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. 2009 Feb;174(2):574-85.
doi: 10.2353/ajpath.2009.080557. Epub 2008 Dec 30.

Glycinergic innervation of motoneurons is deficient in amyotrophic lateral sclerosis mice: a quantitative confocal analysis

Qing Chang  1 , Lee J Martin
Affiliations

Glycinergic innervation of motoneurons is deficient in amyotrophic lateral sclerosis mice: a quantitative confocal analysis

Qing Chang et al. Am J Pathol. 2009 Feb.

Abstract

Altered motoneuron excitability is involved in amyotrophic lateral sclerosis pathobiology. To test the hypothesis that inhibitory interneuron innervation of spinal motoneurons is abnormal in an amyotrophic lateral sclerosis mouse model, we measured GABAergic, glycinergic, and cholinergic immunoreactive terminals on spinal motoneurons in mice expressing a mutant form of human superoxide dismutase-1 with a Gly93-->Ala substitution (G93A-SOD1) and in controls at different ages. Glutamic acid decarboxylase, glycine transporter-2, and choline acetyltransferase were used as markers for GABAergic, glycinergic, and cholinergic terminals, respectively. Triple immunofluorescent labeling of boutons contacting motoneurons was visualized by confocal microscopy and analyzed quantitatively. Glycine transporter-2-bouton density on lateral motoneurons was decreased significantly in G93A-SOD1 mice compared with controls. This reduction was absent at 6 weeks of age but present in asymptomatic 8-week-old mice and worsened with disease progression from 12 to 14 weeks of age. Motoneurons lost most glycinergic innervation by 16 weeks of age (end-stage) when there was a significant decrease in the numbers of motoneurons and choline acetyltransferase-positive boutons. No significant differences in glutamic acid decarboxylase-bouton densities were found in G93A-SOD1 mice. Reduction of glycinergic innervation preceded mitochondrial swelling and vacuolization. Calbindin-positive Renshaw cell number was decreased significantly at 12 weeks of age in G93A-SOD1 mice. Thus, either the selective loss of inhibitory glycinergic regulation of motoneuron function or glycinergic interneuron degeneration contributes to motoneuron degeneration in amyotrophic lateral sclerosis.

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Figures

Figure 1
Figure 1
A: Confocal images of motoneurons in lumbar spinal cord sections triple labeled with GlyT2 (green), GAD (blue), and ChAT (red) in wild-type mice (WT) and G93A-SOD1 mice at 8, 10, and 12 weeks (W) of age. Each panel corresponds to a single optical section. Scale bar = 10 μm. B-E: Quantitative analysis of the GlyT2- (C), GAD- (D), and ChAT- (E) immunopositive boutons contacting the ChAT-immunopositive motoneurons and the number of motoneurons (B) from wild-type and G93A-SOD1 mice at 6, 8, 10, 12, 14, and 16 weeks of age. A significant decrease in the density of GlyT2-boutons was observed at asymptomatic 8-week-old G93A-SOD1 mice (C), before the decrease of the number of motoneurons (B). Motoneurons lost the majority of GlyT2-boutons at 16 weeks of age (C). No significant difference was observed in the density of GAD-boutons at all ages (D). Significant differences in the density of ChAT-boutons were not found until end-stage (16 weeks) in G93A-SOD1 mice (E). Data represent the mean ± SEM (*P < 0.05, **P < 0.01, Student’s t-test). n = 3 to 5 mice per group. Eight to ten motoneurons per spinal cord and two to three lumbar spinal cord sections per animal were quantified.
Figure 2
Figure 2
A: Confocal images of lumbar spinal motoneurons showing colocalization (yellow, arrows) of GlyT2 (left, green) or GAD (right, green) with GAP43 (red) in apposition to ChAT-positive neurons (blue) in wild-type and 10-week-old G93A-SOD1 mice. Scale bar = 10 μm. B: Quantitative analysis of colocalization. Graphics show the average numbers of GAP43/GlyT2 (left) or GAP43/GAD (right) colocalized pixels representing the axonal growth in wild-type and G93A-SOD1 mice at 6, 8, and 10 weeks (W) of age. Analysis of GAP43/GlyT2 colocalization revealed statistically significant increases in the area of immunofluorescent colocalization in 10-week-old G93A-SOD1 mice compared with control. Data represent the mean ± SEM (*P < 0.05, Student’s t-test). n = 3 mice per group.
Figure 3
Figure 3
Optical sections of lumbar motoneurons illustrating GlyT2- (green), GAD- (B; blue), and ChAT- (A; blue) positive boutons colocalized with synaptophysin (SYN; red) immunoreactive presynaptic terminals. A: Colocalization (yellow) of GlyT2 and synaptophysin, and colocalization (pink) of ChAT and synaptophysin around the soma and proximal dendrites of ChAT-positive motoneurons. B: Colocalization (yellow) of GlyT2 and synaptophysin, and colocalization (pink) of GAD and synaptophysin around the putative motoneurons. Three-way colocalization (white) of GlyT2-, GAD-, and synaptophysin immunoreactivity was also observed. Scale bar = 10 μm. The area delineated by white rectangular is shown of higher magnification at right. Scale bar = 2 μm.
Figure 4
Figure 4
Unbiased quantification of all surviving lateral pool motoneurons from single spinal cord optical sections of wild-type mice (WT) and G93A-SOD1 mice at 6, 8, 10, 12, and 14 weeks (W) of age. The average size of motoneurons gradually increased from 10 weeks of age, reached maximum at 12 weeks, and started to decrease at 14 weeks in G93A-SOD1 mice (A). Motoneurons were divided into two groups based on their Feret’s diameters: 20 to 30 μm group and >30 μm group, and the number of motoneurons in wild-type and G93A-SOD1 mice at different ages was shown in (B). The number of GlyT2- (C), GAD- (D), and ChAT- (E) immunopositive boutons contacting all surviving lateral motoneurons was also shown. Data represent the mean ± SEM (*P < 0.05, **P < 0.01, Student’s t-test). n = 3 to 5 mice per group.
Figure 5
Figure 5
Quantification of GlyT2- (A), GAD- (B), and ChAT- (C) immunopositive boutons contacting all medial pool motoneurons from single spinal cord optical sections of wild-type mice (WT) and G93A-SOD1 mice at 6, 8, 10, 12, and 14 weeks (W) of age. No significant decrease in GlyT2-bouton number on medial motoneurons was observed until 14 weeks of age in G93A-SOD1 mice (A). Data represent the mean ± SEM (**P < 0.01, Student’s t-test). n = 3 to 5 mice per group.
Figure 6
Figure 6
Representative images showing MnSOD labeling (green) within ChAT- (red) positive neurons in wild-type and G93A-SOD1 mice at 8, 10, and 12 weeks of age. No obvious MnSOD-positive mitochondrial swelling was observed until 10 weeks of age in G93A-SOD1 mice. Scale bar = 10 μm.
Figure 7
Figure 7
A: Immunocytochemistry shows calbindin-immunopositive Renshaw cells in the ventral horn of spinal cord sections from wild-type and G93A-SOD1 mice. Scale bar = 20 μm. B: Quantitative analysis of Renshaw cell number from wild-type and G93A-SOD1 mice at 6, 8, 10, 12, and 14 weeks (W) of age. Data represent the mean ± SEM (*P < 0.05, Student’s t-test). n = 3 to 5 mice per group. Two to three lumbar spinal cord sections per animal were quantified.
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