Figure 2.
Activity-dependent transport of AMPARs is mediated by KIF13A. (A) Left: Representative images of hippocampal neuronal cultures after 7 d in vitro, infected either with a control (top row) or the shKIF13A-expressing lentivirus (bottom row; red channel) for 7 d. Cultures were fixed and stained for surface GluA1 (nonpermeabilizing conditions, magenta) and total GluA1 (after permeabilization, cyan). Scale bars, 20 µm. Right: Quantifications of fluorescence intensity, measured separately in the somatic and dendritic compartments for both surface (magenta) and total (cyan) GluA1 channels. Plots show mean fluorescence intensity for the individual experiments and mean ± SEM for each pool of receptors in the different compartments. n = 32 for both control neurons and shKIF13A-expressing neurons from six independent experiments. (B) Left: Representative images for dissociated hippocampal neurons infected with lentivirus, as described for A. Immunocytochemistry was performed after baseline or 15 min of cLTP induction. Scale bars, 2.5 µm. Dashed lines outline neuron morphology (from mask of the mCherry channel). Arrowheads indicate individual spines used for the quantification of both surface (left) and total (right) GluA1 immunostaining. Plots on the right show cumulative probability distributions of fluorescence intensity from individual spines for each condition described above. Gray traces represent spines from neurons infected with a control lentivirus, and red traces show spines from neurons infected with a lentivirus expressing shKIF13A. Dashed lines represent baseline values, while continuous lines represent values after cLTP induction. P values compare cLTP versus baseline condition (black for control and red for shKIF13A), according to the Kolmogorov–Smirnov test (left graph, surface GluA1) or control versus shKIF13A in baseline condition (right graph, total GluA1). n = 1,229 spines (24 neurons) for baseline conditions and n = 1,183 spines (26 neurons) for cLTP conditions, from control neurons. n = 750 spines (25 neurons) for baseline conditions and n = 608 spines (24 neurons) for cLTP conditions from shKIF13A-expressing neurons, analyzed from three independent experiments. (C) Quantifications of fluorescence intensity in the adjacent dendritic shaft from each condition described in B. Plots show mean fluorescence intensity after cLTP normalized to mean baseline intensity for each condition for the individual experiments and mean ± SEM for each pool of receptors (surface GluA1, magenta; total GluA1, cyan). n = 26 control neurons and n = 24 shKIF13A-expressing neurons from three independent experiments. (D) Left: Representative images of spine morphology from mCherry channel of the experiments described in B. Scale bars, 2.5 µm. Right, cumulative probability distribution of fluorescence intensity at spines over the value at the adjacent dendrite from the different conditions described for B. Dashed lines represent baseline values, while continuous lines represent values after cLTP induction. P values compare cLTP versus baseline condition (black for control and red for shKIF13A) according to the Kolmogorov–Smirnov test.

Activity-dependent transport of AMPARs is mediated by KIF13A. (A) Left: Representative images of hippocampal neuronal cultures after 7 d in vitro, infected either with a control (top row) or the shKIF13A-expressing lentivirus (bottom row; red channel) for 7 d. Cultures were fixed and stained for surface GluA1 (nonpermeabilizing conditions, magenta) and total GluA1 (after permeabilization, cyan). Scale bars, 20 µm. Right: Quantifications of fluorescence intensity, measured separately in the somatic and dendritic compartments for both surface (magenta) and total (cyan) GluA1 channels. Plots show mean fluorescence intensity for the individual experiments and mean ± SEM for each pool of receptors in the different compartments. n = 32 for both control neurons and shKIF13A-expressing neurons from six independent experiments. (B) Left: Representative images for dissociated hippocampal neurons infected with lentivirus, as described for A. Immunocytochemistry was performed after baseline or 15 min of cLTP induction. Scale bars, 2.5 µm. Dashed lines outline neuron morphology (from mask of the mCherry channel). Arrowheads indicate individual spines used for the quantification of both surface (left) and total (right) GluA1 immunostaining. Plots on the right show cumulative probability distributions of fluorescence intensity from individual spines for each condition described above. Gray traces represent spines from neurons infected with a control lentivirus, and red traces show spines from neurons infected with a lentivirus expressing shKIF13A. Dashed lines represent baseline values, while continuous lines represent values after cLTP induction. P values compare cLTP versus baseline condition (black for control and red for shKIF13A), according to the Kolmogorov–Smirnov test (left graph, surface GluA1) or control versus shKIF13A in baseline condition (right graph, total GluA1). n = 1,229 spines (24 neurons) for baseline conditions and n = 1,183 spines (26 neurons) for cLTP conditions, from control neurons. n = 750 spines (25 neurons) for baseline conditions and n = 608 spines (24 neurons) for cLTP conditions from shKIF13A-expressing neurons, analyzed from three independent experiments. (C) Quantifications of fluorescence intensity in the adjacent dendritic shaft from each condition described in B. Plots show mean fluorescence intensity after cLTP normalized to mean baseline intensity for each condition for the individual experiments and mean ± SEM for each pool of receptors (surface GluA1, magenta; total GluA1, cyan). n = 26 control neurons and n = 24 shKIF13A-expressing neurons from three independent experiments. (D) Left: Representative images of spine morphology from mCherry channel of the experiments described in B. Scale bars, 2.5 µm. Right, cumulative probability distribution of fluorescence intensity at spines over the value at the adjacent dendrite from the different conditions described for B. Dashed lines represent baseline values, while continuous lines represent values after cLTP induction. P values compare cLTP versus baseline condition (black for control and red for shKIF13A) according to the Kolmogorov–Smirnov test.

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