Figure 5. Calretinin-expressing neurites undergo progressive alterations in branching complexity from symptom onset in the supragranular SOD1^G93A motor cortex.

(a–d) CR-labelled tissue sections were imaged and immunopositive neurons in the supragranular lamina assessed using cell tracing software to analyse neurite morphology (arrowheads, insets) at 8 weeks (a) and 20 weeks (b) in WT and SOD1^G93A tissue. The division of the neurite structure into primary, secondary, tertiary and quaternary order processes (as observed with CR-labelling) was used for all investigations. (c,d) A proportional analysis was conducted to determine the extent of CR-labelled processes remaining on neurons in the supragranular motor cortex at both time points, showing a significant increase in neurons with no, or fewer, CR-labelled processes visible in early symptomatic (8 week) SOD1^G93A mice and in 20 week end-stage SOD1^G93A mice compared to WT. (e–h) The mean branch length (e,f) and mean volume (g,h) of CR-labelled neurites were also assessed, demonstrating a pre-symptomatic increase in the length and volume of distal neurite processes in 8 week SOD1^G93A mice (e,g). Analysis of 20 week end-stage SOD1^G93A mice showed a significant reduction in the length and volume of CR-labelled processes compared to WT (f,h). Values in graphs represent means ± SEM. *P < 0.05, two-way ANOVA, Bonferonni’s multiple-comparison test with n = 6 mice per group. Scale bar in (b) 20 μm.