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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

Immunostaining of C-boutons and M2 muscarinic receptors in wild type and SOD1 lumbar motoneurons.Lamina IX confocal microscopy photomicrographs of double-immunofluorescence labeling directed against CholineAcetylTransferase (ChAT, red channel) and M2 muscarinic receptors (green channel) in wild type (WT) and SOD1 postnatal day 1 (P1, A), P10 (B), P21 (C) and P100 (D) mice. Arrowheads point to cholinergic large synapses juxtaposed to motoneurons: the C-boutons. Note the absence of C-boutons in P1 motoneurons in these experimental conditions. Scale bar: 15 μm.
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pone.0135525.g001: Immunostaining of C-boutons and M2 muscarinic receptors in wild type and SOD1 lumbar motoneurons.Lamina IX confocal microscopy photomicrographs of double-immunofluorescence labeling directed against CholineAcetylTransferase (ChAT, red channel) and M2 muscarinic receptors (green channel) in wild type (WT) and SOD1 postnatal day 1 (P1, A), P10 (B), P21 (C) and P100 (D) mice. Arrowheads point to cholinergic large synapses juxtaposed to motoneurons: the C-boutons. Note the absence of C-boutons in P1 motoneurons in these experimental conditions. Scale bar: 15 μm.

Mentions: In the first part of this study, we examined the evolution of C-bouton terminals juxtaposed to Mns throughout the life span of WT and SOD1 mice. For this purpose, ChAT-immunolabeling was performed in the lumbar spinal cords from male SOD1 mice and WT littermates at key developmental stages and over the course of the disease, in P1, P10, P21, P40, P75 and P100 animals (Fig 1A–1D; red channel; data not shown for P40 and P75 mice). Regardless of the mouse genotype, we found that, as previously reported [24], C-boutons are absent or too small to be detected in P1 animals in our experimental conditions (Fig 1A) but are easily distinguishable as large ChAT-immunopositive structures surrounding Mn somata and proximal dendrites in P10 mice (arrowheads in Fig 1B). The mean C-bouton number and the mean C-bouton area and position on Mn soma and proximal dendrites were then computed in P10, P21, P40, P75 and P100 WT (Fig 2, black bars) and SOD1 (Fig 2, white bars) mice. As C-bouton number can vary with Mn size, we measured Mn soma perimeter (Fig 2B) and expressed the results as the density of C-boutons on Mns (C-bouton number/Mn perimeter, Fig 2C). This analysis showed, first, that in juvenile stages (P1-P10), the perimeter of lumbar SOD1 Mns were significantly smaller than age-matched WT Mns (Fig 2B). We also observed that in WT animals, the density of C-boutons juxtaposed to Mns increased from P10 to P21, stabilized until P75, and then increased again in P100 mice (Fig 2C), while the mean C-bouton area exhibited a progressive increase with age (Fig 2D). This temporal profile was different in SOD1 mice where C-bouton density strongly increased from P10 to P40 as in WT but decreased at P75 (Fig 2C). In parallel, the mean C-bouton area progressively augmented with age until P75 and decreased at P100 (Fig 2D). Interestingly, pair-wise comparison of data obtained in each age group between WT and SOD1 mice revealed that the density of C-terminals was significantly higher at P21 and P40 in SOD1 mice and significantly lower at P100 compared to WT mice (Fig 2C). In addition, the mean C-bouton area was found to be significantly lower in P10 and P100 and significantly higher in P21 SOD1 mice compared to WT animals (Fig 2D). In a next step, we computed the distance between C-bouton centroids and Mn centroid to see whether the spatial distribution of C-boutons differs between SOD1 and WT MNs and whether C-boutons juxtaposed to SOD1 MNs exhibit a particular topographic pattern of vulnerability during ALS pathogenesis. We observed that this parameter did not evolve with age in either WT or SOD1 mice (Fig 2E). C-boutons were located within a radius of 20–23 μm around the Mn centroid as early as P10. Because Mns were smaller in size in P10 SOD1 mice compared to WT, the C-boutons appeared more clustered in these neurons (Fig 2E). In contrast, although P21 and P100 Mns exhibited similar perimeter values in both genotypes, C-boutons appeared significantly closer to the Mn centroid in SOD1 P21 and P100 animals compared to WT mice.


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)

Immunostaining of C-boutons and M2 muscarinic receptors in wild type and SOD1 lumbar motoneurons.Lamina IX confocal microscopy photomicrographs of double-immunofluorescence labeling directed against CholineAcetylTransferase (ChAT, red channel) and M2 muscarinic receptors (green channel) in wild type (WT) and SOD1 postnatal day 1 (P1, A), P10 (B), P21 (C) and P100 (D) mice. Arrowheads point to cholinergic large synapses juxtaposed to motoneurons: the C-boutons. Note the absence of C-boutons in P1 motoneurons in these experimental conditions. Scale bar: 15 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135525.g001: Immunostaining of C-boutons and M2 muscarinic receptors in wild type and SOD1 lumbar motoneurons.Lamina IX confocal microscopy photomicrographs of double-immunofluorescence labeling directed against CholineAcetylTransferase (ChAT, red channel) and M2 muscarinic receptors (green channel) in wild type (WT) and SOD1 postnatal day 1 (P1, A), P10 (B), P21 (C) and P100 (D) mice. Arrowheads point to cholinergic large synapses juxtaposed to motoneurons: the C-boutons. Note the absence of C-boutons in P1 motoneurons in these experimental conditions. Scale bar: 15 μm.
Mentions: In the first part of this study, we examined the evolution of C-bouton terminals juxtaposed to Mns throughout the life span of WT and SOD1 mice. For this purpose, ChAT-immunolabeling was performed in the lumbar spinal cords from male SOD1 mice and WT littermates at key developmental stages and over the course of the disease, in P1, P10, P21, P40, P75 and P100 animals (Fig 1A–1D; red channel; data not shown for P40 and P75 mice). Regardless of the mouse genotype, we found that, as previously reported [24], C-boutons are absent or too small to be detected in P1 animals in our experimental conditions (Fig 1A) but are easily distinguishable as large ChAT-immunopositive structures surrounding Mn somata and proximal dendrites in P10 mice (arrowheads in Fig 1B). The mean C-bouton number and the mean C-bouton area and position on Mn soma and proximal dendrites were then computed in P10, P21, P40, P75 and P100 WT (Fig 2, black bars) and SOD1 (Fig 2, white bars) mice. As C-bouton number can vary with Mn size, we measured Mn soma perimeter (Fig 2B) and expressed the results as the density of C-boutons on Mns (C-bouton number/Mn perimeter, Fig 2C). This analysis showed, first, that in juvenile stages (P1-P10), the perimeter of lumbar SOD1 Mns were significantly smaller than age-matched WT Mns (Fig 2B). We also observed that in WT animals, the density of C-boutons juxtaposed to Mns increased from P10 to P21, stabilized until P75, and then increased again in P100 mice (Fig 2C), while the mean C-bouton area exhibited a progressive increase with age (Fig 2D). This temporal profile was different in SOD1 mice where C-bouton density strongly increased from P10 to P40 as in WT but decreased at P75 (Fig 2C). In parallel, the mean C-bouton area progressively augmented with age until P75 and decreased at P100 (Fig 2D). Interestingly, pair-wise comparison of data obtained in each age group between WT and SOD1 mice revealed that the density of C-terminals was significantly higher at P21 and P40 in SOD1 mice and significantly lower at P100 compared to WT mice (Fig 2C). In addition, the mean C-bouton area was found to be significantly lower in P10 and P100 and significantly higher in P21 SOD1 mice compared to WT animals (Fig 2D). In a next step, we computed the distance between C-bouton centroids and Mn centroid to see whether the spatial distribution of C-boutons differs between SOD1 and WT MNs and whether C-boutons juxtaposed to SOD1 MNs exhibit a particular topographic pattern of vulnerability during ALS pathogenesis. We observed that this parameter did not evolve with age in either WT or SOD1 mice (Fig 2E). C-boutons were located within a radius of 20–23 μm around the Mn centroid as early as P10. Because Mns were smaller in size in P10 SOD1 mice compared to WT, the C-boutons appeared more clustered in these neurons (Fig 2E). In contrast, although P21 and P100 Mns exhibited similar perimeter values in both genotypes, C-boutons appeared significantly closer to the Mn centroid in SOD1 P21 and P100 animals compared to WT mice.

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