Mns (Fig 2C) and a return to immature (P10-like) features with the dispersion of M2 receptors under these synaptic contacts.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: INCIA, Université de Bordeaux, CNRS UMR5287, Bordeaux, France.

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.

No MeSH data available.


Related in: MedlinePlus