Figure 7

Casp6N73T is less damaging to neuronal function and neurodegeneration than Casp6WT. (a) Representative two-photon images of 10 pg Casp6C163A- (Alexa 488, green) and 10 pg Casp6WT- or Casp6N73T- (Alexa 594, red) patched hippocampal CA1 pyramidal neurons. White arrows indicate basal dendrite degeneration. Scale bar: 25 µm. Maximum-intensity projection of two-photon stacks was compiled using ImageJ, and imaging montage of entire neurons was performed using Affinity Designer 1.7. (b) Sample EPSP time course plots from Casp6C163A- (green) and Casp6WT- (red) patched neurons in (a), showing reduction of neurotransmission for Casp6WT (1.53 ± 0.32 mV, n = 8 vs. 0.14 ± 0.07 mV, n = 8, p < 0.01) but not for Casp6C163A (1.94 ± 0.32 mV, n = 10 vs. 1.91 ± 0.29 mV, n = 10, p = 0.76) when comparing the last 10 traces (light thick line) to the first 10 traces (dark thick line). Open circles: EPSP amplitude recorded every 30 s. Closed circles: EPSP amplitude binned and averaged across 10 traces. Inset: representative EPSP traces highlight the paired-pulse ratio (PPR). Scale bars: 2 mV, 25 ms. Resting membrane potential and input resistance remained stable throughout experiment. (c) EPSP time courses for Casp6C163A- (n = 10), Casp6N73T- (n = 8), or Casp6WT- (n = 8) patched neurons. (d) PPR from Casp6WT-, Casp6C163A- or Casp6N73T-patched neurons. One-way ANOVA (p = 0.0043), followed by Tukey’s post-hoc test (**p < 0.01 vs. C163A; #p < 0.05 vs. WT). (e) CV analysis of Casp6N73T- and Casp6WT-patched neurons. Casp6C163A was unaltered. (f) Representative reconstructions of Casp6C163A-, Casp6N73T-, or Casp6WT-patched neurons. Image stacks were used for manual reconstruction of 3D morphologies using the Neuromantic freeware (http://www.reading.ac.uk/neuromantic/body_index.php). (g) Dendritic density maps of Casp6C163A- (n = 11), Casp6N73T- (n = 8), or Casp6WT- (n = 8) patched neurons generated using custom software running in Igor Pro 8 v8.04 (https://www.wavemetrics.com, https://github.com/pj-sjostrom/qMorph). Dotted lines show the convex hull of the maximum extent. (h) Cumulative dendritic length of reconstructed neurons in layers SO, PCL, SR, and SLM. (i) Casp6C163A-, Casp6N73T- and Casp6WT-patched CA1 pyramidal neurons beading basal dendrites. One-way ANOVA (p = 0.0001), followed by Tukey’s test (****p < 0.0001 vs Casp6C163A, ##p < 0.01 vs Casp6WT).