Scope 3

At 35 days after injury and transplantation, animals were anesthetized using isoflurane. Compound muscle action potential (CMAP) recordings from the left and right hemidiaphragm were separately obtained, as previously described (Lepore et al., 2010 (link)). Briefly, a single stimulation (0.5 ms; 6 mV) of the phrenic nerve was performed by needle electrodes placed transcutaneously near the passage of the nerve in the neck. A ground electrode was placed in the tail, and reference electrode was inserted in the abdominal region. A surface strip electrode was placed along the costal margin of the hemi-diaphragm. CMAP recordings were obtained using an ADI Powerlab 8/30 stimulator and BioAMP amplifier (ADInstruments, Colorado Springs, CO), followed by computer-assisted data analysis (Scope 3.5.6, ADInstruments). For each animal, 10–20 tracings were averaged. Following CMAP recording, a right upper quadrant laparotomy was performed to expose the inferior surface of the hemi-diaphragm. Bipolar electrodes spaced 3 mm apart were introduced to obtain electromyography (EMG) recordings from the three diaphragmatic subregions (for at least 2 min each) during normal eupnic breathing (Li, Javed, Scura, et al., 2015b (link)). The EMG signal was amplified, filtered through a band-pass filter (50–3,000 Hz) and integrated using LabChart 7 software (ADInstruments).

Rats were anesthetized in the same manner described above. Phrenic nerve conduction studies were performed with single stimulation (0.5 ms duration; 6 mV amplitude) at the neck via near nerve needle electrodes placed along the phrenic nerve (Li et al., 2014b (link); Nicaise et al., 2012 (link)). The ground needle electrode was placed in the tail, and the reference electrode was placed subcutaneously in the right abdominal region. Recording was obtained via a surface strip along the costal margin of the diaphragm, and CMAP amplitude was measured baseline to peak. Recordings were made using an ADI Powerlab 8/30 stimulator and BioAMP amplifier (ADInstruments, Colorado Springs, CO), followed by computer-assisted data analysis (Scope 3.5.6, ADInstruments). For each animal, 10–20 tracings were averaged to ensure reproducibility.

Isoflurane (2.0-2.5% diluted in oxygen; Piramal Healthcare, Bethlehem, PA) was used to anesthetize the rats. Positive and negative stimulating needle electrodes were inserted at the neck close to the phrenic nerve either ipsilateral or contralateral to the injury and spaced 0.5cm apart (Lepore et al., 2008 (link); Lepore et al., 2010 (link)). A ground needle electrode was subcutaneously placed into the tail, and a reference electrode was inserted subcutaneously into the right abdominal region. A recording electrode with a surface strip was placed along the costal margin of the diaphragm The phrenic nerve was then stimulated (0.5 ms duration; 6 mV amplitude), and 10-20 recordings were obtained with 5 sec intervals between stimulations. CMAP amplitude was measured baseline to peak. An ADI Powerlab 8/30 stimulator and BioAMP amplifier (ADInstruments, Colorado Springs, CO) were used for recordings, and Scope 3.5.6 (ADInstruments) was used to analyze data. CMAPs were measured for each animal weekly for three weeks following SCI.

After mice were anesthetized with isofluorane, stimulating needle electrodes were placed in the neck along the phrenic nerve, as described by Li et. al. (K. Li et al., 2015 (link)). A ground needle electrode was placed in the tail, a reference needle electrode was placed subcutaneously in the contralateral abdomen, and a recording surface strip was placed on the costal margin of the hemi-diaphragm, ipsilateral to stimulation. Stimulation pulses of 0.5 ms and up to 6 mV amplitude were injected via the stimulating electrode and the resulting CMAP was recorded using an ADI Powerlab 8/30 stimulator and BioAMP amplifier (ADInstruments, Colorado Springs, CO), followed by computer-assisted data analysis (Scope 3.5.6, ADInstruments). The baseline to peak CMAP amplitude was measured and averaged over at least 10 pulses. Non-ALS mice with EAAT2^WT/WT, EAAT2^WT/CR or EAAT2^CR/CR were assessed at P90 (N = 16, 8, 14 respectively), P120 (N = 20, 12, 19) and P150 (N = 8, 8, 8) to identify potential confounders. Mice without SOD1-G93A and SOD1-G93A mice with EAAT2^WT/WT, EAAT2^WT/CR or EAAT2^CR/CR were assessed at P90 (N = 42, 14, 15, 14, respectively), P120 (N = 51, 18, 29, 28) and P150 (N = 34, 10, 14, 17).