Figure 1.
Increased oxidative stress in Sod1−/− mice leads to early onset limb muscle atrophy and weakness.
(A) Gastrocnemius and extensor digitorum longus muscle mass expressed as % of body mass from in wild-type (WT, N = 47, 7 months old, white) and Sod1−/− (N = 32, 7 months old, gray) mice. (B, left) Chart showing the average (10 trials) grip strength in WT (N = 14, 5–6 months old, white) and Sod1−/− (N = 13, 5 months old, gray) mice. (B, right) Chart showing time to fall in the wire hanging assay for WT (N = 10, 5–6 months old, white) and Sod1−/− (N = 8, 4–5 months old, gray) mice. All mice are between 4 to 10 months of age. Data are plotted as mean with error bars representing standard error. Statistics: t-test, *p<0.05, **p<0.01.
Figure 2.
Aberrant electromyographic characteristics in Sod1−/− muscle imply deficits in neurotransmission and denervation.
(A) Representative traces of gastrocnemius compound muscle action potential (CMAP) when stimulated at 0.2 Hz and 10 Hz. Dashed line shows the amplitude of the initial peak. Traces were arbitrarily spaced 1ms apart for easy visualization. (B) Quantitative result of CMAP decrement in WT (N = 17, 7 months old, white) and Sod1−/− (N = 11, 7 months old, gray) mice in response to repetitive nerve stimulation. Data are plotted as mean with error bars representing standard error. All mice are between 4 to 10 months of age. Statistics: t-test, *p<0.05, **p<0.01.
Figure 3.
Spontaneous and evoked synaptic release are impaired at Sod1−/− EDL neuromuscular junctions (NMJs).
(A, top) Representative traces of spontaneous mEPPs in wild-type (WT) and Sod1−/− EDL muscles. (A, bottom) Amplitude-frequency histogram of spontaneous mEPPs for WT (white) and Sod1−/− (gray). Data were analyzed from pooled results from 22 individual records (∼1000 mEPPs) and binned in 0.1 mV intervals. (B) Representative traces of evoked EPPs recorded in WT (black) and Sod1−/− (gray) EDL muscle fibers. (C) EPP rundown analysis over 5s at 10 Hz and 40 Hz (EPP amplitude, top; normalized result, bottom) for WT (n = 34∼36 NMJs from N = 5 mice, 5 months old, triangles) and Sod1−/− (n = 22∼28 NMJs from N = 5 mice, 5–6 months old, diamonds).
Table 1.
Summary of EDL muscle electrophysiological properties in wild-type (WT) and Sod1−/− mice.
Figure 4.
Degenerative morphological alterations in neuromuscular junctions (NMJs) in wild-type and Sod1−/− muscles.
NMJs in EDL and gastrocnemius muscles were visualized via Thy1-YFP fluorescence in the presynaptic axon and nerve terminal (green), as described in detail in Materials and Methods, and by staining acetylcholine receptors (nAChRs) with fluorophore conjugated α-bungarotoxin (red). Partially innervated and completely denervated NMJs in Sod1−/− muscle were indicated (arrows). Axon thinning was also frequently observed in Sod1−/− mice (arrow heads). Scale bars: 50µm.
Figure 5.
Morphology-synaptic function relationship in wild-type and Sod1−/− EDL neuromuscular junctions (NMJs).
(A) Scatter plot showing the relationship between AChRs total area and its occupancy by axon shown as % of overlap over the AChRs total area in wild-type (WT, white) and Sod1−/− (gray) mice. NMJs labeled as “#” and “1, 2 and 3” are examples of individual NMJs in WT and Sod1−/− mice which were recorded for electrophysiology shown in (C). (B) Quantification of average AChR density (Fint/Area) in EDL muscle NMJs from WT (n = 30, white) and Sod1−/− (n = 30, gray) mice. Data are plotted as mean with error bars representing standard error. (C) Representative images of individual EDL NMJs showing the overlap (yellow) between axonal (green) and AChRs (red) fluorescence and their respective spontaneous mEPP and evoked EPP electrophysiological responses in WT and Sod1−/− mice. The areas and corresponding occupancies for the NMJs are shown by numbered circles in (A).
Table 2.
Quantification of neuromuscular junction (NMJ) denervation in wild-type and Sod1−/− muscles.
Figure 6.
Presynaptic potentiation via potassium channel blocker 3,4-diaminopyridine (DAP) reduces compound muscle action potential (CMAP) deficit and improves grip strength in Sod1−/− mice.
(A) Decrement of CMAP in response to 10 Hz repetitive stimulation was measured before (white) and 20 minutes after DAP administration (8 mg/kg, i.p., gray) in wild-type (WT, N = 7, 7-8 months old) and Sod1−/− (N = 17, 5–6 months old) mice as described in detail in Materials and Methods. (B) Grip strength was tested in wild type (WT, N = 13, 5 months old) and Sod1−/− (N = 11, 5 months old) mice after injection of DAP (1 mg/kg, i.p., gray) or saline (white). Data are plotted as mean with error bars representing standard error. Statistics: t-test, *p<0.05, **p<0.01.