Figure 1. Spinal motor neurons are lost presymptomatically, before corticospinal motor neuron loss and before significant axon and muscle degeneration in the SOD1G93A rat. P90 and P120 were designated “presymptomatic” time points based on Kaplan–Meier analyses showing that disease onset for rats in this study ranged from 140 to 201 d, with a median of 154 d (n = 21) (A) and survival ranged from 147 to 218 d, with a median of 172 d (n = 13) (B). C, Immunostaining for ChAT+ spinal motor neurons in the lumbar spinal cord at P90, P120, early symptomatic, and endpoint (EP) showed qualitative changes between WT rats and SOD1G93A rats over time. D, Quantification revealed a significant reduction of large (>700 μm) ChAT+ spinal motor neurons starting at presymptomatic time point P120. E, Osmium tetroxide and toludine blue staining provided qualitative analysis of (L5) ventral root axons in WT versus SOD1G93A rats over time. Stereological quantification revealed that the number of total axons was not significantly reduced until rats reach endpoint (F), whereas the number of healthy axons is first significantly reduced when rats appear symptomatic (G) and a slight yet significant increase in degenerating (“unhealthy”) axons is seen starting at P120 (H). I, Immunostaining for α-bungarotoxin and SV2 in the muscle of WT versus SOD1G93A rats. J, Quantification revealed that NMJs do not become significantly denervated until the early symptomatic stages, after spinal motor neuron loss. K, Immunostaining of motor cortex layer V sections using CTIP2 that specifically stains nuclei of motor neurons, and Neurotrace, a fluorescent Nissl stain, in WT and SOD1G93A rats. L, Quantification revealed that the number of large corticospinal motor neurons was declined at early symptomatic stages and was significantly reduced at disease endpoint. Scale bars: C, K, 50 μm; E, I, 10 μm. *p < 0.05; error bars indicate SEM. M, Summary of the time course of degeneration in SOD1G93A rats showing the percentage of corticospinal motor neuron loss in the brain, spinal motor neuron loss, loss of healthy ventral root axons, and degenerated NMJs: *p < 0.05, **p < 0.001, ***p < 0.0001, ## indicates moderate degeneration designated attributable to a slight yet significant increase in numbers of unhealthy axons. N.C., No change; N.S., not significant.