Journal: The European Journal of Neuroscience

Article Title: MAP6 interacts with Tctex1 and Ca v 2.2/N‐type calcium channels to regulate calcium signalling in neurons

doi: 10.1111/ejn.13766

Figure Lengend Snippet: Specificity of Ca v 2‐type calcium channels deficit in MAP6 KO neurons. (A) Left panel, examples of KCl‐stimulated fluo‐4‐loaded WT cortical neurons in the absence (black line) or presence (yellow line) of 20 μ m nimodipine. Right panels, ratios of KCl‐elicited fluorescence intensity peaks, recorded from WT (white squares) and MAP6 KO (grey squares) cortical neurons in the presence or absence of 20 μ m nimodipine (yellow curves), 180 n m ω‐agatoxin IVA (red curves) or 320 n m ω‐conotoxin GVIA (green curves). n represents the total number of wells recorded from eight independent neuronal cultures. ns, P > 0.05, *, P < 0.05 and **, P < 0.01 against the corresponding WT, using paired nonparametric t ‐tests. Note that Wilcoxon t ‐tests have been performed to account for technical data pairing between neighbouring MAP6 KO and WT culture wells and this pairing was deemed as highly significant ( P < 0.01) for each panel. (B) Left panel, example of identified axons (yellow arrowheads) from a neuronal culture with immunolabelled microtubules (red), tau (green) and nuclei (blue). Right panel, measurements of axonal length of WT (white symbols) and MAP6 KO (grey symbols) hippocampal neurons after 48 h in the absence (sham, discs) or presence (conotox, diamonds) of 320 n m ω‐conotoxin GVIA. n represents the total number of neurons measured from three independent neuronal cultures. ns, P > 0.05 and *, P < 0.05 as compared to corresponding sham, using unpaired nonparametric t ‐tests. Scale bar = 10 μm. (C) Left panel, examples of spontaneous calcium activity recorded from fluo‐4‐loaded WT (black line) and MAP6 KO (grey line) hippocampal neurons. Right panel, quantification of mean peak intensity during spontaneous calcium activity of fluo‐4‐loaded WT (white discs) and MAP6 KO (grey discs) hippocampal neurons, recorded in cell bodies and neurites, independently, normalized by that of WT (dashed grey line = 100%). n represents the total number of fields recorded from six independent neuronal cultures. ns, P > 0.05 and *, P < 0.05 as compared to corresponding WT, using unpaired parametric t ‐tests.[Colour figure can be viewed at wileyonlinelibrary.com ].

Article Snippet: Twenty‐four‐well plates of cortical cultures after 7–8 DIV were incubated at 37 °C without CO 2 , in the presence of 1 μ m Fluo‐4 (Thermo Fisher Scientific, France) in warm aCSF containing LiCl + KA, in the absence [nimo] or presence [toxins] of 20 μ m nimodipine (see above, ), during 20 min. Each well was rinsed with aCSF containing LiCl + KA, without [nimo] or with [toxins] 20 m m nimodipine and 450 μL of fresh medium containing also DMSO or 20 μ m nimodipine [nimo] on the one hand or DMSO, 320 n m ω‐conotoxin GVIA (STC‐750, Alomone Labs, Israel, diluted to 160 μ m in DMSO) or 180 n m ω‐agatoxin IVA (STC‐750, Alomone Labs, Israel, diluted to 90 μ m in DMSO) [toxins] on the other hand, was added to the corresponding wells.

Techniques: Fluorescence, Activity Assay