Retrograde Labeling of Corticospinal and Callosal Projection Neurons

CSMN were retrogradely labeled via stereotactic FluoroGold injections (2% FG, 250 nl/mouse) into the cervical region (C4–C6) of the corticospinal tract within the dorsal funiculus of the spinal cord at distinct, phenotypically distinguishable times defined by others previously (Gurney et al., 1994 (link); Tu et al., 1996 (link); Hall et al., 1998 (link); Cleveland and Rothstein, 2001 (link); Wengenack et al., 2004 (link); Hegedus et al., 2007 (link)) postnatal day 20 (P20), “early”; P50, “symptomatic”; and P110, “end stage”. In a subset of experiments, mice injected at P50 were perfused at P120 to distinguish between genuine CSMN degeneration and potential appearance of reduced FG labeling due to defects in axonal transport (Fig. 1A, “#”). 10 days after FluoroGold injection, mice were deeply anesthetized and perfused with cold 0.1M PBS supplemented with heparin, followed by cold 4% paraformaldehyde (PFA) in 0.1M PBS. To further investigate whether degeneration of other neocortical projection neurons with equivalently long axons (most notably, interhemispheric callosal projection neurons (CPN)) occurs, in a subset of experiments dual CPN and CSMN retrograde labeling was performed in hSOD1^G93A and WT mice at P30, and mice were perfused at P120. Callosal projection neurons (CPN) were retrogradely labeled via stereotactic injection of green fluorescent microspheres into contralateral cortex (250 nl/mouse), and CSMN were retrogradely labeled via stereotactic injection of red fluorescent microspheres (250 nl/mouse) into the cervical region (C4–C6) of the corticospinal tract within the dorsal funiculus of the spinal cord. Brains were postfixed in 4% PFA overnight, and 40 µm thick coronal sections were cut on a Leica VT 1000S vibrating microtome.