rat dorsal root ganglion neurons (drgs)  (Procell Inc)

Journal: The Journal of Neuroscience

Article Title: MAP7 Prevents Axonal Branch Retraction by Creating a Stable Microtubule Boundary to Rescue Polymerization

doi: 10.1523/JNEUROSCI.0775-19.2019

Figure Lengend Snippet: Loss of MAP7 decreases axon branching in DRG neurons. A, In situ analysis of E15.5 mouse spinal cords from WT and Map7−/− animals labeled with a Map7-specific DIG-labeled probe. B, Cultured WT and Map7−/− DRG neurons colabeled by antibodies for NF and MAP7. C, D, Inverted fluorescence images of dissociated WT (C) or Map7−/− (D) neurons cultured for 24 h and labeled with NF antibodies. E–H, Comparison between WT and mutant (−/−) neurons of branch number per 100 μm axon length (E; total p = 0.0046, interstitial p = 0.0001, terminal p = 0.6081, n = 35 for WT and n = 34 for −/−, Mann–Whitney test), primary axon length (F; p = 0.0298, n = 35 for WT and n = 34 for −/−,unpaired t test), branch length (G; p = 0.0002, n = 30 for WT; n = 25 for −/−, Mann–Whitney test), and primary neurite number (H; p = 0.8894, n = 35 for WT, n = 39 for −/−, Mann–Whitney test). Data are reported as mean ± SEM in E–G, and median in H. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. Scale bars, 100 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various MAP7 or tau constructs by Nucleofection (Lonza) using reagent P3 and the CU-133 program.

Techniques: In Situ, Labeling, Cell Culture, Fluorescence, Mutagenesis, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: MAP7 Prevents Axonal Branch Retraction by Creating a Stable Microtubule Boundary to Rescue Polymerization

doi: 10.1523/JNEUROSCI.0775-19.2019

Figure Lengend Snippet: Loss of MAP7 increases axonal branch retraction in DRG neurons. A, Formation of filopodia (arrows) is shown by sequential phase contrast images of an axonal segment. B–D, Comparisons between WT and Map7−/− (−/−) neurons in filopodium formation frequency (B; p = 0.8026, n = 8 for WT and n = 9 for −/−, unpaired t test), filopodium lifetime (C; p = 0.7047, n = 9 for WT and n = 8 for −/−, unpaired t test), and filopodium length (D; p = 0.2575, n = 15 for WT and n = 21 for −/−, unpaired t test). E–G, Examples of sequential phase contrast images (60 min apart) to show branch growth (yellow arrows) and branch retraction (red arrows) of WT (E, F) and Map7−/− (G) neurons. H–K, Comparison between WT and mutant (−/−) neurons of branch growth (H; total p = 0.0169, bifurcation p = 0.1318, collateral p = 0.0510; n = 18 cells for WT and n = 10 cells for −/−; unpaired t test), branch retraction (I; total p = 0.0157, bifurcation p = 0.0244, collateral p = 0.0204; n = 18 cells for WT and n = 10 cells for −/−; Mann–Whitney test), branch length before retraction (J; total p = 0.1092, n = 19 for WT and n = 27 for −/−; bifurcation, p = 0.2948, n = 7 for WT and n = 11 for −/−; collateral, p = 0.2344, n = 12 for WT and n = 16 for −/−; Mann–Whitney test), and branch lifetime before retraction (K; total p = 0.3267, n = 20 for WT and n = 20 for −/−; bifurcation, p = 0.3590, n = 6 for WT and n = 7 for −/−; for collateral, p = 0.5808, n = 14 for WT and n = 13 for −/−; unpaired t test). Data are reported as mean ± SEM. *p < 0.05; ***p < 0.001; ns: not significant (only shown for the total in H–K). Scale bars: 20 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various MAP7 or tau constructs by Nucleofection (Lonza) using reagent P3 and the CU-133 program.

Techniques: Mutagenesis, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: MAP7 Prevents Axonal Branch Retraction by Creating a Stable Microtubule Boundary to Rescue Polymerization

doi: 10.1523/JNEUROSCI.0775-19.2019

Figure Lengend Snippet: Loss or overexpression of MAP7 affects axonal microtubule stability. A, B, Fluorescence intensity images of WT and Map7−/− neurons labeled for tubulin (tub, red hot) and NF (cyan hot) in axon or branch regions after CtrlF or NocF. C, D, Analysis of tubulin/NF fluorescence signal ratios between WT and Map7−/− (−/−) neurons after CtrlF and NocF in axon (C) or branch (D) regions. E, F, Quantification of tubulin/NF fluorescence signal ratios in axons (E) or branch (F) regions of DRG neurons expressing control (ctrl), MAP7 or tau. All comparisons were done by unpaired t tests. C, WT, p = 0.0029, n = 13 for CtrlF, and n = 21 for NocF; −/−, p ≤ 0.0001, n = 19 for CtrlF, and n = 21 for NocF; D, WT, p = 0.1397, n = 24 for CtrlF, and n = 28 for NocF; −/−, p ≤ 0.0001, n = 34 for CtrlF; n = 22 for NocF; E, Ctrl, p = 0.0238, n = 20 for CtrlF, and n = 21 for NocF; MAP7, p = 0.6780, n = 10 for CtrlF, and n = 18 for NocF; Tau, p = 0.8018, n = 12 for CtrlF, and n = 13 for NocF; F, Ctrl, p = 0.5822, n = 11 for CtrlF, and n = 16 for NocF; MAP7, p = 0.3004, n = 15 for CtrlF and n = 8 for NocF; and Tau, p = 0.6715, n = 16 for CtrlF and n = 14 for NocF. Data are reported as mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant. Scale bar, 20 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various MAP7 or tau constructs by Nucleofection (Lonza) using reagent P3 and the CU-133 program.

Techniques: Over Expression, Fluorescence, Labeling, Expressing

Journal: The Journal of Neuroscience

Article Title: MAP7 Prevents Axonal Branch Retraction by Creating a Stable Microtubule Boundary to Rescue Polymerization

doi: 10.1523/JNEUROSCI.0775-19.2019

Figure Lengend Snippet: MAP7 prevents branch retraction caused by laser-induced severing or nocodazole-induced microtubule destabilization. A, Phase contrast (top row) and EGFP (bottom row) images of DRG axons before (pre) and after laser-induced severing in neurons expressing EGFP, MAP7-EGFP, or tau-EGFP. The injured sites are marked by asterisks and the proximal branch terminals are marked by arrows. B, The extent of retraction is plotted as distance retracted over time after severing. C, Quantification and comparison of the axon retraction rate derived from B (EGFP-MAP7; p = 0.0002, EGFP-tau; p = 0.0918; MAP7-tau; p = 0.0561; n = 6 for EGFP, n = 10 for MAP7 and n = 6 tau; one-way ANOVA). D, E, Time courses (D) and rates (E) of retraction after nocodazole (15 μm) addition to WT and Map7−/− (−/−) DRG neurons (p = 0.0222, n = 6 for WT and n = 9 for −/−, t test). F, G, Time courses (F) and rates (G) of retraction in rat DRG neurons overexpressing MAP7 or tau after nocodazole (15 μm) addition (EGFP-MAP7; p = 0.0302, EGFP-tau; p = 0.7490; MAP7-tau; p = 0.2378, n = 15 for EGFP, n = 12 for MAP7 and n = 9 for tau, one-way ANOVA). Data are reported as mean ± SEM. ns, not significant. *p < 0.05, ***p < 0.001. Scale bar, 20 μm.

Article Snippet: Dissociated rat DRG neurons (∼7.5 × 10 5 cells) were transfected with various MAP7 or tau constructs by Nucleofection (Lonza) using reagent P3 and the CU-133 program.

Techniques: Expressing, Derivative Assay