Early and Persistent Abnormal Decoding by Glial Cells at the Neuromuscular Junction in an ALS Model

Altered PSC excitability at presymptomatic stage of the disease. A, False color images of PSCs (*) loaded with fluorescent Ca²⁺ indicator Fluo-4 AM, before (baseline), during (stimulation), and after (recovery) motor nerve stimulation at NMJs of WT, SOD1^WT, and SOD1^G37R. Representative changes in fluorescence are illustrated on the right for the PSCs indicated by the arrowhead. B, Histogram depicting the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSCs by transmitter release evoked by motor nerve stimulation (50 Hz, 5 s). PSCs at NMJs from SOD1^G37R mice had larger Ca²⁺ responses (one-way ANOVA, p = 0.0004) C, Examples of an NMJ imaged during Ca²⁺ imaging experiment (top) and the same NMJ imaged after MHC and BTX staining (bottom). This NMJ was associated with a fiber Type I (MHC-I, blue) and not a fiber Type IIa (MHC-IIa, green). D, Histogram illustrating the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSCs by motor nerve stimulation as a function of each fiber type. The altered PSC excitability did not correlate with motor neuron vulnerability at a presymptomatic stage of the disease. Scale bars: A, 10 μm; C, 20 μm. *p < 0.05, **p < 0.01.

Enhanced synaptic transmission at NMJs from SOD1^G37R mice at presymptomatic stage of the disease. A, Examples of spontaneous MEPP recordings from WT, SOD1^WT, and SOD1^G37R NMJs. Histogram showing the mean ± SEM of the amplitude (B) and the frequency (C) of the MEPP. D, Examples of EPPs evoked by paired-pulse stimulation of the motor nerve (10 ms interval) from WT, SOD1^WT, and SOD1^G37R NMJs. E, Histogram showing the mean ± SEM of the amplitude of the first EPP. F, Calculated quantal content obtained from electrophysiological recordings of synaptic transmission and determined as EPP amplitude/mEPP amplitude. NMJs from SOD1^G37R mice had higher quantal content. G, Calculated PPF obtained from electrophysiological recordings of synaptic transmission and determined as the mean amplitude of the second EPPs divided by the mean amplitude of the first EPPs. *p < 0.05, **p < 0.01, ***p < 0.001.

PSC decoding ability is unaffected by an increase in transmitter release. A, Changes of EPP amplitude before and during (red bar) bath application of TEA (0.2 mm). Insets, Examples of EPPs (black) before and 30 min after TEA application (red). TEA increased synaptic transmission of both WT and SOD1^G37R nerve terminals. Effect of TEA (0.2 mm) bath application on corresponding PSC Ca²⁺ responses induced by synaptic activity (50 Hz, 5 s) from WT (B) and SOD1^G37R (C) animals. Gray zones represent the mean ± SEM of the amplitude of the Ca²⁺ responses elicited by motor nerve stimulation in control (without TEA). PSC Ca²⁺ responses elicited by potentiated WT nerve terminals remain smaller than those triggered by the SOD1^G37R and were not significantly different from the ones without TEA (unpaired t test, p > 0.05). Ca²⁺ responses elicited in PSCs by local applications of ATP in WT (D) and SOD1^G37R NMJs (E). The ability of PSCs to produce larger Ca²⁺ responses was not affected by TEA because local application of ATP (red arrow) elicited larger Ca²⁺ responses in PSC from WT and SOD1^G37R mice. In B–D, black and grey traces represent the mean and SEM, respectively.

PSC Ca²⁺ responses to local application of ATP and muscarine. A, False color confocal images of PSCs (*) loaded with fluorescent Ca²⁺ indicator Fluo-4 AM, before (baseline), during (ATP application), and after (recovery) local ATP applications at NMJs of WT, SOD1^WT, and SOD1^G37R. Representative changes in fluorescence are illustrated on the right for the PSCs indicated by the arrowhead. Higher magnification of PSCs marked with the arrowhead in left are illustrated on the three other images for each group. B, Histogram depicting the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSC by ATP application. C, Histograms depicting the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSC by ATP application as a function of fiber type. D–F, Similar representation as in A–C, but illustrating the muscarine application. There was no statistical difference between the different animal groups either for the ATP or the muscarine application (one-way ANOVA, p > 0.05). Scale bar, 10 μm.

Larger contribution of mAChR activation of PSCs during synaptic transmission in SOD1^G37R mice. PSC Ca²⁺ responses evoked by motor nerve stimulation in the presence of atropine (5–20 μm) for WT (A) and SOD1^G37R (B) mice. No Ca²⁺ responses were elicited in PSCs by local application of muscarine in the presence of atropine while responses were still elicited by local application of ATP. Dark trace represents the average of PSC Ca²⁺ responses. Dotted line indicates the SEM. Gray boxes represent the mean ± SEM of the amplitude of the Ca²⁺ responses elicited by nerve stimulation or agonist application without atropine. Histogram depicting the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSCs by transmitter release evoked by motor nerve stimulation (50 Hz, 5 s) during bath application of mAChR antagonist (atropine, 5–20 μm) for WT (C) and SOD1^G37R (D) mice. Histogram depicting the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSC by motor nerve stimulation in presence of atropine as a function of each fiber type for WT (E) and SOD1^G37R (F) mice. *p < 0.05, **p < 0.01, ***p < 0.001.

Enhanced synaptic transmission at NMJs from SOD1^G37R mice at preonset stage of the disease. A, Traces of spontaneous activity (MEPPs) recorded from WT and SOD1^G37R NMJs. Histogram showing the mean ± SEM of the amplitude (B) and the frequency (C) of MEPP events. D, Examples of EPPs evoked by paired-pulse stimulation (10 ms interval) from WT and SOD1^G37R NMJs. E, Histogram showing the mean ± SEM of the amplitude of the first EPP. F, Histogram showing the quantal content determined as the ratio of EPP amplitude/mEPP amplitude. NMJs from SOD1^G37R mice had a larger quantal content. G, Histogram showing the PPF determined as the mean amplitude of the second EPPs divided by the mean amplitude of the first EPPs. *p < 0.05.

Altered PSC excitability at preonset stage of the disease. A, Dark traces represent the mean ± SEM of the Ca²⁺ responses elicited in PSCs by transmitter release evoked by motor nerve stimulation (50 Hz, 5 s) at NMJs of WT and SOD1^G37R. Larger Ca²⁺ responses were elicited in PSCs of NMJs from SOD1^G37R mice (unpaired t test, p = 0.0006). Blue traces represent the mean ± SEM of the PSC nerve-evoked (50 Hz, 5 s) Ca²⁺ responses in presence of atropine (20 μm) for WT and SOD1^G37R mice. PSC Ca²⁺ responses of WT (unpaired t test, p = 0.0129) and SOD1 mice (unpaired t test, p = 0.0016) were significantly smaller in the presence of atropine; this represents a 42% and 85% contribution of muscarinic signaling during synaptic transmission, respectively. B, Histograms depicting the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSCs by motor nerve stimulation in the presence of atropine depending of each type of fiber for WT and SOD1^G37R (C) mice. The altered PSC-detecting ability did not correlate with MN vulnerability at a preonset stage of the disease. *p < 0.05, **p < 0.01.

PSC Ca²⁺ responses to local application of ATP and muscarine at preonset stage of the disease. A, False color confocal images of PSCs (*) loaded with fluorescent Ca²⁺ indicator Fluo-4 AM, before (baseline), during (ATP application), and after (recovery) local ATP applications at NMJs of WT and SOD1^G37R. Representative changes in fluorescence are illustrated on the right for the PSCs indicated by the arrowhead. Higher magnification of PSCs marked with the arrowhead in left are illustrated on the three other images for each group. B, Histogram depicting the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSCs by ATP application. C, Histograms depicting the mean ± SEM of the amplitude of the Ca²⁺ responses elicited in PSC by ATP as a function of each fiber type. D–F, Similar representation as in A–C, but for muscarine application. Note the statistical difference between the different animal groups for the muscarine application and for the PSC Ca²⁺ responses associated with fiber Type IIa for ATP and muscarine application. Scale bar, 10 μm. ***p < 0.001.