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Dendritic Morphology of CA1 and CA3 Hippocampal Pyramidal Neurons

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Dendritic Morphology of CA1 and CA3 Hippocampal Pyramidal Neurons Supplemental figure legends Supplemental figure 1: Dendritic morphology of CA1 and CA3 hippocampal pyramidal neurons. (A) ApoTome images, consisting of stacks of multiple optical sections showing the normal morphology of fEGFP labeled CA1 (left) and CA3 (right) hippocampal pyramidal neurons. Note the complete filling of dendrites as well as the absence of any sign of degeneration. Note the highly significant differences in dendritic complexity. Scale bar 100µm. (B) Sholl analysis comparing the apical (B) and basal (B’) dendrite of CA1 and CA3 pyramidal neurons. Note the highly significant differences in dendritic complexity. * indicate p<0.05; ** indicate p<0.01. Supplemental figure 2: Comparison between fEGFP expressing non treated and fEGFP expressing mIgG1 treated hippocampal neurons (A) Sholl analysis comparing the apical and basal dendrite of fEGFP expressing non treated (closed circles) and fEGFP expressing mIgG1 treated (open circles) CA1 pyramidal neurons. The inserts show the total dendritic complexity for the apical (left) and basal (right) dendrites of fEGFP expressing non treated (solid bar) and fEGFP expressing mIgG1 treated (open bar) CA1 pyramidal neurons. (B) Sholl analysis comparing the apical and basal dendrite of fEGFP expressing not treated (closed circles) and fEGFP expressing mIgG1 treated (open circles) CA3 pyramidal neurons. The inserts show the total dendritic complexity for the apical (left) and basal (right) dendrites of fEGFP expressing non treated (solid bar) and fEGFP expressing mIgG1 treated (open bar) CA3 pyramidal neurons. (C) Graph comparing the dendritic spine density values for fEGFP expressing non treated (solid bars) and fEGFP expressing mIgG1 treated (open bar) CA1 pyramidal neurons. (D) Graph comparing the dendritic spine type distribution in fEGFP expressing non treated (solid bars) and fEGFP expressing mIgG1 treated (open bar) CA1 pyramidal neurons. Note the significant higher proportion of mushroom (Type 2) spines in the mid-apical and basal dendrites of the mIgG1 treated cells. The * indicate p<0.05. (E) Graph comparing the dendritic spine density values for fEGFP expressing non treated (solid bars) and fEGFP expressing mIgG1 treated (open bar) CA3 pyramidal neurons. (F) Graph comparing the dendritic spine type distribution in fEGFP expressing non treated (solid bars) and fEGFP expressing mIgG1 treated (open bar) CA3 pyramidal neurons. (G) Sholl analysis comparing axonal complexity of fEGFP expressing non treated and fEGFP expressing mIgG1 treated CA3 pyramidal neurons. The inserts show the total length (left) and number of nodes (right) of fEGFP expressing non treated (solid bar) and fEGFP expressing mIgG1 treated (open bar) CA3 pyramidal neurons. Supplemental figure 3: Comparison between fEGFP/control shRNA expressing and fEGFP expressing hippocampal neurons (A) Sholl analysis comparing the apical and basal dendrites of fEGFP expressing and fEGFP/control shRNA expressing CA3 pyramidal neurons. The inserts show the total dendritic complexity for the apical (left) and basal (right) dendrites of fEGFP expressing (light green) and fEGFP/control shRNA expressing (dark green) CA3 pyramidal neurons. (B) Sholl analysis comparing axonal complexity of fEGFP expressing and fEGFP/control shRNA expressing CA3 pyramidal neurons. The inserts show the total length (left) and number of nodes (right) of fEGFP expressing (light green) and fEGFP/control shRNA expressing (dark green) CA3 pyramidal neurons. (C) Graph comparing the dendritic spine density values for fEGFP expressing (light green) and fEGFP/control shRNA expressing (dark green) CA3 pyramidal neurons. (D) Graph comparing the dendritic spine type distribution in fEGFP expressing (light green) and fEGFP/control shRNA expressing (dark green) CA3 pyramidal neurons. Supplemental figure 4: Effect of Nogo-A over-expression on the dendritic architecture of CA3 hippocampal neurons. (A) Sholl analysis comparing the apical dendrite of fEGFP expressing and fEGFP/Nogo-A over-expressing CA3 pyramidal neurons. The inserts show the total dendritic complexity for the apical dendrite of fEGFP expressing and fEGFP/Nogo-A over-expressing CA3 pyramidal neurons. Note the significant shift of complexity proximally of the Nogo-A over-expressing neurons when compared to the fEGFP only expressing neurons (A’) Sholl analysis comparing the basal dendrite of fEGFP expressing and fEGFP/Nogo-A over-expressing CA3 pyramidal neurons. The inserts show the total dendritic complexity for the basal dendrite of fEGFP expressing and fEGFP/Nogo-A over-expressing CA3 pyramidal neurons. (B) Spine density counts for CA3 hippocampal neurons showing no significant differences in spine density between fEGFP/Nogo-A and fEGFP expressing neurons. Both total spine density and the density in the different dendritic compartments are shown. (B’) The chart shows the proportions of the three spine types for fEGFP/Nogo-A (orange) and fEGFP (green) expressing CA3 neurons plotted for the total neuron or separated between distal-apical, mid-apical and basal compartments. In each dendritic compartments as well as in the total count the proportion of stubby spines (type-I), mushroom spines (type-II) and thin spines (Type-III) are not significantly different between the two neuron groups. Supplemental figure 5: Nogo-B up regulation in nogo-A knockout hippocampus. (A) The Western Blot confirms the absence of Nogo-A in the knockout mice and shows a strong up regulation of Nogo-B when compared to the WT hippocampus. βTubulin was used as a loading control. (B) Sholl analysis comparing the apical dendrite of fEGFP expressing and fEGFP/Nogo-B over-expressing CA3 pyramidal neurons. The inserts show the total dendritic complexity for the apical dendrite of fEGFP expressing and fEGFP/Nogo-B over expressing CA3 pyramidal neurons. Note the absence of significant differences in dendritic complexity in Nogo-B over-expressing when compared to control neurons. (B’) Sholl analysis comparing the basal dendrite of fEGFP expressing and fEGFP/Nogo-B over-expressing CA3 pyramidal neurons. The inserts show the total dendritic complexity for the apical dendrite of fEGFP expressing and fEGFP/Nogo-B over expressing CA3 pyramidal neurons. Note the absence of significant differences in dendritic complexity in Nogo-B over-expressing when compared to control neurons. Supplemental figure 6: (A) and (A’) Micrograph showing the immunohistochemistry for NgR1 in thin sections of the P28 mouse hippocampus in WT and nogo-A KO mice respectively. Note NgR1 expression in the neuropil in the stratum oriens and radiatum of CA1 and CA3. The scale bar is 1mm. (B) The graph shows the relative intensity of NgR1 staining within the different regions of the P28 WT (black) and nogo-A KO (grey) mouse hippocampus. Note the lower levels of NgR1 expression in all regions of the nogo-A knockout when compared to the WT hippocampus. (B’) The graph shows the relative intensity of NgR1 staining within the different layers of CA1, CA3 and DG in P28 WT (black) and nogo-A KO (grey) mouse hippocampus. Note the generally reduced levels of NgR1 in all layers of the nogo-A KO hippocampus. (C) The Western Blot shows a reduction in the levels of NgR1 protein in the hippocampus of nogo-A KO mice when compared to the WT hippocampus. βTubulin was used as a loading control. (C’) The graph shows reduced relative intensity for the NgR1 band in the nogo-A KO (grey) when compared to the WT (black) mouse hippocampus. Supplemental figure 7: (A) and (A’) Micrograph showing the morphology of dendritic spines of fEGFP expressing primary hippocampal neurons treated for 15 minutes with a control (A) or a Nogo-A-Δ20 containing solution (A’). The scale bar is 10 µm. (B) Spine density counts for primary hippocampal neurons showing no significant differences in spine density between fEGFP expressing control (green) and Nogo-A-Δ20 (purple) treated neurons. (B’) The chart shows the proportions of the three spine types for fEGFP expressing primary hippocampal neurons treated with or without Nogo-A-Δ20. Note that while the proportion of stubby spines (type-I) is significantly lower in Nogo-A-Δ20 treated neurons, in these neurons the proportion of mushroom spines (type-II) is significantly increased. CA1 CA3 A Control Control B B’ Apical Basal 12 25 ** m * µ m 0 µ 20 1 0 / 1 * s / n s 15 o n i t o i c t 6 e c s e * r 10 s e r t e n t I n I 5 0 0 0 200 400 600 800 0 50 100 150 200 250 300 350 Distance from the cell body (µm) Distance from the cell body (µm) CA1 n=29 CA3 n=20 CA1 n=29 CA3 n=20 Zagrebelsky et al., Suppl. Fig 1 A B 20 25 s s s 300 250 200 300 s n n n Apical Basal Basal Apical n o o o o i i i i t t t 200 t c c c 150 c e e e m 200 e 200 m s s s 20 150 s µ r r r µ 15 r e e e 0 e 100 0 t t t t 1 1 n n n 100 n i i i i / 100 100 / f f f f s s o o o 50 o 15 50 n n # # # # o o i i t 10 0 0 t 0 0 c non mIgG1 non mIgG1 c non mIgG1 non mIgG1 e e s treated treated treated s 10 treated r r e e t t n n I 5 I 5 0 0 0 200 400 600 800 0 200 400 600 800 Distance from the cell body (µm) Distance from the cell body (µm) non treated n=29 mIgG1 n=22 non treated n=20 mIgG1 n=19 C D 1.4 80 * * 60 s m e µ n / i p s s 40 e 0.7 f n i o p S % 20 0.0 0 Apical Mid-ap. Basal Total 1 2 3 1 2 3 1 2 3 1 2 3 non treated n=18 mIgG1 n=7 Dist-ap.
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