Figure S5.
Definition of AIS length and distance from the cell body or axon carrying dendrite and illustration of the workflow to induce AIS plasticity. (A) Schematic of AIS short-term and chronic plasticity experiment. (B) Definition of AIS length and AIS distance in nonAcD and AcD neurons as read out of AIS plasticity. Orange dashed line indicates the border of stem dendrite of AcD neurons; black dashed line indicates the border of axon origin. (C) Illustration of intensity-based AIS length and AIS distance (AIS dist.) measurement. (D) Cumulative of AIS length in nonAcD and AcD neuron upon induction of short-term plasticity. Mean ± SEM, three independent cultures, nonAcD: n (KCl) = 295 cells, n (NaCl) = 261 cells, n (TTX) = 303 cells, AcD: n (KCl) = 47 cells, n (NaCl) = 38 cells, n (TTX) = 41 cells. One-way ANOVA with Tukey’s multiple comparisons test, no significance (n.s.) P > 0.05. (E and F) Measurement of AIS length in nonAcD (E) and AcD (F) neurons upon induction of chronic plasticity. Mean ± SEM, four independent cultures, nonAcD: n (KCl) = 185 cells, n (NaCl) = 186 cells, n (TTX) = 199 cells, AcD: n (KCl) = 86 cells, n (NaCl) = 75 cells, n (TTX) = 65 cells. Grey dot indicates number of individual cells, cyan (E) and orange (F) triangle indicates mean of each experiment. One-way ANOVA with Tukey’s multiple comparisons test, no significance (n.s.) P > 0.05, *P < 0.05, ***P < 0.001. (G) Left panel: representative image of GAD1 negative (GAD1−) AcD neuron and GAD1 positive inhibitory interneuron. White arrowhead indicates GAD1 positive interneuron, orange arrowhead indicates GAD1 negative (GAD1−) AcD neuron. Scale bar is 20 µm. Right panel: Measurement of AIS distance in GAD1 negative AcD neurons upon chronic plasticity induction. Mean ± SEM, four independent cultures, KCl: n = 42 cells, NaCl: n = 34 cells. Grey dot indicates number of individual cells, orange triangle indicates mean of each experiment. Mann–Whitney test (two-sided): not significant (n.s.) P > 0.05. (H) Measurement of stem dendrite length in AcD neuron upon induction of chronic plasticity. Mean ± SEM, three independent cultures, AcD: n (KCl) = 66 cells, n (NaCl) = 55 cells. Grey dot indicates number of individual cells, orange triangle indicates mean of each experiment. Mann–Whitney test (two-sided), no significance (n.s.) P > 0.05. (I) Two-way ANOVA (type II) analysis of nonAcD and AcD neurons treated with KCl for 48 h to induce chronic plasticity, NaCl and TTX as control. Mean ± SEM, nonAcD neuron: three independent cultures, n (KCl) = 174 cells, n (NaCl) = 184 cells, n (TTX) = 192 cells, AcD neuron: four independent cultures, n (KCl) = 82 cells, n (NaCl) = 72 cells, n (TTX) = 63 cells. Blue * and “n.s.” indicates significance for nonAcD neurons between treatment, dark orange * and “n.s.” indicates significance for AcD neurons between treatment, black * and “.” Indicates significance of interaction between morphology and treatment, no significance (n.s. or “.”) P > 0.05, ***P < 0.001.

Definition of AIS length and distance from the cell body or axon carrying dendrite and illustration of the workflow to induce AIS plasticity. (A) Schematic of AIS short-term and chronic plasticity experiment. (B) Definition of AIS length and AIS distance in nonAcD and AcD neurons as read out of AIS plasticity. Orange dashed line indicates the border of stem dendrite of AcD neurons; black dashed line indicates the border of axon origin. (C) Illustration of intensity-based AIS length and AIS distance (AIS dist.) measurement. (D) Cumulative of AIS length in nonAcD and AcD neuron upon induction of short-term plasticity. Mean ± SEM, three independent cultures, nonAcD: n (KCl) = 295 cells, n (NaCl) = 261 cells, n (TTX) = 303 cells, AcD: n (KCl) = 47 cells, n (NaCl) = 38 cells, n (TTX) = 41 cells. One-way ANOVA with Tukey’s multiple comparisons test, no significance (n.s.) P > 0.05. (E and F) Measurement of AIS length in nonAcD (E) and AcD (F) neurons upon induction of chronic plasticity. Mean ± SEM, four independent cultures, nonAcD: n (KCl) = 185 cells, n (NaCl) = 186 cells, n (TTX) = 199 cells, AcD: n (KCl) = 86 cells, n (NaCl) = 75 cells, n (TTX) = 65 cells. Grey dot indicates number of individual cells, cyan (E) and orange (F) triangle indicates mean of each experiment. One-way ANOVA with Tukey’s multiple comparisons test, no significance (n.s.) P > 0.05, *P < 0.05, ***P < 0.001. (G) Left panel: representative image of GAD1 negative (GAD1−) AcD neuron and GAD1 positive inhibitory interneuron. White arrowhead indicates GAD1 positive interneuron, orange arrowhead indicates GAD1 negative (GAD1−) AcD neuron. Scale bar is 20 µm. Right panel: Measurement of AIS distance in GAD1 negative AcD neurons upon chronic plasticity induction. Mean ± SEM, four independent cultures, KCl: n = 42 cells, NaCl: n = 34 cells. Grey dot indicates number of individual cells, orange triangle indicates mean of each experiment. Mann–Whitney test (two-sided): not significant (n.s.) P > 0.05. (H) Measurement of stem dendrite length in AcD neuron upon induction of chronic plasticity. Mean ± SEM, three independent cultures, AcD: n (KCl) = 66 cells, n (NaCl) = 55 cells. Grey dot indicates number of individual cells, orange triangle indicates mean of each experiment. Mann–Whitney test (two-sided), no significance (n.s.) P > 0.05. (I) Two-way ANOVA (type II) analysis of nonAcD and AcD neurons treated with KCl for 48 h to induce chronic plasticity, NaCl and TTX as control. Mean ± SEM, nonAcD neuron: three independent cultures, n (KCl) = 174 cells, n (NaCl) = 184 cells, n (TTX) = 192 cells, AcD neuron: four independent cultures, n (KCl) = 82 cells, n (NaCl) = 72 cells, n (TTX) = 63 cells. Blue * and “n.s.” indicates significance for nonAcD neurons between treatment, dark orange * and “n.s.” indicates significance for AcD neurons between treatment, black * and “.” Indicates significance of interaction between morphology and treatment, no significance (n.s. or “.”) P > 0.05, ***P < 0.001.

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