Journal: Frontiers in Cellular Neuroscience

Article Title: Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

doi: 10.3389/fncel.2021.640709

Figure Lengend Snippet: Na V 1.7 α1 subunit expression in wild-type (WT) and R192Q KI TG cultures. (A) Representative immunofluorescent examples of Na V 1.7 α1 expression in WT and R192Q KI mouse trigeminal ganglia (TG) cultures. Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note extensive co-staining of Na V 1.7 α1 (red) and β-tubulin III (green) protein in both WT and KI samples. (B) Western blot example showing protein expression of Na V 1.7 α1 in TG from P12 mice. β-actin was used as a loading control. Histograms representing Na V 1.7 α1 relative optical density values for each group (* p = 0.46, Mann-Whitney test; n = 8 experiments). Note the significant difference between WT and KI groups. (C) The histograms quantify the cell diameter distribution of Na V 1.7 α1 immunofluorescence in different subgroups of WT and KI cultures ( n = 3 experiments). The percentages of positive neurons for Na V 1.7 were calculated by considering the total number of neurons labeled by β-Tubulin III as standard.

Article Snippet: Cells were then incubated with primary antibodies against Na V 1.7 α1 subunit (mouse anti-SCN9A/PN1, monoclonal, 1:500 in blocking buffer; Acris Antibodies GmbH Cat# AM12054PU-N, RRID:AB_10654661 , Herford, Germany) and β-tubulin III (mouse T5076, 1:1,000; Sigma, Milan) was used to specifically mark neurons.

Techniques: Expressing, Staining, Western Blot, MANN-WHITNEY, Immunofluorescence, Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

doi: 10.3389/fncel.2021.640709

Figure Lengend Snippet: Na V 1.7 α1 subunit expression in WT and R192Q KI TG cultures after pretreatment with ω-agatoxin IVA. (A) Representative immunofluorescent examples of Na V 1.7 α1 expression in WT and R192Q KI mouse TG cultures. ω-agatoxin IVA (200 nM, overnight: in this and subsequent Figures the toxin is abbreviated as ω-Agatoxin) was used to specifically block Ca V 2.1 channels (that are mutated in the KI model). Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note extensive co-staining of Na V 1.7 α1 (red) and β-tubulin III (green) protein in both WT and KI samples. (B) Histograms represent the percentage of Na V 1.7-positive cells in WT and KI cultures. Note significant difference between WT and KI control groups (* p = 0.015, Mann–Whitney test; n = 7 experiments), and the decrease in Na V 1.7 positive neurons in the KI after application of ω-agatoxin IVA ( p = 0.035; n = 3 experiments). (C) The histograms quantify the cell diameter distribution of Na V 1.7 α1 immunofluorescence in different subgroups in WT and KI ω-agatoxin IVA-treated cultures.

Article Snippet: Cells were then incubated with primary antibodies against Na V 1.7 α1 subunit (mouse anti-SCN9A/PN1, monoclonal, 1:500 in blocking buffer; Acris Antibodies GmbH Cat# AM12054PU-N, RRID:AB_10654661 , Herford, Germany) and β-tubulin III (mouse T5076, 1:1,000; Sigma, Milan) was used to specifically mark neurons.

Techniques: Expressing, Blocking Assay, Staining, MANN-WHITNEY, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

doi: 10.3389/fncel.2021.640709

Figure Lengend Snippet: Co-expression of Na V 1.7 α1 subunit and P2X3R in WT and R192Q KI cultures. (A) Representative examples of Na V 1.7 α1–P2X3 immunostaining in WT and R192Q KI mouse TG cultures. Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note the extensive co-staining of Na V 1.7 α1 (red) and P2X3 (green) protein in both WT and KI samples. (B) Histograms represent percentage co-expression of Na V 1.7 α1–P2X3 proteins in WT and KI cultures ( p = 0.95, Mann–Whitney test; n = 3 experiments). (C) The histograms quantify the cell diameter distribution of Na V 1.7 α1–P2X3R immunofluorescence in different subgroups in WT and KI cultures.

Article Snippet: Cells were then incubated with primary antibodies against Na V 1.7 α1 subunit (mouse anti-SCN9A/PN1, monoclonal, 1:500 in blocking buffer; Acris Antibodies GmbH Cat# AM12054PU-N, RRID:AB_10654661 , Herford, Germany) and β-tubulin III (mouse T5076, 1:1,000; Sigma, Milan) was used to specifically mark neurons.

Techniques: Expressing, Immunostaining, Staining, MANN-WHITNEY, Immunofluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

doi: 10.3389/fncel.2021.640709

Figure Lengend Snippet: Na V 1.7 current in WT and R192Q KI TG neurons. (A) Mean amplitudes of the currents evoked by a square pulse 100 ms depolarizing step from –75 to –45 mV, in WT and R192Q KI TG neurons under control conditions and after application of 1 μM TTX. Note larger control current in KI (* p = 0.039, two-sample Student’s t -test). Number of cells in each group: n = 29 (WT, control), n = 20 (WT, TTX), n = 43 (KI, control), i = 24 (KI, TTX). (B) Representative traces of inward currents recorded from one WT neuron in response to the same stimulation in control and after 1 μM TTX; the trace of TTX-sensitive current was obtained by subtraction of the TTX-resistant current from the control. (C) Superimposed representative traces of WT and KI TTX-sensitive currents obtained by subtraction; note larger KI current. (D) Histograms represent the calculated mean amplitudes of the currents evoked by the same stimulus (100-ms step from –75 to –45 mV) in control and after Tp1a (7 nM, 30 min). *Indicates statistically significant change (two-sample Student’s t -test, p -values are given in the text body). Number of cells in each group: n = 29 (WT, control), n = 28 (WT, Tp1a), n = 43 (KI, control), n = 38 (KI, Tp1a). (E) Superimposed representative traces of WT and KI Tp1a-sensitive (Na V 1.7) currents obtained by subtraction of the TTX-resistant current from the control; note larger Na V 1.7 current in KI.

Article Snippet: Cells were then incubated with primary antibodies against Na V 1.7 α1 subunit (mouse anti-SCN9A/PN1, monoclonal, 1:500 in blocking buffer; Acris Antibodies GmbH Cat# AM12054PU-N, RRID:AB_10654661 , Herford, Germany) and β-tubulin III (mouse T5076, 1:1,000; Sigma, Milan) was used to specifically mark neurons.

Techniques: