Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Effect of Aβ 1–42 exposure on Na V 1.6 protein expression and activity in primary hippocampal neurons at 10–12 DIV. ( A ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ( B ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control. ( D ) Representative traces of Na + currents recorded under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min) in primary hippocampal neurons. (E) Representative traces of Na + currents recorded after 5 μM Aβ 1–42 (24 h) alone, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min) in primary hippocampal neurons. ( F ) Normalization of Na + current densities, at −20 mV, represented in panel D and E. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus control, # p < 0.001 versus control Aβ 1–42 . (G) Representative western blot of Na V 1.6 protein expression (top) in the presence of siNa V 1.6 (50 nM; 48 h) in primary hippocampal neurons at 12 DIV. Quantification of siNav1.6 inhibition in primary hippocampal neurons (bottom). Values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control neurons.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay, Western Blot, Inhibition

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Expression and activity of Na V 1.6 channels in Tg2576 primary hippocampal neurons. ( A ) Representative traces of Na + currents recorded in WT and Tg2576 primary hippocampal neurons after 8 and 12 DIV. ( B ) Normalization of Na + current densities at −20 mV represented in panel A. Values are expressed as mean ± SEM of current densities of 3 independent experimental sessions. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus WT. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( D ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( E ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. (F) Representative confocal images displaying Na V 1.6 distribution in WT (left) and Tg2576 (right) primary hippocampal neurons after 12 DIV. Scale bars: 20 μm.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Effect of siNa V 1.6 and anisomycin on Na V 1.6 protein expression and activity in Tg2576 primary hippocampal neurons. ( A ) Representative traces of Na + currents recorded in WT primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( B ) Representative traces of Na + currents recorded in Tg2576 primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( C ) Normalization of Na + current densities at −20 mV represented in panel A and B. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control WT, *** p < 0.001 versus control WT, # p < 0.001 versus control Tg2576. ( D ) Representative current tracings recorded in the gap-free mode in WT and Tg2576 hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( E ) Quantification of spike frequency recorded in WT and Tg2576 hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus WT. # p < 0.001 versus control Tg2576. ( F ) Quantification of membrane depolarization recorded in WT and Tg2576 primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. # p < 0.001 versus control Tg2576

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Immunocytochemical analysis of Na V 1.6 protein expression after anisomycin treatment in Tg2576 primary hippocampal neurons at 12 DIV. ( A ) Confocal double immunofluorescence images displaying Na V 1.6 (green) and MAP2 (red) distribution in WT (a-c) and Tg2576 primary hippocampal neurons in the absence (d-f) or in the presence (g-i) of anisomycin. Scale bars in a-i: 20 μm. ( B ) Quantitative analyses of Na V 1.6-positive puncta within the soma of WT and Tg2576 primary hippocampal neurons in the absence or in the presence of anisomycin. Scale bars: 5 μm. Data are expressed as mean ± SEM of values obtained from 20 cells per group in 3 independent experimental sessions. ** p < 0.01 versus WT; # p < 0.001 versus Tg2576.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Evaluation of Na V 1.6 protein expression in the hippocampus of 3-month-old WT and Tg2576 mice. ( A ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( B ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. ( D ) Confocal double immunofluorescence images displaying Na V 1.6 (green) and MAP2 (red) distribution in the hippocampus of 3-month-old WT (a-c) and Tg2576 mice (d-f). Scale bars in a-f: 20 μm. (E) Quantitative analyses of Na V 1.6-positive puncta within the soma of WT and Tg2576 neurons in the hippocampus of 3-month-old mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. **p < 0.01 versus WT.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: The Journal of Physiology

Article Title: Visceral and somatic pain modalities reveal Na V 1.7‐independent visceral nociceptive pathways

doi: 10.1113/JP272837

Figure Lengend Snippet: A, proportions of thoracolumbar and lumbosacral colonic sensory neurons expressing transcripts for NaV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.7, NaV1.8 and NaV1.9. B, relative expression of NaV transcripts in thoracolumbar and lumbosacral colonic sensory neurons. C, co‐expression analysis of voltage‐gated sodium channels in both thoracolumbar (left) and lumbosacral (right) colonic sensory neuronal populations. Each segment in the wheel‐diagrams is representative of a single cell, with a coloured segment signifying positive expression. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: After dilution (1:5 TE buffer), Taqman qPCR assays were run for each gene of interest (Taqman Assay ID: Na V 1.1, Mm00450580_m1; Na V 1.2, Mm01270359_m1; Na V 1.3, Mm00658167_m1; Na V 1.4, Mm00500103_m1; Na V 1.5, Mm01342518_m1; Na V 1.6, Mm00488110_m1; Na V 1.7, Mm00450762_s1; Na V 1.8, Mm00501467_m1; Na V 1.9, Mm00449367_m1; GAPDH, Mm99999915_g1; Applied Biosystems) using the following cycling protocol: 50°C for 2 min, 95°C for 10 min, then 40 cycles of (95°C for 15 s, 60°C for 1 min).

Techniques: Expressing