anti scn1a nav1 1 antibody  (Alomone Labs)


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    Alomone Labs anti scn1a nav1 1 antibody
    Cultures from two isogenic pairs contain a similar proportion of GABAergic and glutamatergic neurons. (A) Representative micrographs from cultures of two isogenic pairs of iPSC-derived neurons stained with neuronal subtype markers GABA or CaMKII antibodies to identify GABAergic and glutamatergic neurons at D21-24 post plating. (B) The percentage of GABAergic and glutamatergic neurons (white arrows) in neuronal cultures at D21-24 post plating in each line. There was no significant difference between cell lines (p = 0.1, one-way ANOVA for GABAergic neurons and p = 0.6 Kruskal-Wallis test for glutamatergic neurons). (C) Identification of inhibitory (top) and excitatory (bottom) neurons at D21-24 post plating labeled with plasmids via transient transfection during live recording (white arrows). At least 3 coverslips of 2-3 individual platings were evaluated for each line. (D) Localization of <t>Nav1.1</t> in GABAergic and glutamatergic neurons of the control line (white arrows). Data represented as mean + s.e.m. Ctrl = control, mut ctrl = mutated control, corr pt = corrected patient, and pt = patient. Scale bar represents 50 µ m.
    Anti Scn1a Nav1 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Comparisons of dual isogenic human iPSC pairs identify functional alterations directly caused by an epilepsy associated SCN1A mutation"

    Article Title: Comparisons of dual isogenic human iPSC pairs identify functional alterations directly caused by an epilepsy associated SCN1A mutation

    Journal: bioRxiv

    doi: 10.1101/524835

    Cultures from two isogenic pairs contain a similar proportion of GABAergic and glutamatergic neurons. (A) Representative micrographs from cultures of two isogenic pairs of iPSC-derived neurons stained with neuronal subtype markers GABA or CaMKII antibodies to identify GABAergic and glutamatergic neurons at D21-24 post plating. (B) The percentage of GABAergic and glutamatergic neurons (white arrows) in neuronal cultures at D21-24 post plating in each line. There was no significant difference between cell lines (p = 0.1, one-way ANOVA for GABAergic neurons and p = 0.6 Kruskal-Wallis test for glutamatergic neurons). (C) Identification of inhibitory (top) and excitatory (bottom) neurons at D21-24 post plating labeled with plasmids via transient transfection during live recording (white arrows). At least 3 coverslips of 2-3 individual platings were evaluated for each line. (D) Localization of Nav1.1 in GABAergic and glutamatergic neurons of the control line (white arrows). Data represented as mean + s.e.m. Ctrl = control, mut ctrl = mutated control, corr pt = corrected patient, and pt = patient. Scale bar represents 50 µ m.
    Figure Legend Snippet: Cultures from two isogenic pairs contain a similar proportion of GABAergic and glutamatergic neurons. (A) Representative micrographs from cultures of two isogenic pairs of iPSC-derived neurons stained with neuronal subtype markers GABA or CaMKII antibodies to identify GABAergic and glutamatergic neurons at D21-24 post plating. (B) The percentage of GABAergic and glutamatergic neurons (white arrows) in neuronal cultures at D21-24 post plating in each line. There was no significant difference between cell lines (p = 0.1, one-way ANOVA for GABAergic neurons and p = 0.6 Kruskal-Wallis test for glutamatergic neurons). (C) Identification of inhibitory (top) and excitatory (bottom) neurons at D21-24 post plating labeled with plasmids via transient transfection during live recording (white arrows). At least 3 coverslips of 2-3 individual platings were evaluated for each line. (D) Localization of Nav1.1 in GABAergic and glutamatergic neurons of the control line (white arrows). Data represented as mean + s.e.m. Ctrl = control, mut ctrl = mutated control, corr pt = corrected patient, and pt = patient. Scale bar represents 50 µ m.

    Techniques Used: Derivative Assay, Staining, Labeling, Transfection

    Isogenic pairs of iPSCs generated by CRISPR/Cas9 editing. (A) The K1270T mutation is located in the second transmembrane segment of the third homologous domain of Nav1.1 alpha subunit. (B) CRISPR/Cas9 editing was used to generate two isogenic pairs from iPSCs derived from two siblings in the GEFS+ family (control and patient) followed by differentiation into functional neurons. (C) Scheme of CRISPR/Cas9 editing design. Additional silent mutation introduced by ssODN1 resulted in an EcoRV restriction site for clone screening when knocking in the K1270T mutation. (D) Sequencing of two isogenic iPSC pairs confirmed the absence and the presence of the mutation. (E) All four lines had normal karyotypes (46, XY). (F) iPSCs of two isogenic pairs were stained with nuclei marker DAPI and pluripotency markers OCT-3/4, SOX2 and NANOG individually. The percentage of cells positive for each of the pluripotency markers was not significantly different between the 4 lines (p = 0.1, 0.06 and 0.2 for OCT-3/4, SOX2 and NANOG respectively, one-way ANOVA). Scale bar represents 50 µ m. Data represented as mean + s.e.m. Data represent counts from three fields in three coverslips from three platings for each genotype.
    Figure Legend Snippet: Isogenic pairs of iPSCs generated by CRISPR/Cas9 editing. (A) The K1270T mutation is located in the second transmembrane segment of the third homologous domain of Nav1.1 alpha subunit. (B) CRISPR/Cas9 editing was used to generate two isogenic pairs from iPSCs derived from two siblings in the GEFS+ family (control and patient) followed by differentiation into functional neurons. (C) Scheme of CRISPR/Cas9 editing design. Additional silent mutation introduced by ssODN1 resulted in an EcoRV restriction site for clone screening when knocking in the K1270T mutation. (D) Sequencing of two isogenic iPSC pairs confirmed the absence and the presence of the mutation. (E) All four lines had normal karyotypes (46, XY). (F) iPSCs of two isogenic pairs were stained with nuclei marker DAPI and pluripotency markers OCT-3/4, SOX2 and NANOG individually. The percentage of cells positive for each of the pluripotency markers was not significantly different between the 4 lines (p = 0.1, 0.06 and 0.2 for OCT-3/4, SOX2 and NANOG respectively, one-way ANOVA). Scale bar represents 50 µ m. Data represented as mean + s.e.m. Data represent counts from three fields in three coverslips from three platings for each genotype.

    Techniques Used: Generated, CRISPR, Mutagenesis, Derivative Assay, Functional Assay, Sequencing, Staining, Marker

    (A) Sequences of the top five predicted off-target sites in SCN7A, FLII, SCN2A, SCN3A and SCN9A . Off-target mutation was identified in the SCN7A gene of the mutated control line. Regions highlighted in blue are the sites predicted to be modified by non-specific CRISPR/Cas9 editing. (B) Expression of SCN1A, SCN7A and the housekeeping gene ACTB in the derived neurons of the mutated control line at D21 post plating. Expected band sizes of SCN1A, SCN7A and ACTB are 220, 283 and 177 bp. (C) Isolated sodium current recording in the derived neurons of the control line at D54 post plating. Sodium currents were completely eliminated in the presence of 1 μM TTX.
    Figure Legend Snippet: (A) Sequences of the top five predicted off-target sites in SCN7A, FLII, SCN2A, SCN3A and SCN9A . Off-target mutation was identified in the SCN7A gene of the mutated control line. Regions highlighted in blue are the sites predicted to be modified by non-specific CRISPR/Cas9 editing. (B) Expression of SCN1A, SCN7A and the housekeeping gene ACTB in the derived neurons of the mutated control line at D21 post plating. Expected band sizes of SCN1A, SCN7A and ACTB are 220, 283 and 177 bp. (C) Isolated sodium current recording in the derived neurons of the control line at D54 post plating. Sodium currents were completely eliminated in the presence of 1 μM TTX.

    Techniques Used: Mutagenesis, Modification, CRISPR, Expressing, Derivative Assay, Isolation

    2) Product Images from "MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion"

    Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-015-0032-9

    MiR-9 induced the increased of total Nav1.1 and Nav1.2 in primary cultured neonatal rat neurons (NRNs). a , b Effects of miR-9 on total protein levels of endogenous Nav1.1 ( a ), Nav1.2 ( b ), in NRNs, using western blot analysis and . Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. * P
    Figure Legend Snippet: MiR-9 induced the increased of total Nav1.1 and Nav1.2 in primary cultured neonatal rat neurons (NRNs). a , b Effects of miR-9 on total protein levels of endogenous Nav1.1 ( a ), Nav1.2 ( b ), in NRNs, using western blot analysis and . Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. * P

    Techniques Used: Cell Culture, Western Blot, Transfection

    MiR-9 produces the disturbance of trafficking, cellular distribution of Nav1.1 and Nav1.2 in rats. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. Rats were transfected with lenti-pre- miR-9, lenti-pre-miR-9 + lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P
    Figure Legend Snippet: MiR-9 produces the disturbance of trafficking, cellular distribution of Nav1.1 and Nav1.2 in rats. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. Rats were transfected with lenti-pre- miR-9, lenti-pre-miR-9 + lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P

    Techniques Used: Injection, Quantitative RT-PCR, Transfection

    AMO-miR-9 prevented the disturbed trafficking of Nav1.1/Nav1.2 induced by 2VO. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. 2VO rats were transfected with lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P
    Figure Legend Snippet: AMO-miR-9 prevented the disturbed trafficking of Nav1.1/Nav1.2 induced by 2VO. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. 2VO rats were transfected with lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P

    Techniques Used: Injection, Quantitative RT-PCR, Transfection

    Nav1.1 and Nav1.2 trafficking were disturbed in hippocampi and cortices after chronic brain hypoperfusion (CBH). a , western-blot analysis of the surface protein levels of Nav1.1 and Nav1.2 in sham and 2VO rats, upper: representative immunoblots of Nav1.1 and Nav1.2; lower: the quantitative analysis data of the immunoblots. The optical density was evaluated for each band and values for 2VO rat tissue were normalized to sham group after correction for protein loading with TfR,** P
    Figure Legend Snippet: Nav1.1 and Nav1.2 trafficking were disturbed in hippocampi and cortices after chronic brain hypoperfusion (CBH). a , western-blot analysis of the surface protein levels of Nav1.1 and Nav1.2 in sham and 2VO rats, upper: representative immunoblots of Nav1.1 and Nav1.2; lower: the quantitative analysis data of the immunoblots. The optical density was evaluated for each band and values for 2VO rat tissue were normalized to sham group after correction for protein loading with TfR,** P

    Techniques Used: Western Blot

    3) Product Images from "Overexpression of neuregulin 1 in GABAergic interneurons results in reversible cortical disinhibition"

    Article Title: Overexpression of neuregulin 1 in GABAergic interneurons results in reversible cortical disinhibition

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20552-y

    Inhibition of Na v currents in GABAergic interneurons by NRG1-ICD. a Increased rheobase and depolarized action potential threshold (APT) in the FS-GABAergic interneurons of gto Nrg1 ; Gfp mice, compared with gtoGfp mice. * P = 0.0321, ** P = 0.0015, two-sided t test, n = 17 cells from five gtoGfp mice, n = 21 cells from four gto Nrg1 ; Gfp mice. b Representative traces of a single AP evoked from a suprathreshold current injection (left) and corresponding phase plots (dV/dt vs voltage) (right) recorded in FS-GABAergic interneurons from gtoGfp and gto Nrg1 ; Gfp mice. c Reduced Na + current density in GABAergic interneurons from gto Nrg1 ; Gfp PFC. Representative current traces of Na v channels in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. Currents were elicited by step depolarizations from −80 to +30 mV in 5 mV increments from a holding potential of −80 mV. The tracts shown are for depolarizations from −80 to +20 mV. d I/V curves of Na v channel activation in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. **Genotype F (1, 28) = 8.264, P = 0.0076, two-way ANOVA, n = 13 cells from three gto Nrg1 ; Gfp mice, n = 17 cells from four gtoGfp mice. Data are presented as mean values + /− SEM. e Similar voltage-dependent activation curves of Na v channels in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. Genotype F (1, 28) = 0.023, P = 0.879, two-way ANOVA, n = 13 cells from three gto Nrg1 ; Gfp mice, n = 17 cells from four gtoGfp mice. Data are presented as mean values + /− SEM. f Transcriptional levels of Scn1a is higher than Scn2a1, Scn3a, and Scn8a in PV and SST-positive GABAergic interneurons ( n = 2688 cells). Shown are transcripts of Scn genes per 100 k total transcripts from single-cell RNA sequencing. g Transcriptional levels of Scn1a were lower than Scn2a1 and Scn8a in layer 2/3 PN ( n = 41827 cells). Shown are transcripts of Scn genes per 100 k total transcripts from single-cell RNA sequencing. h Diagram showing the structure of SCN1A. The SCN1A protein was composed of four transmembrane domains (I–IV) and two major cytoplasmic loops (CL1 and CL2). The NRG1-ICD could interact with the CL1 of SCN1A. i Coomassie blue staining of 30 μg GST and GST-NRG1-ICD proteins. Asterisks indicated degradation product of GST-NRG1-ICD proteins. Four independent experiments were repeated to get similar results. j Interaction of NRG1-ICD with His-CL1. The recombinant GST-NRG1-ICD and His-CL1, or His-CL2 proteins were used for GST pulldown experiments. Four independent experiments were repeated to get similar results. k Diagram showing delivery of GST-NRG1-ICD or GST proteins into GABAergic interneurons in gtoGfp slices. l Reduced Na + current density in GABAergic interneurons treated with GST-NRG1-ICD. Representative current traces of Na v channels in GABAergic interneurons treated with recombinant GST or GST-NRG1-ICD proteins. Currents were elicited by step depolarizations from −80 to +30 mV in 5 mV increments from a holding potential of −80 mV. The tracts shown are for depolarizations from −80 to +20 mV. m I/V curves of Na v channel activation in GABAergic interneurons treated with recombinant GST or GST-NRG1-ICD proteins. **Treatment F (1, 24) = 10.89, P = 0.003, two-way ANOVA, n = 14 cells treated with GST-NRG1-ICD, n = 12 cells treated with GST. Data are presented as mean values + /− SEM. n Similar voltage-dependent activation curves of Na v channels in GABAergic interneurons treated with GST or GST-NRG1-ICD. Genotype F (1, 24) = 0.059, P = 0.81, two-way ANOVA, n = 14 cells treated with GST-NRG1-ICD, n = 12 cells treated with GST. Data are presented as mean values + /− SEM.
    Figure Legend Snippet: Inhibition of Na v currents in GABAergic interneurons by NRG1-ICD. a Increased rheobase and depolarized action potential threshold (APT) in the FS-GABAergic interneurons of gto Nrg1 ; Gfp mice, compared with gtoGfp mice. * P = 0.0321, ** P = 0.0015, two-sided t test, n = 17 cells from five gtoGfp mice, n = 21 cells from four gto Nrg1 ; Gfp mice. b Representative traces of a single AP evoked from a suprathreshold current injection (left) and corresponding phase plots (dV/dt vs voltage) (right) recorded in FS-GABAergic interneurons from gtoGfp and gto Nrg1 ; Gfp mice. c Reduced Na + current density in GABAergic interneurons from gto Nrg1 ; Gfp PFC. Representative current traces of Na v channels in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. Currents were elicited by step depolarizations from −80 to +30 mV in 5 mV increments from a holding potential of −80 mV. The tracts shown are for depolarizations from −80 to +20 mV. d I/V curves of Na v channel activation in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. **Genotype F (1, 28) = 8.264, P = 0.0076, two-way ANOVA, n = 13 cells from three gto Nrg1 ; Gfp mice, n = 17 cells from four gtoGfp mice. Data are presented as mean values + /− SEM. e Similar voltage-dependent activation curves of Na v channels in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. Genotype F (1, 28) = 0.023, P = 0.879, two-way ANOVA, n = 13 cells from three gto Nrg1 ; Gfp mice, n = 17 cells from four gtoGfp mice. Data are presented as mean values + /− SEM. f Transcriptional levels of Scn1a is higher than Scn2a1, Scn3a, and Scn8a in PV and SST-positive GABAergic interneurons ( n = 2688 cells). Shown are transcripts of Scn genes per 100 k total transcripts from single-cell RNA sequencing. g Transcriptional levels of Scn1a were lower than Scn2a1 and Scn8a in layer 2/3 PN ( n = 41827 cells). Shown are transcripts of Scn genes per 100 k total transcripts from single-cell RNA sequencing. h Diagram showing the structure of SCN1A. The SCN1A protein was composed of four transmembrane domains (I–IV) and two major cytoplasmic loops (CL1 and CL2). The NRG1-ICD could interact with the CL1 of SCN1A. i Coomassie blue staining of 30 μg GST and GST-NRG1-ICD proteins. Asterisks indicated degradation product of GST-NRG1-ICD proteins. Four independent experiments were repeated to get similar results. j Interaction of NRG1-ICD with His-CL1. The recombinant GST-NRG1-ICD and His-CL1, or His-CL2 proteins were used for GST pulldown experiments. Four independent experiments were repeated to get similar results. k Diagram showing delivery of GST-NRG1-ICD or GST proteins into GABAergic interneurons in gtoGfp slices. l Reduced Na + current density in GABAergic interneurons treated with GST-NRG1-ICD. Representative current traces of Na v channels in GABAergic interneurons treated with recombinant GST or GST-NRG1-ICD proteins. Currents were elicited by step depolarizations from −80 to +30 mV in 5 mV increments from a holding potential of −80 mV. The tracts shown are for depolarizations from −80 to +20 mV. m I/V curves of Na v channel activation in GABAergic interneurons treated with recombinant GST or GST-NRG1-ICD proteins. **Treatment F (1, 24) = 10.89, P = 0.003, two-way ANOVA, n = 14 cells treated with GST-NRG1-ICD, n = 12 cells treated with GST. Data are presented as mean values + /− SEM. n Similar voltage-dependent activation curves of Na v channels in GABAergic interneurons treated with GST or GST-NRG1-ICD. Genotype F (1, 24) = 0.059, P = 0.81, two-way ANOVA, n = 14 cells treated with GST-NRG1-ICD, n = 12 cells treated with GST. Data are presented as mean values + /− SEM.

    Techniques Used: Inhibition, Mouse Assay, Injection, Activation Assay, RNA Sequencing Assay, Staining, Recombinant

    4) Product Images from "Lateral habenula dysfunctions in Tm4sf2−/y mice model for neurodevelopmental disorder"

    Article Title: Lateral habenula dysfunctions in Tm4sf2−/y mice model for neurodevelopmental disorder

    Journal: Neurobiology of Disease

    doi: 10.1016/j.nbd.2020.105189

    Decreased VGNC expression and increased PKC and ERK activity in Tm4sf2 −/y mice. A) Western blot of crude membrane preparation from Tm4sf2+/y and Tm4sf2−/y mice habenulae detected using ChemiDoc XRS+ System (BioRad) and quantification of voltage-gated sodium and potassium channel protein levels normalized on tubulin. You can see a downward trend and a significant reduced expression for NaV1.1 and NaV1.6 respectively in Tm4sf2−/y mice, while no changes for Kv4.2 has been detected; B) Quantification of PKC brain activity obtained using a PKC Kinase Activity Assay Kit showing a hyperactivity in Tm4sf2−/y compared to Tm4sf2+/y mice; C) Western blot of habenulae homogenates from Tm4sf2+/y and Tm4sf2−/y mice and quantification of expression levels represented as pERK/ERK ratio. α-tubulin was used as loading control.
    Figure Legend Snippet: Decreased VGNC expression and increased PKC and ERK activity in Tm4sf2 −/y mice. A) Western blot of crude membrane preparation from Tm4sf2+/y and Tm4sf2−/y mice habenulae detected using ChemiDoc XRS+ System (BioRad) and quantification of voltage-gated sodium and potassium channel protein levels normalized on tubulin. You can see a downward trend and a significant reduced expression for NaV1.1 and NaV1.6 respectively in Tm4sf2−/y mice, while no changes for Kv4.2 has been detected; B) Quantification of PKC brain activity obtained using a PKC Kinase Activity Assay Kit showing a hyperactivity in Tm4sf2−/y compared to Tm4sf2+/y mice; C) Western blot of habenulae homogenates from Tm4sf2+/y and Tm4sf2−/y mice and quantification of expression levels represented as pERK/ERK ratio. α-tubulin was used as loading control.

    Techniques Used: Expressing, Activity Assay, Mouse Assay, Western Blot, Kinase Assay

    5) Product Images from "MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion"

    Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-015-0032-9

    MiR-9 induced the increased of total Nav1.1 and Nav1.2 in primary cultured neonatal rat neurons (NRNs). a , b Effects of miR-9 on total protein levels of endogenous Nav1.1 ( a ), Nav1.2 ( b ), in NRNs, using western blot analysis and . Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. * P
    Figure Legend Snippet: MiR-9 induced the increased of total Nav1.1 and Nav1.2 in primary cultured neonatal rat neurons (NRNs). a , b Effects of miR-9 on total protein levels of endogenous Nav1.1 ( a ), Nav1.2 ( b ), in NRNs, using western blot analysis and . Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. * P

    Techniques Used: Cell Culture, Western Blot, Transfection

    MiR-9 produces the disturbance of trafficking, cellular distribution of Nav1.1 and Nav1.2 in rats. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. Rats were transfected with lenti-pre- miR-9, lenti-pre-miR-9 + lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P
    Figure Legend Snippet: MiR-9 produces the disturbance of trafficking, cellular distribution of Nav1.1 and Nav1.2 in rats. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. Rats were transfected with lenti-pre- miR-9, lenti-pre-miR-9 + lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P

    Techniques Used: Injection, Quantitative RT-PCR, Transfection

    AMO-miR-9 prevented the disturbed trafficking of Nav1.1/Nav1.2 induced by 2VO. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. 2VO rats were transfected with lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P
    Figure Legend Snippet: AMO-miR-9 prevented the disturbed trafficking of Nav1.1/Nav1.2 induced by 2VO. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. 2VO rats were transfected with lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P

    Techniques Used: Injection, Quantitative RT-PCR, Transfection

    Nav1.1 and Nav1.2 trafficking were disturbed in hippocampi and cortices after chronic brain hypoperfusion (CBH). a , western-blot analysis of the surface protein levels of Nav1.1 and Nav1.2 in sham and 2VO rats, upper: representative immunoblots of Nav1.1 and Nav1.2; lower: the quantitative analysis data of the immunoblots. The optical density was evaluated for each band and values for 2VO rat tissue were normalized to sham group after correction for protein loading with TfR,** P
    Figure Legend Snippet: Nav1.1 and Nav1.2 trafficking were disturbed in hippocampi and cortices after chronic brain hypoperfusion (CBH). a , western-blot analysis of the surface protein levels of Nav1.1 and Nav1.2 in sham and 2VO rats, upper: representative immunoblots of Nav1.1 and Nav1.2; lower: the quantitative analysis data of the immunoblots. The optical density was evaluated for each band and values for 2VO rat tissue were normalized to sham group after correction for protein loading with TfR,** P

    Techniques Used: Western Blot

    6) Product Images from "Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment"

    Article Title: Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00005

    Co-localization of FGF14 and Nav1.6 in mouse cortex using 1% formaldehyde and 0.5% MeOH fixation . (A–D) The gray and red channels represent FGF14 immunoreactivity visualized with an Alexa 568-conjugated secondary antibody, the green channel represents PanNav (Sigma-Aldrich, rabbit anti PanNav, catalog number S6936) in (A) , Nav1.1 (Alomone Labs) in (B) , Nav1.2 (Alomone Labs) in (C) , and Nav1.6 (Alomone Labs) in (D) visualized with an Alexa 488-conjugated secondary antibody and the blue represents Topro3 nuclear staining in the cortex. Right panels represent overlaid images (third column from the left) and high magnification of boxed ROI from the merged images. Scale bars represent 20 μm.
    Figure Legend Snippet: Co-localization of FGF14 and Nav1.6 in mouse cortex using 1% formaldehyde and 0.5% MeOH fixation . (A–D) The gray and red channels represent FGF14 immunoreactivity visualized with an Alexa 568-conjugated secondary antibody, the green channel represents PanNav (Sigma-Aldrich, rabbit anti PanNav, catalog number S6936) in (A) , Nav1.1 (Alomone Labs) in (B) , Nav1.2 (Alomone Labs) in (C) , and Nav1.6 (Alomone Labs) in (D) visualized with an Alexa 488-conjugated secondary antibody and the blue represents Topro3 nuclear staining in the cortex. Right panels represent overlaid images (third column from the left) and high magnification of boxed ROI from the merged images. Scale bars represent 20 μm.

    Techniques Used: Staining

    7) Product Images from "Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scn1a-associated epileptic encephalopathyThe dose makes the poison – novel insights into Dravet Syndrome and SCN1A regulation through non-productive splicing"

    Article Title: Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scn1a-associated epileptic encephalopathyThe dose makes the poison – novel insights into Dravet Syndrome and SCN1A regulation through non-productive splicing

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1009195

    Increased inclusion of Exon 20N in Scn1a+/KI brains. (A) The positions of qPCR amplicons to quantify Scn1a mRNA transcripts. Amplicon 1 detects two isoforms (56bp and 120bp) of the Scn1a transcript, with the longer isoform reflecting exon 20N inclusion. Amplicon 2 quantifies only the Exon 20N-containing transcript. Amplicon 3 quantifies the total Scn1a mRNA levels including the transcript with Exon 20N. (B) Bioanalyzer evaluation of RNA from Scn1a+/+ mouse brain amplified with amplicon 1, showing a single Scn1a peak at 56 bp. The peaks at 15-bp and 1500-bp are size markers recommended and supplied by the manufacturer. ( C) Bioanalyzer evaluation of RNA from Scn1a+/KI mouse brain amplified with amplicon 1, showing a second peak at 120 bp, representing inclusion of exon 20N. The 120-bp amplicon containing the 64-bp exon 20N is denoted with a red asterisk. (D) Scn1a+/KI mice had increased levels of the exon 20N–containing Scn1a transcript, measured using amplicon 2. The levels of exon 20N transcript (amplicon 2) expressed as a percentage of the total Scn1a levels (amplicon 3) using the formula (amplicon 2 levels)/(amplicon 3 levels)*100. (n = 4, 11.64 ± 2.90 months, Student's unpaired t-test, p = 2e-4). ** p
    Figure Legend Snippet: Increased inclusion of Exon 20N in Scn1a+/KI brains. (A) The positions of qPCR amplicons to quantify Scn1a mRNA transcripts. Amplicon 1 detects two isoforms (56bp and 120bp) of the Scn1a transcript, with the longer isoform reflecting exon 20N inclusion. Amplicon 2 quantifies only the Exon 20N-containing transcript. Amplicon 3 quantifies the total Scn1a mRNA levels including the transcript with Exon 20N. (B) Bioanalyzer evaluation of RNA from Scn1a+/+ mouse brain amplified with amplicon 1, showing a single Scn1a peak at 56 bp. The peaks at 15-bp and 1500-bp are size markers recommended and supplied by the manufacturer. ( C) Bioanalyzer evaluation of RNA from Scn1a+/KI mouse brain amplified with amplicon 1, showing a second peak at 120 bp, representing inclusion of exon 20N. The 120-bp amplicon containing the 64-bp exon 20N is denoted with a red asterisk. (D) Scn1a+/KI mice had increased levels of the exon 20N–containing Scn1a transcript, measured using amplicon 2. The levels of exon 20N transcript (amplicon 2) expressed as a percentage of the total Scn1a levels (amplicon 3) using the formula (amplicon 2 levels)/(amplicon 3 levels)*100. (n = 4, 11.64 ± 2.90 months, Student's unpaired t-test, p = 2e-4). ** p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Mouse Assay

    Scn1a+/KI mice exhibit premature mortality and a hyperactivity phenotype. (A) Kaplan-Meier analysis showed severe premature mortality in Scn1a+/KI mice ( n = 22–93, Log-rank (Mantel-Cox) test, p
    Figure Legend Snippet: Scn1a+/KI mice exhibit premature mortality and a hyperactivity phenotype. (A) Kaplan-Meier analysis showed severe premature mortality in Scn1a+/KI mice ( n = 22–93, Log-rank (Mantel-Cox) test, p

    Techniques Used: Mouse Assay

    Inverse relationship between poison exon usage and expression of multiple sodium channels during mouse brain development. (A) Scn1a transcripts including exon 20N are highly expressed in the developing mouse brain and decrease dramatically after birth (aqua bars), with a corresponding developmental increase in Scn1a expression (blue bars). (B) The poison exon in Scn8a previously described by Plummer et al.[ 29 ]. Scn1a exon 20N and Scn8a exon 18N are 37.5% identical (57% in human), and the amino acid sequences shown at exon boundaries are identical. The amino acid sequences shown are fully identical between mouse and human for both genes. (C) Scn8a transcripts including exon 18N are highly expressed in the developing mouse brain and decrease dramatically after birth (aqua bars), with a corresponding developmental increase in Scn8a expression (blue bars).
    Figure Legend Snippet: Inverse relationship between poison exon usage and expression of multiple sodium channels during mouse brain development. (A) Scn1a transcripts including exon 20N are highly expressed in the developing mouse brain and decrease dramatically after birth (aqua bars), with a corresponding developmental increase in Scn1a expression (blue bars). (B) The poison exon in Scn8a previously described by Plummer et al.[ 29 ]. Scn1a exon 20N and Scn8a exon 18N are 37.5% identical (57% in human), and the amino acid sequences shown at exon boundaries are identical. The amino acid sequences shown are fully identical between mouse and human for both genes. (C) Scn8a transcripts including exon 18N are highly expressed in the developing mouse brain and decrease dramatically after birth (aqua bars), with a corresponding developmental increase in Scn8a expression (blue bars).

    Techniques Used: Expressing

    The non-coding Dravet Syndrome–causing variant, NM_006920.4(SCN1A):c.3969+2451G > C, is present in a highly conserved region. (A) The alternate exon 20N (shaded rectangle) is highly conserved, with GERP scores that are comparable to canonical exons in SCN1A . ( B) Multiple alignment in the 64bp SCN1A 20N region of human, mouse, opossum, alligator, and duck, modified from the Multiz Alignment of 100 Vertebrates track from the UCSC Genome Browser (Fig 1). The red box indicates the position of the variant NM_006920.4(SCN1A):c.3969+2451G > C in our index patient, orthologous to NC_000068.7:g.66293870C > G (GRCm38.p6) in the mouse genome. The red line indicates the position of the guide RNA used for CRISPR/Cas9 gene editing. ( C) Alternative splicing of intron 20 in SCN1A . Inclusion of exon 20N (bottom) results in a frame shift and hence a premature termination codon (PTC) in exon 21. The c.3969+2451G > C variant also results in a Gly-Ala (red) substitution within exon 20N. (D) Exon 20N would be in the intracellular loop connecting the fourth and fifth transmembrane voltage sensing regions of the third SCN1A homologous domain (D3) but brings a premature termination codon (PTC) in frame resulting in nonsense-mediated RNA decay.
    Figure Legend Snippet: The non-coding Dravet Syndrome–causing variant, NM_006920.4(SCN1A):c.3969+2451G > C, is present in a highly conserved region. (A) The alternate exon 20N (shaded rectangle) is highly conserved, with GERP scores that are comparable to canonical exons in SCN1A . ( B) Multiple alignment in the 64bp SCN1A 20N region of human, mouse, opossum, alligator, and duck, modified from the Multiz Alignment of 100 Vertebrates track from the UCSC Genome Browser (Fig 1). The red box indicates the position of the variant NM_006920.4(SCN1A):c.3969+2451G > C in our index patient, orthologous to NC_000068.7:g.66293870C > G (GRCm38.p6) in the mouse genome. The red line indicates the position of the guide RNA used for CRISPR/Cas9 gene editing. ( C) Alternative splicing of intron 20 in SCN1A . Inclusion of exon 20N (bottom) results in a frame shift and hence a premature termination codon (PTC) in exon 21. The c.3969+2451G > C variant also results in a Gly-Ala (red) substitution within exon 20N. (D) Exon 20N would be in the intracellular loop connecting the fourth and fifth transmembrane voltage sensing regions of the third SCN1A homologous domain (D3) but brings a premature termination codon (PTC) in frame resulting in nonsense-mediated RNA decay.

    Techniques Used: Variant Assay, Modification, CRISPR

    Scn1a mRNA and Na v 1.1 protein levels are reduced in Scn1a+/KI mice. (A) Sanger sequence confirmation of Scn1a+/KI mouse with the gene-edited intron 20 C > G variant. ( B) Brain mRNA levels in Scn1a+/+ and Scn1a+/KI mice using qRT-PCR. Relative expression of Scn1a vs. the control gene Tbp ( n = 4−4, 11.64 ± 2.90 months, Student's unpaired t-test, p = 0.0251). Scn1a+/KI mice have ~50% less Scn1a mRNA than Scn1a +/+ mice. ( C) RNA-seq counts (normalized to sequencing library size by DEseq2) of Scn1a mRNA in whole brains of Scn1a+/+ and Scn1a+/KI mice (n = 4−4, 11.64 ± 2.90 months, Student's unpaired t-test, p = 0.0541). Scn1a+/KI mice have about 42% less Scn1a mRNA than Scn1a+/+ mice. (D) Levels of Na v 1.1, the sodium channel encoded by Scn1a , are reduced in frontal cortex of Scn1a+/KI vs. Scn1a+/+ mice using rabbit anti-Na v 1.1 antibody from Alomone Labs, which recognizes an N-terminal epitope. GAPDH served as a loading control. (E) Quantification of Na v 1.1 levels from the blot in D ( n = 2–3, 17.7 ± 0.96 months, Student's unpaired t-test, p = 0.0142). (F) Quantification GAPDH protein levels from the blot in D ( n = 2–3, 17.7 ± 0.96 months, Student's unpaired t-test, p = 0.4459). (G) Na v 1.1 levels using anti-Na v 1.1 Antibodies Incorporated antibody, which recognizes a C-terminal epitope. Actin served as a loading control. (H) Quantification of Na v 1.1 protein levels from the blot in G (n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.0059). (I) Quantification of actin protein levels from the blot in G (n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.9859, respectively). * p
    Figure Legend Snippet: Scn1a mRNA and Na v 1.1 protein levels are reduced in Scn1a+/KI mice. (A) Sanger sequence confirmation of Scn1a+/KI mouse with the gene-edited intron 20 C > G variant. ( B) Brain mRNA levels in Scn1a+/+ and Scn1a+/KI mice using qRT-PCR. Relative expression of Scn1a vs. the control gene Tbp ( n = 4−4, 11.64 ± 2.90 months, Student's unpaired t-test, p = 0.0251). Scn1a+/KI mice have ~50% less Scn1a mRNA than Scn1a +/+ mice. ( C) RNA-seq counts (normalized to sequencing library size by DEseq2) of Scn1a mRNA in whole brains of Scn1a+/+ and Scn1a+/KI mice (n = 4−4, 11.64 ± 2.90 months, Student's unpaired t-test, p = 0.0541). Scn1a+/KI mice have about 42% less Scn1a mRNA than Scn1a+/+ mice. (D) Levels of Na v 1.1, the sodium channel encoded by Scn1a , are reduced in frontal cortex of Scn1a+/KI vs. Scn1a+/+ mice using rabbit anti-Na v 1.1 antibody from Alomone Labs, which recognizes an N-terminal epitope. GAPDH served as a loading control. (E) Quantification of Na v 1.1 levels from the blot in D ( n = 2–3, 17.7 ± 0.96 months, Student's unpaired t-test, p = 0.0142). (F) Quantification GAPDH protein levels from the blot in D ( n = 2–3, 17.7 ± 0.96 months, Student's unpaired t-test, p = 0.4459). (G) Na v 1.1 levels using anti-Na v 1.1 Antibodies Incorporated antibody, which recognizes a C-terminal epitope. Actin served as a loading control. (H) Quantification of Na v 1.1 protein levels from the blot in G (n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.0059). (I) Quantification of actin protein levels from the blot in G (n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.9859, respectively). * p

    Techniques Used: Mouse Assay, Sequencing, Variant Assay, Quantitative RT-PCR, Expressing, RNA Sequencing Assay

    8) Product Images from "Tau Reduction Prevents Disease in a Mouse Model of Dravet Syndrome"

    Article Title: Tau Reduction Prevents Disease in a Mouse Model of Dravet Syndrome

    Journal: Annals of Neurology

    doi: 10.1002/ana.24230

    Tau ablation ameliorates deficits of Scn1a RX/+ mice in the Barnes maze and in a fear conditioning task. (A–E) Mice (n = 6–7 mice per genotype) were tested in the Barnes maze at 4 months of age. (A) Learning curves in the Barnes maze did not differ significantly among genotypes (linear mixed effects model analysis). (B, C) Latency (B) and distance traveled (C) to reach the target location during a probe trial 5 days after training in the Barnes maze. Interaction between Scn1a and Tau genotypes by 2-way analysis of variance: (B) p = 0.020, F 1,22 = 6.3 and (C) p = 0.0073, F 1,22 = 8.7. * p
    Figure Legend Snippet: Tau ablation ameliorates deficits of Scn1a RX/+ mice in the Barnes maze and in a fear conditioning task. (A–E) Mice (n = 6–7 mice per genotype) were tested in the Barnes maze at 4 months of age. (A) Learning curves in the Barnes maze did not differ significantly among genotypes (linear mixed effects model analysis). (B, C) Latency (B) and distance traveled (C) to reach the target location during a probe trial 5 days after training in the Barnes maze. Interaction between Scn1a and Tau genotypes by 2-way analysis of variance: (B) p = 0.020, F 1,22 = 6.3 and (C) p = 0.0073, F 1,22 = 8.7. * p

    Techniques Used: Mouse Assay

    Tau ablation improves alterations in nest building, open field behaviors and social function in Scn1a RX/+ mice. Mice of the indicated genotypes were tested for nest building at 1 to 7 months (n = 11–22 mice per genotype) or open field activity and social approach at 2 to 3 months (n = 8–13 mice per genotype). See Supplementary Table S2 for age balance among groups. (A) Nest building behavior was monitored for up to 8 days and scored as described in Materials and Methods. A linear mixed effects model was used to fit the data and to obtain estimates of the area under the curve as a measure of nest building performance. Scn1a RX/+ / Tau +/+ mice differed from Scn1a +/+ / Tau +/+ ( p = 0.0000004) and Scn1a RX/+ / Tau −/− ( p = 0.00063) mice, whereas Scn1a RX/+ / Tau −/− mice did not differ from Scn1a +/+ / Tau +/+ mice ( p = 0.21). A gene–dose effect of Tau deletion was present ( p = 0.00063). Exploratory post hoc analyses without multiple comparison correction indicated that Scn1a RX/+ / Tau +/− mice differed more from Scn1a RX/+ / Tau +/+ ( p = 0.014) than Scn1a +/+ / Tau +/+ ( p = 0.069) mice. (B–D) Open field behavior. (B) Circling was recorded for 30 minutes and (C) rearing and (D) total movements during the first 5 minutes. Interaction between Scn1a and Tau genotypes by 2-way analysis of variance: (B) p = 0.015, F 1,37 = 6.5; (C) p = 0.0022, F 1,38 = 10.8; (D) p = 0.34, F 1,38 = 0.93. * p
    Figure Legend Snippet: Tau ablation improves alterations in nest building, open field behaviors and social function in Scn1a RX/+ mice. Mice of the indicated genotypes were tested for nest building at 1 to 7 months (n = 11–22 mice per genotype) or open field activity and social approach at 2 to 3 months (n = 8–13 mice per genotype). See Supplementary Table S2 for age balance among groups. (A) Nest building behavior was monitored for up to 8 days and scored as described in Materials and Methods. A linear mixed effects model was used to fit the data and to obtain estimates of the area under the curve as a measure of nest building performance. Scn1a RX/+ / Tau +/+ mice differed from Scn1a +/+ / Tau +/+ ( p = 0.0000004) and Scn1a RX/+ / Tau −/− ( p = 0.00063) mice, whereas Scn1a RX/+ / Tau −/− mice did not differ from Scn1a +/+ / Tau +/+ mice ( p = 0.21). A gene–dose effect of Tau deletion was present ( p = 0.00063). Exploratory post hoc analyses without multiple comparison correction indicated that Scn1a RX/+ / Tau +/− mice differed more from Scn1a RX/+ / Tau +/+ ( p = 0.014) than Scn1a +/+ / Tau +/+ ( p = 0.069) mice. (B–D) Open field behavior. (B) Circling was recorded for 30 minutes and (C) rearing and (D) total movements during the first 5 minutes. Interaction between Scn1a and Tau genotypes by 2-way analysis of variance: (B) p = 0.015, F 1,37 = 6.5; (C) p = 0.0022, F 1,38 = 10.8; (D) p = 0.34, F 1,38 = 0.93. * p

    Techniques Used: Mouse Assay, Activity Assay

    Tau reduction improves survival of Scn1a RX/+ mice in a gene dose-dependent manner. Survival plots of 292 Scn1a RX/+ mice and littermate controls with 2, 1, or no Tau alleles (n = 20–98 mice per genotype) indicate the percentage of live mice between 22 and 150 days postnatally. Scn1a RX/+ / Tau +/+ mice differed from Scn1a +/+ / Tau +/+ ( p = 0.0048, hazard ratio [HR] = 31.0), Scn1a RX/+ / Tau +/− ( p = 0.00011, HR = 6.1), and Scn1a RX/+ / Tau −/− mice ( p = 0.026, HR 17.1). Scn1a +/+ / Tau +/+ mice did not differ from Scn1a RX/+ / Tau +/− mice ( p = 0.37, HR = 5.1) or Scn1a RX/+ / Tau −/− mice ( p = 1.0, HR = 1.8). Gene–dose effect: p = 0.00005, HR = 0.018 for each Tau deletion (Cox proportional hazards regression).
    Figure Legend Snippet: Tau reduction improves survival of Scn1a RX/+ mice in a gene dose-dependent manner. Survival plots of 292 Scn1a RX/+ mice and littermate controls with 2, 1, or no Tau alleles (n = 20–98 mice per genotype) indicate the percentage of live mice between 22 and 150 days postnatally. Scn1a RX/+ / Tau +/+ mice differed from Scn1a +/+ / Tau +/+ ( p = 0.0048, hazard ratio [HR] = 31.0), Scn1a RX/+ / Tau +/− ( p = 0.00011, HR = 6.1), and Scn1a RX/+ / Tau −/− mice ( p = 0.026, HR 17.1). Scn1a +/+ / Tau +/+ mice did not differ from Scn1a RX/+ / Tau +/− mice ( p = 0.37, HR = 5.1) or Scn1a RX/+ / Tau −/− mice ( p = 1.0, HR = 1.8). Gene–dose effect: p = 0.00005, HR = 0.018 for each Tau deletion (Cox proportional hazards regression).

    Techniques Used: Mouse Assay

    Na v 1.1 levels are reduced in Scn1a RX/+ mice, and this reduction is not prevented by tau ablation. Levels of Na v 1.1 and total sodium channels (pan Na v ) in the parietal cortex of 8-month-old mice were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype). The average Na v 1.1 to pan Na v ratio in Scn1a +/+ / Tau +/+ mice was arbitrarily defined as 1.0. ***p
    Figure Legend Snippet: Na v 1.1 levels are reduced in Scn1a RX/+ mice, and this reduction is not prevented by tau ablation. Levels of Na v 1.1 and total sodium channels (pan Na v ) in the parietal cortex of 8-month-old mice were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype). The average Na v 1.1 to pan Na v ratio in Scn1a +/+ / Tau +/+ mice was arbitrarily defined as 1.0. ***p

    Techniques Used: Mouse Assay, Western Blot

    Cortical levels of total and phosphorylated tau are not altered in Scn1a RX/+ mice. Levels of phospho-tau (PHF-1, Ser396/Ser404; AT8, Ser202/Thr205; CP9, Thr231) and total tau (Tau-5, EP2456Y) in the parietal cortex of 8-month-old mice of the indicated genotypes were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype) revealed no statistically significant differences between Scn1a RX/+ /Tau +/+ and Scn1a +/+ / Tau +/+ mice (Student t test). Average phospho-tau to EP2456Y ratios (PHF-1, AT8, CP9) or average total tau levels (Tau-5, EP2456Y) in Scn1a +/+ / Tau +/+ mice were arbitrarily defined as 1.0. Values represent mean ± standard error of the mean.
    Figure Legend Snippet: Cortical levels of total and phosphorylated tau are not altered in Scn1a RX/+ mice. Levels of phospho-tau (PHF-1, Ser396/Ser404; AT8, Ser202/Thr205; CP9, Thr231) and total tau (Tau-5, EP2456Y) in the parietal cortex of 8-month-old mice of the indicated genotypes were determined by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (A) Representative Western blot. (B) Quantification of Western blot signals (n = 5–7 mice per genotype) revealed no statistically significant differences between Scn1a RX/+ /Tau +/+ and Scn1a +/+ / Tau +/+ mice (Student t test). Average phospho-tau to EP2456Y ratios (PHF-1, AT8, CP9) or average total tau levels (Tau-5, EP2456Y) in Scn1a +/+ / Tau +/+ mice were arbitrarily defined as 1.0. Values represent mean ± standard error of the mean.

    Techniques Used: Mouse Assay, Western Blot

    Tau ablation improves hippocampal abnormalities in neuropeptide Y (NPY) and calbindin expression in Scn1a RX/+ mice. Coronal brain sections of 6- to 10-month-old mice (n = 11–14 per genotype) were immunostained for NPY (A–C) or calbindin (D–F). (A) Photomicrographs illustrating NPY alterations in the hippocampus of Scn1a RX/+ mice and improvement of this measure in mice with tau ablation. (B, C) Densitometric quantitation of NPY in the mossy fiber pathway (B) and the molecular layer of the dentate gyrus (C). (D) Photomicrographs illustrating calbindin alterations in the hippocampus of Scn1a RX/+ mice and improvement of this measure in mice with tau ablation. (E, F) Densitometric quantitation of calbindin in the stratum radiatum of hippocampal region CA1 (E) and the molecular layer of the dentate gyrus (F). Interaction between the Scn1a and Tau genotypes by 2-way analysis of variance: (B) p = 4.66E−07, F 1,46 = 34.1; (C) p = 0.00034, F 1,46 = 14.9; (E) p = 0.00039, F 1,46 = 16.3; and (F) p = 0.0014, F 1,46 = 11.6. * p
    Figure Legend Snippet: Tau ablation improves hippocampal abnormalities in neuropeptide Y (NPY) and calbindin expression in Scn1a RX/+ mice. Coronal brain sections of 6- to 10-month-old mice (n = 11–14 per genotype) were immunostained for NPY (A–C) or calbindin (D–F). (A) Photomicrographs illustrating NPY alterations in the hippocampus of Scn1a RX/+ mice and improvement of this measure in mice with tau ablation. (B, C) Densitometric quantitation of NPY in the mossy fiber pathway (B) and the molecular layer of the dentate gyrus (C). (D) Photomicrographs illustrating calbindin alterations in the hippocampus of Scn1a RX/+ mice and improvement of this measure in mice with tau ablation. (E, F) Densitometric quantitation of calbindin in the stratum radiatum of hippocampal region CA1 (E) and the molecular layer of the dentate gyrus (F). Interaction between the Scn1a and Tau genotypes by 2-way analysis of variance: (B) p = 4.66E−07, F 1,46 = 34.1; (C) p = 0.00034, F 1,46 = 14.9; (E) p = 0.00039, F 1,46 = 16.3; and (F) p = 0.0014, F 1,46 = 11.6. * p

    Techniques Used: Expressing, Mouse Assay, Quantitation Assay

    Tau ablation reduces epileptic activity in Scn1a RX/+ mice. (A–C) Subdural electroencephalographic (EEG) recordings were obtained in freely behaving mice of the indicated genotypes at 2 to 3 months of age. For the traces in (A) and (B), the low and high frequency filters were set at 5Hz and 40Hz, respectively. (A) Representative EEG trace depicting a seizure in an Scn1a RX/+ mouse. This seizure (highlighted in gray) lasted 30 seconds and had a severity score of 4 (see Materials and Methods). Scale bars = 3 sec (horizontal), 0.25V (vertical). (B) Representative traces of interictal EEG activity. Note the spikes (arrowheads) in Scn1a RX/+ / Tau +/+ mice. Scale bars = 0.3 sec (horizontal), 0.5V (vertical). (C) Quantitation of epileptic spikes during 1 hour of a 24-hour recording session (n = 6–24 mice per genotype). Linear regression: p = 0.0030, F 1,58 = 11.08 for an interaction between Scn1a and Tau genotypes. Gene–dose effect of Tau deletion in Scn1a RX/+ mice: p = 0.0000054 (Wald test). Exploratory post hoc 1-tailed t tests without multiple comparison correction indicated that Scn1a RX/+ / Tau +/− mice differed from both Scn1a RX/+ / Tau +/+ ( p = 0.0085) and Scn1a +/+ / Tau +/+ mice ( p = 0.0087). *** p
    Figure Legend Snippet: Tau ablation reduces epileptic activity in Scn1a RX/+ mice. (A–C) Subdural electroencephalographic (EEG) recordings were obtained in freely behaving mice of the indicated genotypes at 2 to 3 months of age. For the traces in (A) and (B), the low and high frequency filters were set at 5Hz and 40Hz, respectively. (A) Representative EEG trace depicting a seizure in an Scn1a RX/+ mouse. This seizure (highlighted in gray) lasted 30 seconds and had a severity score of 4 (see Materials and Methods). Scale bars = 3 sec (horizontal), 0.25V (vertical). (B) Representative traces of interictal EEG activity. Note the spikes (arrowheads) in Scn1a RX/+ / Tau +/+ mice. Scale bars = 0.3 sec (horizontal), 0.5V (vertical). (C) Quantitation of epileptic spikes during 1 hour of a 24-hour recording session (n = 6–24 mice per genotype). Linear regression: p = 0.0030, F 1,58 = 11.08 for an interaction between Scn1a and Tau genotypes. Gene–dose effect of Tau deletion in Scn1a RX/+ mice: p = 0.0000054 (Wald test). Exploratory post hoc 1-tailed t tests without multiple comparison correction indicated that Scn1a RX/+ / Tau +/− mice differed from both Scn1a RX/+ / Tau +/+ ( p = 0.0085) and Scn1a +/+ / Tau +/+ mice ( p = 0.0087). *** p

    Techniques Used: Activity Assay, Mouse Assay, Quantitation Assay

    9) Product Images from "Transfer of SCN1A to the brain of adolescent mouse model of Dravet syndrome improves epileptic, motor, and behavioral manifestations"

    Article Title: Transfer of SCN1A to the brain of adolescent mouse model of Dravet syndrome improves epileptic, motor, and behavioral manifestations

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.08.003

    Functional validation of the SCN1A transgene (A) Schematic representation of the HCA-CAG-SCN1A and HCA-EF-SCN1A-GFP genomes. (B) Representative sodium current traces from HEK-293 cells infected with HCA-EF-SCN1A-GFP before (left) and after addition of the selective Nav1.1 opener Hm1a (50 nM, right). (C) Average current densities (±SD) of cells expressing HCA-EF-SCN1A-GFP (filled symbols, n = 10) and uninfected cells (empty symbols, n = 4). (D) Voltage dependence of activation (right curves: V 1/2 of −27.7 ± 2 mV) and the voltage dependence for steady-state fast inactivation (left curves, V 1/2 of −59.6 ± 2.2 mV).
    Figure Legend Snippet: Functional validation of the SCN1A transgene (A) Schematic representation of the HCA-CAG-SCN1A and HCA-EF-SCN1A-GFP genomes. (B) Representative sodium current traces from HEK-293 cells infected with HCA-EF-SCN1A-GFP before (left) and after addition of the selective Nav1.1 opener Hm1a (50 nM, right). (C) Average current densities (±SD) of cells expressing HCA-EF-SCN1A-GFP (filled symbols, n = 10) and uninfected cells (empty symbols, n = 4). (D) Voltage dependence of activation (right curves: V 1/2 of −27.7 ± 2 mV) and the voltage dependence for steady-state fast inactivation (left curves, V 1/2 of −59.6 ± 2.2 mV).

    Techniques Used: Functional Assay, High Content Screening, Infection, Expressing, Activation Assay

    Intracerebral administration of HCA-CAG-SCN1A improves survival and attenuates sensitivity to hyperthermia-induced seizures in DS mice Five-week-old DS mice received bilateral stereotaxic injections of the vector (2 × 10 7 vg/injection) in basal ganglia and cerebellum (BG/Cb) or basal ganglia, cerebellum, and prefrontal cortex (BG/Cb/pCtx). Control DS mice were left untreated (ut) or were injected with the reporter vector HCA-CAG-Luc. (A) Survival after treatment. p = 0.02 in BG/Cb/pCtx versus ut; p = 0.04 in BG/Cb versus ut, log-rank test. (B) Mice were subjected to controlled hyperthermia 1 month after treatment. The graphs represent the average seizure threshold temperature with indication of individual values (left). Right: the cumulative fraction of mice suffering a generalized tonic-clonic seizure for each body temperature before (pre) and 1 month after treatment (post). More than 80% WT mice experienced no seizures at the maximal temperature tested (42.5°C). ∗∗p
    Figure Legend Snippet: Intracerebral administration of HCA-CAG-SCN1A improves survival and attenuates sensitivity to hyperthermia-induced seizures in DS mice Five-week-old DS mice received bilateral stereotaxic injections of the vector (2 × 10 7 vg/injection) in basal ganglia and cerebellum (BG/Cb) or basal ganglia, cerebellum, and prefrontal cortex (BG/Cb/pCtx). Control DS mice were left untreated (ut) or were injected with the reporter vector HCA-CAG-Luc. (A) Survival after treatment. p = 0.02 in BG/Cb/pCtx versus ut; p = 0.04 in BG/Cb versus ut, log-rank test. (B) Mice were subjected to controlled hyperthermia 1 month after treatment. The graphs represent the average seizure threshold temperature with indication of individual values (left). Right: the cumulative fraction of mice suffering a generalized tonic-clonic seizure for each body temperature before (pre) and 1 month after treatment (post). More than 80% WT mice experienced no seizures at the maximal temperature tested (42.5°C). ∗∗p

    Techniques Used: High Content Screening, Mouse Assay, Plasmid Preparation, Injection

    Intracerebral administration of HCA-CAG-SCN1A in adolescent DS mice improves motor skills and ameliorates some behavioral manifestations but not hyperactivity and learning Five-week-old DS mice received bilateral stereotaxic injections of the HCA-CAG-SCN1A vector (2 × 10 7 vg/injection) in basal ganglia, cerebellum, and prefrontal cortex (BG/Cb/pCtx). Control mice were left untreated (ut) or were injected with the reporter vector HCA-CAG-Luc (Luc). Wild-type littermates (WT) are included as a reference of normal score for each test. Mice were subjected to the following tests: NOR (A); MWM (B); OF (C); marble burying (D); nest building (E); rotarod (F); and clasping (G). Bars represent the average value of each group, and individual scores are indicated by symbols (WT, gray inverted triangles; ut, empty orange diamonds; Luc, red circles; SCN1A, green triangles). In the MWM (B), the visible and invisible platform phases of the test (VP and IP, respectively, mean ±SEM) and the 60-s probe tests (P60) are represented at left, center, and right, respectively. (C) In the OF the mean velocity, time spent in the center of the arena, and number of stereotypies/min are represented at left, center, and right, respectively. ∗p
    Figure Legend Snippet: Intracerebral administration of HCA-CAG-SCN1A in adolescent DS mice improves motor skills and ameliorates some behavioral manifestations but not hyperactivity and learning Five-week-old DS mice received bilateral stereotaxic injections of the HCA-CAG-SCN1A vector (2 × 10 7 vg/injection) in basal ganglia, cerebellum, and prefrontal cortex (BG/Cb/pCtx). Control mice were left untreated (ut) or were injected with the reporter vector HCA-CAG-Luc (Luc). Wild-type littermates (WT) are included as a reference of normal score for each test. Mice were subjected to the following tests: NOR (A); MWM (B); OF (C); marble burying (D); nest building (E); rotarod (F); and clasping (G). Bars represent the average value of each group, and individual scores are indicated by symbols (WT, gray inverted triangles; ut, empty orange diamonds; Luc, red circles; SCN1A, green triangles). In the MWM (B), the visible and invisible platform phases of the test (VP and IP, respectively, mean ±SEM) and the 60-s probe tests (P60) are represented at left, center, and right, respectively. (C) In the OF the mean velocity, time spent in the center of the arena, and number of stereotypies/min are represented at left, center, and right, respectively. ∗p

    Techniques Used: High Content Screening, Mouse Assay, Plasmid Preparation, Injection

    Nav1.1 expression from HC-AdV vectors SH-SY5Y cells and primary mouse neurons were infected with the vectors at the indicated multiplicities of infection (MOIs), and Nav1.1 was detected by qRT-PCR (A) and IF (B). Scale bars: 50 μm. Bars indicate averages for each group, and individual values are represented by small circles. ITR, inverted terminal repeats; pA, polyadenylation signal; Ѱ, packaging signal. ∗p
    Figure Legend Snippet: Nav1.1 expression from HC-AdV vectors SH-SY5Y cells and primary mouse neurons were infected with the vectors at the indicated multiplicities of infection (MOIs), and Nav1.1 was detected by qRT-PCR (A) and IF (B). Scale bars: 50 μm. Bars indicate averages for each group, and individual values are represented by small circles. ITR, inverted terminal repeats; pA, polyadenylation signal; Ѱ, packaging signal. ∗p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    Plasmids encoding the codon-optimized SCN1A cDNA achieve efficient expression of Nav1.1. in HEK-293 cells (A) Schematic representation of the plasmids expressing SCN1A (not drawn to scale). The plasmids were transfected in HEK-293 cells. The pCDNA3 empty plasmid was used as a negative control. Transgene expression was analyzed 48 h after transfection. (B). Quantification of SCN1A mRNA was performed by qRT-PCR. The values correspond to copies of SCN1A mRNA per cell. (C). Detection of Nav1.1 protein by western blot in membrane-enriched protein extracts. GAPDH in total extracts is shown as a housekeeping control. (D). Detection of Nav1.1 by IF. Bars indicate averages for each group, and individual values are represented by small circles. IRES, internal ribosomal entry site; pA, polyadenylation signal. ∗∗p
    Figure Legend Snippet: Plasmids encoding the codon-optimized SCN1A cDNA achieve efficient expression of Nav1.1. in HEK-293 cells (A) Schematic representation of the plasmids expressing SCN1A (not drawn to scale). The plasmids were transfected in HEK-293 cells. The pCDNA3 empty plasmid was used as a negative control. Transgene expression was analyzed 48 h after transfection. (B). Quantification of SCN1A mRNA was performed by qRT-PCR. The values correspond to copies of SCN1A mRNA per cell. (C). Detection of Nav1.1 protein by western blot in membrane-enriched protein extracts. GAPDH in total extracts is shown as a housekeeping control. (D). Detection of Nav1.1 by IF. Bars indicate averages for each group, and individual values are represented by small circles. IRES, internal ribosomal entry site; pA, polyadenylation signal. ∗∗p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Western Blot

    Intracerebral administration of HCA-CAG-SCN1A increases the expression of functional Nav1.1 in DS mice Two groups of 5-week-old DS mice received the vector by stereotaxic injection in basal ganglia at 4.6 × 10 6 or 2 × 10 7 vg/injection (DS+SCN1A (LD) and (HD), respectively). Control mice were injected with saline solution in the same location. Electrodes were placed close to the injection site and in the prefrontal area during the same surgical session. (A) One week later, electrophysiological signals were recorded from awake, freely moving animals. Representative signals from deep and superficial electrodes are shown at top and bottom, respectively. The number of IEDs per min was quantified and is represented at right. ∗p
    Figure Legend Snippet: Intracerebral administration of HCA-CAG-SCN1A increases the expression of functional Nav1.1 in DS mice Two groups of 5-week-old DS mice received the vector by stereotaxic injection in basal ganglia at 4.6 × 10 6 or 2 × 10 7 vg/injection (DS+SCN1A (LD) and (HD), respectively). Control mice were injected with saline solution in the same location. Electrodes were placed close to the injection site and in the prefrontal area during the same surgical session. (A) One week later, electrophysiological signals were recorded from awake, freely moving animals. Representative signals from deep and superficial electrodes are shown at top and bottom, respectively. The number of IEDs per min was quantified and is represented at right. ∗p

    Techniques Used: High Content Screening, Expressing, Functional Assay, Mouse Assay, Plasmid Preparation, Injection

    10) Product Images from "Notoginsenoside R1–Induced Neuronal Repair in Models of Alzheimer Disease Is Associated With an Alteration in Neuronal Hyperexcitability, Which Is Regulated by Nav"

    Article Title: Notoginsenoside R1–Induced Neuronal Repair in Models of Alzheimer Disease Is Associated With an Alteration in Neuronal Hyperexcitability, Which Is Regulated by Nav

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2020.00280

    Effect of R1 on the expression, location, and cleavage status of sodium channel proteins after Aβ1-42 treatment. Representative Western blot (A) and densitometry quantification of the total Nav1.1α (B) , extracellular Nav1.1α (C) , intracellular Nav1.1α (D) , Navβ2 full-length (E) , Navβ2-CTF (F) , and BACE1 (G) expression. Data are shown as mean ± SD ( n = 5). * p
    Figure Legend Snippet: Effect of R1 on the expression, location, and cleavage status of sodium channel proteins after Aβ1-42 treatment. Representative Western blot (A) and densitometry quantification of the total Nav1.1α (B) , extracellular Nav1.1α (C) , intracellular Nav1.1α (D) , Navβ2 full-length (E) , Navβ2-CTF (F) , and BACE1 (G) expression. Data are shown as mean ± SD ( n = 5). * p

    Techniques Used: Expressing, Western Blot

    Notoginsenoside R1 altered the distribution of Nav1.1α and cleavage of Navβ2 in the brains of APP/PS1 mice. Representative expression (A,E) and densitometric quantification of BACE1 (B) , Navβ2 full-length (C) , Navβ2-CTF (D) , total expression of Nav1.1α (F) extracellular Nav1.1α (G) , and intracellular Nav1.1α (H) protein in the cortex and hippocampus lysates from WT mice, APP/PS1 mice, or APP/PS1 + R1 mice, respectively. Data are shown as mean ± SD ( n = 15). * p
    Figure Legend Snippet: Notoginsenoside R1 altered the distribution of Nav1.1α and cleavage of Navβ2 in the brains of APP/PS1 mice. Representative expression (A,E) and densitometric quantification of BACE1 (B) , Navβ2 full-length (C) , Navβ2-CTF (D) , total expression of Nav1.1α (F) extracellular Nav1.1α (G) , and intracellular Nav1.1α (H) protein in the cortex and hippocampus lysates from WT mice, APP/PS1 mice, or APP/PS1 + R1 mice, respectively. Data are shown as mean ± SD ( n = 15). * p

    Techniques Used: Mouse Assay, Expressing

    11) Product Images from "Inhibitory Interneuron Deficit Links Altered Network Activity and Cognitive Dysfunction in Alzheimer Model"

    Article Title: Inhibitory Interneuron Deficit Links Altered Network Activity and Cognitive Dysfunction in Alzheimer Model

    Journal: Cell

    doi: 10.1016/j.cell.2012.02.046

    Increasing Levels of Nav1.1 in hAPPJ20 Mice by Nav1.1-BAC Transgene Overexpression
    Figure Legend Snippet: Increasing Levels of Nav1.1 in hAPPJ20 Mice by Nav1.1-BAC Transgene Overexpression

    Techniques Used: Mouse Assay, BAC Assay, Over Expression

    Reduced Nav1.1 Levels in PV Cells of hAPPJ20 Mice and in AD Brains
    Figure Legend Snippet: Reduced Nav1.1 Levels in PV Cells of hAPPJ20 Mice and in AD Brains

    Techniques Used: Mouse Assay

    Enhancing Nav1.1 Levels Increases Inhibitory Synaptic Currents and Gamma Activity and Reduces Epileptiform Discharges in hAPPJ20 Mice
    Figure Legend Snippet: Enhancing Nav1.1 Levels Increases Inhibitory Synaptic Currents and Gamma Activity and Reduces Epileptiform Discharges in hAPPJ20 Mice

    Techniques Used: Activity Assay, Mouse Assay

    12) Product Images from "Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury"

    Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

    Journal: Acta Pharmaceutica Sinica. B

    doi: 10.1016/j.apsb.2020.05.002

    Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P
    Figure Legend Snippet: Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P

    Techniques Used: Mouse Assay, Expressing, Immunostaining, Staining, Marker, MANN-WHITNEY

    A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.
    Figure Legend Snippet: A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.

    Techniques Used: Binding Assay

    13) Product Images from "Aberrant regulation of a poison exon caused by a non-coding variant in Scn1a-associated epileptic encephalopathy"

    Article Title: Aberrant regulation of a poison exon caused by a non-coding variant in Scn1a-associated epileptic encephalopathy

    Journal: bioRxiv

    doi: 10.1101/2020.06.21.163428

    Scn1a mRNA and Na v 1.1 protein levels are reduced in Scn1a+/KI mice. (A) Sanger sequence confirmation of Scn1a+/KI mouse with the gene-edited intron 20 G > C variant. (B) Brain mRNA levels in Scn1a +/+ and Scn1a+/KI mice using qRT-PCR. Relative expression of Scn1a vs. the control gene Tbp ( n = 4, 11.64 ± 2.90 months, Student’s unpaired t -test, p = 0.0251). Scn1a+/KI mice have ~50% less Scn1a mRNA than Scn1a +/+ mice. ( C) RNA-seq counts (normalized to sequencing library size by DEseq2) of Scn1a mRNA in whole brains of Scn1a +/+ and Scn1a+/KI mice (n = 4, 11.64 ± 2.90 months, Student’s unpaired t-test, p = 0.0541). Scn1a+/KI mice have about 42% less Scn1a mRNA than Scn1a +/+ mice. (D) Levels of Na v 1.1, the sodium channel encoded by Scn1a , are reduced in frontal cortex of Scn1a+/KI vs. Scn1a +/+ mice using rabbit anti-Na v 1.1 antibody from Alomone Labs, which recognizes an N-terminal epitope. GAPDH served as a loading control. (E) Quantification of Na v 1.1 levels from the blot in D ( n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.0142). (F) Quantification GAPDH protein levels from the blot in D ( n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.4459). (G) Na v 1.1 levels using anti-Na v 1.1 Antibodies Incorporated antibody, which recognizes a C-terminal epitope. Actin served as a loading control. (H) Quantification of Na v 1.1 protein levels from the blot in G (n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.0059). (I) Quantification of actin protein levels from the blot in E (n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.9859, respectively). * p
    Figure Legend Snippet: Scn1a mRNA and Na v 1.1 protein levels are reduced in Scn1a+/KI mice. (A) Sanger sequence confirmation of Scn1a+/KI mouse with the gene-edited intron 20 G > C variant. (B) Brain mRNA levels in Scn1a +/+ and Scn1a+/KI mice using qRT-PCR. Relative expression of Scn1a vs. the control gene Tbp ( n = 4, 11.64 ± 2.90 months, Student’s unpaired t -test, p = 0.0251). Scn1a+/KI mice have ~50% less Scn1a mRNA than Scn1a +/+ mice. ( C) RNA-seq counts (normalized to sequencing library size by DEseq2) of Scn1a mRNA in whole brains of Scn1a +/+ and Scn1a+/KI mice (n = 4, 11.64 ± 2.90 months, Student’s unpaired t-test, p = 0.0541). Scn1a+/KI mice have about 42% less Scn1a mRNA than Scn1a +/+ mice. (D) Levels of Na v 1.1, the sodium channel encoded by Scn1a , are reduced in frontal cortex of Scn1a+/KI vs. Scn1a +/+ mice using rabbit anti-Na v 1.1 antibody from Alomone Labs, which recognizes an N-terminal epitope. GAPDH served as a loading control. (E) Quantification of Na v 1.1 levels from the blot in D ( n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.0142). (F) Quantification GAPDH protein levels from the blot in D ( n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.4459). (G) Na v 1.1 levels using anti-Na v 1.1 Antibodies Incorporated antibody, which recognizes a C-terminal epitope. Actin served as a loading control. (H) Quantification of Na v 1.1 protein levels from the blot in G (n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.0059). (I) Quantification of actin protein levels from the blot in E (n = 2–3, 17.7 ± 0.96 months, Student’s unpaired t-test, p = 0.9859, respectively). * p

    Techniques Used: Mouse Assay, Sequencing, Variant Assay, Quantitative RT-PCR, Expressing, RNA Sequencing Assay

    Multiple alignment of organisms with conservation in the SCN1A 20N region from the Multiz Alignment of 100 Vertebrates track from the UCSC Genome Browser.
    Figure Legend Snippet: Multiple alignment of organisms with conservation in the SCN1A 20N region from the Multiz Alignment of 100 Vertebrates track from the UCSC Genome Browser.

    Techniques Used:

    Scn1a+/KI mice exhibit premature mortality and a hyperactivity phenotype. (A) Kaplan-Meier analysis showed severe premature mortality in Scn1a+/KI mice ( n = 22–93, Log-rank (Mantel-Cox) test, p
    Figure Legend Snippet: Scn1a+/KI mice exhibit premature mortality and a hyperactivity phenotype. (A) Kaplan-Meier analysis showed severe premature mortality in Scn1a+/KI mice ( n = 22–93, Log-rank (Mantel-Cox) test, p

    Techniques Used: Mouse Assay

    Full length Western blots of protein levels in Scn1a +/KI and Scn1a +/+ mouse brains. (A) Brain (frontal lobe) Scn1a protein levels in Scn1a+/KI vs. Scn1a +/+ mice using rabbit anti-Na v 1.1 (Scn1a) antibody from Alomone Labs. (B) GAPDH using anti-GAPDH antibody from Millipore was used as loading control. (C) Scn1a protein levels using anti-Na v 1.1 (SCN1A) UC-Davis antibody. (D) Actin using anti-Actin antibodies from Cell Signaling was used as loading control. (E) RNA-seq counts of Gapdh mRNA from DEseq2 analysis in whole brains of Scn1a+/ KI and Scn1a Scn1a +/+ mice (n = 4, 11.64 ± 2.90 months, Student’s unpaired t-test, p = 0.67).
    Figure Legend Snippet: Full length Western blots of protein levels in Scn1a +/KI and Scn1a +/+ mouse brains. (A) Brain (frontal lobe) Scn1a protein levels in Scn1a+/KI vs. Scn1a +/+ mice using rabbit anti-Na v 1.1 (Scn1a) antibody from Alomone Labs. (B) GAPDH using anti-GAPDH antibody from Millipore was used as loading control. (C) Scn1a protein levels using anti-Na v 1.1 (SCN1A) UC-Davis antibody. (D) Actin using anti-Actin antibodies from Cell Signaling was used as loading control. (E) RNA-seq counts of Gapdh mRNA from DEseq2 analysis in whole brains of Scn1a+/ KI and Scn1a Scn1a +/+ mice (n = 4, 11.64 ± 2.90 months, Student’s unpaired t-test, p = 0.67).

    Techniques Used: Western Blot, Mouse Assay, RNA Sequencing Assay

    Increased inclusion of Exon 20N in Scn1a+/KI brains. (A) The positions of qPCR amplicons to quantify Scn1a mRNA transcripts. Amplicon 1 detects two isoforms (56bp and 120bp) of the Scn1a transcript, with the longer isoform reflecting exon 20N inclusion. Amplicon 2 quantifies only the Exon 20N-containing transcript. Amplicon 3 quantifies the total Scn1a mRNA levels including the transcript with Exon 20N. (B) Bioanalyzer evaluation of RNA from Scn1a +/+ mouse brain amplified with amplicon 1, showing a single Scn1a peak at 56 bp. The peaks at 15-bp and 1500-bp are size markers recommended and supplied by the manufacturer. ( C) Bioanalyzer evaluation of RNA from Scn1a+/KI mouse brain amplified with amplicon 1, showing a second peak at 120 bp, representing inclusion of exon 20N. The 120-bp amplicon containing the 64-bp exon 20N is denoted with a red asterisk. (D) Scn1a+/KI mice had increased levels of the exon 20N–containing Scn1a transcript, measured using amplicon 2. The levels of exon 20N transcript (amplicon 2) expressed as a percentage of the total Scn1a levels (amplicon 3) using the formula (amplicon 2 levels)/(amplicon 3 levels)*100. (n = 4, 11.64 ± 2.90 months, Student’s unpaired t-test, p = 2e-4). ** p
    Figure Legend Snippet: Increased inclusion of Exon 20N in Scn1a+/KI brains. (A) The positions of qPCR amplicons to quantify Scn1a mRNA transcripts. Amplicon 1 detects two isoforms (56bp and 120bp) of the Scn1a transcript, with the longer isoform reflecting exon 20N inclusion. Amplicon 2 quantifies only the Exon 20N-containing transcript. Amplicon 3 quantifies the total Scn1a mRNA levels including the transcript with Exon 20N. (B) Bioanalyzer evaluation of RNA from Scn1a +/+ mouse brain amplified with amplicon 1, showing a single Scn1a peak at 56 bp. The peaks at 15-bp and 1500-bp are size markers recommended and supplied by the manufacturer. ( C) Bioanalyzer evaluation of RNA from Scn1a+/KI mouse brain amplified with amplicon 1, showing a second peak at 120 bp, representing inclusion of exon 20N. The 120-bp amplicon containing the 64-bp exon 20N is denoted with a red asterisk. (D) Scn1a+/KI mice had increased levels of the exon 20N–containing Scn1a transcript, measured using amplicon 2. The levels of exon 20N transcript (amplicon 2) expressed as a percentage of the total Scn1a levels (amplicon 3) using the formula (amplicon 2 levels)/(amplicon 3 levels)*100. (n = 4, 11.64 ± 2.90 months, Student’s unpaired t-test, p = 2e-4). ** p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Mouse Assay

    The non-coding Dravet Syndrome–causing variant, NM_006920.4(SCN1A):c.3969+2451G > C, is present in a highly conserved region. (A) The alternate exon 20N (shaded rectangle) is highly conserved, with GERP scores that are comparable to canonical exons in SCN1A . (B) Multiple alignment in the 64bp SCN1A 20N region of human, mouse, opossum, alligator, and duck, modified from the Multiz Alignment of 100 Vertebrates track from the UCSC Genome Browser ( SFig.1 ). The red box indicates the position of the variant NM_006920.4(SCN1A):c.3969+2451G > C in our index patient. The red line indicates the position of the guide RNA used for CRISPR/Cas9 gene editing. (C) Alternative splicing of intron 20 in SCN1A . Inclusion of exon 20N (bottom) results in a frame shift and hence a premature termination codon (PTC) in exon 21. The NM_006920.4(SCN1A):c.3969+2451G > C also results in a Gly-Ala (red) substitution within exon 20N. (D) Exon 20N would be in the intracellular loop connecting the fourth and fifth transmembrane voltage sensing regions of the third SCN1A homologous domain (D3) but brings a premature termination codon (PTC) in frame resulting in nonsense-mediated RNA decay.
    Figure Legend Snippet: The non-coding Dravet Syndrome–causing variant, NM_006920.4(SCN1A):c.3969+2451G > C, is present in a highly conserved region. (A) The alternate exon 20N (shaded rectangle) is highly conserved, with GERP scores that are comparable to canonical exons in SCN1A . (B) Multiple alignment in the 64bp SCN1A 20N region of human, mouse, opossum, alligator, and duck, modified from the Multiz Alignment of 100 Vertebrates track from the UCSC Genome Browser ( SFig.1 ). The red box indicates the position of the variant NM_006920.4(SCN1A):c.3969+2451G > C in our index patient. The red line indicates the position of the guide RNA used for CRISPR/Cas9 gene editing. (C) Alternative splicing of intron 20 in SCN1A . Inclusion of exon 20N (bottom) results in a frame shift and hence a premature termination codon (PTC) in exon 21. The NM_006920.4(SCN1A):c.3969+2451G > C also results in a Gly-Ala (red) substitution within exon 20N. (D) Exon 20N would be in the intracellular loop connecting the fourth and fifth transmembrane voltage sensing regions of the third SCN1A homologous domain (D3) but brings a premature termination codon (PTC) in frame resulting in nonsense-mediated RNA decay.

    Techniques Used: Variant Assay, Modification, CRISPR

    Scn1a+/KI mice have no behavioral changes detected in the elevated plus and Y mazes. (A) Time spent in open arms of the elevated plus maze ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.1475). (B) Time spent in closed arms of the elevated plus maze ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.0958). (C) Total arm entries of the elevated plus maze ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.1577). (D) Correct alternations in the Y maze ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.5888). (E) Total distance travelled during 5 min in the Y maze (n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.1242). All data are expressed as mean +/– SEM.
    Figure Legend Snippet: Scn1a+/KI mice have no behavioral changes detected in the elevated plus and Y mazes. (A) Time spent in open arms of the elevated plus maze ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.1475). (B) Time spent in closed arms of the elevated plus maze ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.0958). (C) Total arm entries of the elevated plus maze ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.1577). (D) Correct alternations in the Y maze ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.5888). (E) Total distance travelled during 5 min in the Y maze (n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p = 0.1242). All data are expressed as mean +/– SEM.

    Techniques Used: Mouse Assay

    Inverse relationship between poison exon usage and expression of multiple sodium channels during mouse brain development. (A) Scn1a transcripts including exon 20N are highly expressed in the developing mouse brain and decrease dramatically after birth (aqua bars), with a corresponding developmental increase in Scn1a expression (blue bars). (B) The poison exon in Scn8a previously described by Plummer et al. (ref 38). Scn1a exon 20N and Scn8a exon 18N are 37.5% identical (57% in human), and the amino acid sequences shown at exon boundaries are identical between the two genes. The amino acid sequences shown are fully identical between mouse and human for both genes. (C) Scn8a transcripts including exon 18N are highly expressed in the developing mouse brain and decrease dramatically after birth (aqua bars), with a corresponding developmental increase in Scn8a expression (blue bars).
    Figure Legend Snippet: Inverse relationship between poison exon usage and expression of multiple sodium channels during mouse brain development. (A) Scn1a transcripts including exon 20N are highly expressed in the developing mouse brain and decrease dramatically after birth (aqua bars), with a corresponding developmental increase in Scn1a expression (blue bars). (B) The poison exon in Scn8a previously described by Plummer et al. (ref 38). Scn1a exon 20N and Scn8a exon 18N are 37.5% identical (57% in human), and the amino acid sequences shown at exon boundaries are identical between the two genes. The amino acid sequences shown are fully identical between mouse and human for both genes. (C) Scn8a transcripts including exon 18N are highly expressed in the developing mouse brain and decrease dramatically after birth (aqua bars), with a corresponding developmental increase in Scn8a expression (blue bars).

    Techniques Used: Expressing

    Scn1a+/KI mice have no social behavior deficits detected. (A) Scn1a+/KI mice win equally to the littermate Scn1a +/+ mice in the social dominance tube test ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p > 0.9999). (B) During habituation, mice of both genotypes had no preference to the side (top or bottom) of the three-chamber box ( n = 6–8, 13.81 ± 0.47 months, two-way RM-ANOVA, interaction p = 0.1484, main effect of side p = 0.04828, main effect of genotype p = 0.4806). (C) During testing, mice of both genotypes had no preference to a Lego block or a stranger mouse (S) as measured by time spent in a specific chamber containing a Lego block or a stranger mouse ( n = 6–8, 13.81 ± 0.47 months, two-way RM-ANOVA, interaction p = 0.08994, main effect of stranger mouse p = 0.1339,main effect of genotype p = 0.2311). (D) Scn1a+/KI mice were not significantly different from Scn1a +/+ litter-mate controls in time spent around a cup containing stranger mouse compared to time spent around a cup containing a Lego object (n = 6-8, 13.81 ± 0.47 months, two-way RM-ANOVA, interaction p = 0.1969, main effect of stranger mouse * p = 0.0167,main effect of genotype p = 0.0867). All data are expressed as mean +/– SEM.
    Figure Legend Snippet: Scn1a+/KI mice have no social behavior deficits detected. (A) Scn1a+/KI mice win equally to the littermate Scn1a +/+ mice in the social dominance tube test ( n = 6–8, 13.81 ± 0.47 months, Student’s unpaired t-test, p > 0.9999). (B) During habituation, mice of both genotypes had no preference to the side (top or bottom) of the three-chamber box ( n = 6–8, 13.81 ± 0.47 months, two-way RM-ANOVA, interaction p = 0.1484, main effect of side p = 0.04828, main effect of genotype p = 0.4806). (C) During testing, mice of both genotypes had no preference to a Lego block or a stranger mouse (S) as measured by time spent in a specific chamber containing a Lego block or a stranger mouse ( n = 6–8, 13.81 ± 0.47 months, two-way RM-ANOVA, interaction p = 0.08994, main effect of stranger mouse p = 0.1339,main effect of genotype p = 0.2311). (D) Scn1a+/KI mice were not significantly different from Scn1a +/+ litter-mate controls in time spent around a cup containing stranger mouse compared to time spent around a cup containing a Lego object (n = 6-8, 13.81 ± 0.47 months, two-way RM-ANOVA, interaction p = 0.1969, main effect of stranger mouse * p = 0.0167,main effect of genotype p = 0.0867). All data are expressed as mean +/– SEM.

    Techniques Used: Mouse Assay, Blocking Assay

    14) Product Images from "Mechanism of Differential Cardiovascular Response to Propofol in Dahl Salt-Sensitive, Brown Norway, and Chromosome 13-Substituted Consomic Rat Strains: Role of Large Conductance Ca2+ and Voltage-Activated Potassium Channels"

    Article Title: Mechanism of Differential Cardiovascular Response to Propofol in Dahl Salt-Sensitive, Brown Norway, and Chromosome 13-Substituted Consomic Rat Strains: Role of Large Conductance Ca2+ and Voltage-Activated Potassium Channels

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.109.154104

    Expression of BK α and β 1 subunits in small mesenteric arteries of SS and BN rats. a, representative images of BK α subunit immunofluorescence staining in SS and BN rats. The expression of α and β 1 subunits was assessed by confocal microscopy using selective polyclonal antibodies and the fluorescence-tagged secondary antibody. b, quantification by densitometry demonstrated that expression of α subunit was significantly greater in SS compared with BN rats. Although β 1 subunit expression also varied between SS and BN rats, the difference was not significant.
    Figure Legend Snippet: Expression of BK α and β 1 subunits in small mesenteric arteries of SS and BN rats. a, representative images of BK α subunit immunofluorescence staining in SS and BN rats. The expression of α and β 1 subunits was assessed by confocal microscopy using selective polyclonal antibodies and the fluorescence-tagged secondary antibody. b, quantification by densitometry demonstrated that expression of α subunit was significantly greater in SS compared with BN rats. Although β 1 subunit expression also varied between SS and BN rats, the difference was not significant.

    Techniques Used: Expressing, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence

    15) Product Images from "Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury"

    Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

    Journal: Acta Pharmaceutica Sinica. B

    doi: 10.1016/j.apsb.2020.05.002

    Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P
    Figure Legend Snippet: Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P

    Techniques Used: Mouse Assay, Expressing, Immunostaining, Staining, Marker, MANN-WHITNEY

    A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.
    Figure Legend Snippet: A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.

    Techniques Used: Binding Assay

    16) Product Images from "Transfer of SCN1A to the brain of adolescent mouse model of Dravet syndrome improves epileptic, motor, and behavioral manifestations"

    Article Title: Transfer of SCN1A to the brain of adolescent mouse model of Dravet syndrome improves epileptic, motor, and behavioral manifestations

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.08.003

    Functional validation of the SCN1A transgene (A) Schematic representation of the HCA-CAG-SCN1A and HCA-EF-SCN1A-GFP genomes. (B) Representative sodium current traces from HEK-293 cells infected with HCA-EF-SCN1A-GFP before (left) and after addition of the selective Nav1.1 opener Hm1a (50 nM, right). (C) Average current densities (±SD) of cells expressing HCA-EF-SCN1A-GFP (filled symbols, n = 10) and uninfected cells (empty symbols, n = 4). (D) Voltage dependence of activation (right curves: V 1/2 of −27.7 ± 2 mV) and the voltage dependence for steady-state fast inactivation (left curves, V 1/2 of −59.6 ± 2.2 mV).
    Figure Legend Snippet: Functional validation of the SCN1A transgene (A) Schematic representation of the HCA-CAG-SCN1A and HCA-EF-SCN1A-GFP genomes. (B) Representative sodium current traces from HEK-293 cells infected with HCA-EF-SCN1A-GFP before (left) and after addition of the selective Nav1.1 opener Hm1a (50 nM, right). (C) Average current densities (±SD) of cells expressing HCA-EF-SCN1A-GFP (filled symbols, n = 10) and uninfected cells (empty symbols, n = 4). (D) Voltage dependence of activation (right curves: V 1/2 of −27.7 ± 2 mV) and the voltage dependence for steady-state fast inactivation (left curves, V 1/2 of −59.6 ± 2.2 mV).

    Techniques Used: Functional Assay, High Content Screening, Infection, Expressing, Activation Assay

    Intracerebral administration of HCA-CAG-SCN1A improves survival and attenuates sensitivity to hyperthermia-induced seizures in DS mice Five-week-old DS mice received bilateral stereotaxic injections of the vector (2 × 10 7 vg/injection) in basal ganglia and cerebellum (BG/Cb) or basal ganglia, cerebellum, and prefrontal cortex (BG/Cb/pCtx). Control DS mice were left untreated (ut) or were injected with the reporter vector HCA-CAG-Luc. (A) Survival after treatment. p = 0.02 in BG/Cb/pCtx versus ut; p = 0.04 in BG/Cb versus ut, log-rank test. (B) Mice were subjected to controlled hyperthermia 1 month after treatment. The graphs represent the average seizure threshold temperature with indication of individual values (left). Right: the cumulative fraction of mice suffering a generalized tonic-clonic seizure for each body temperature before (pre) and 1 month after treatment (post). More than 80% WT mice experienced no seizures at the maximal temperature tested (42.5°C). ∗∗p
    Figure Legend Snippet: Intracerebral administration of HCA-CAG-SCN1A improves survival and attenuates sensitivity to hyperthermia-induced seizures in DS mice Five-week-old DS mice received bilateral stereotaxic injections of the vector (2 × 10 7 vg/injection) in basal ganglia and cerebellum (BG/Cb) or basal ganglia, cerebellum, and prefrontal cortex (BG/Cb/pCtx). Control DS mice were left untreated (ut) or were injected with the reporter vector HCA-CAG-Luc. (A) Survival after treatment. p = 0.02 in BG/Cb/pCtx versus ut; p = 0.04 in BG/Cb versus ut, log-rank test. (B) Mice were subjected to controlled hyperthermia 1 month after treatment. The graphs represent the average seizure threshold temperature with indication of individual values (left). Right: the cumulative fraction of mice suffering a generalized tonic-clonic seizure for each body temperature before (pre) and 1 month after treatment (post). More than 80% WT mice experienced no seizures at the maximal temperature tested (42.5°C). ∗∗p

    Techniques Used: High Content Screening, Mouse Assay, Plasmid Preparation, Injection

    Intracerebral administration of HCA-CAG-SCN1A in adolescent DS mice improves motor skills and ameliorates some behavioral manifestations but not hyperactivity and learning Five-week-old DS mice received bilateral stereotaxic injections of the HCA-CAG-SCN1A vector (2 × 10 7 vg/injection) in basal ganglia, cerebellum, and prefrontal cortex (BG/Cb/pCtx). Control mice were left untreated (ut) or were injected with the reporter vector HCA-CAG-Luc (Luc). Wild-type littermates (WT) are included as a reference of normal score for each test. Mice were subjected to the following tests: NOR (A); MWM (B); OF (C); marble burying (D); nest building (E); rotarod (F); and clasping (G). Bars represent the average value of each group, and individual scores are indicated by symbols (WT, gray inverted triangles; ut, empty orange diamonds; Luc, red circles; SCN1A, green triangles). In the MWM (B), the visible and invisible platform phases of the test (VP and IP, respectively, mean ±SEM) and the 60-s probe tests (P60) are represented at left, center, and right, respectively. (C) In the OF the mean velocity, time spent in the center of the arena, and number of stereotypies/min are represented at left, center, and right, respectively. ∗p
    Figure Legend Snippet: Intracerebral administration of HCA-CAG-SCN1A in adolescent DS mice improves motor skills and ameliorates some behavioral manifestations but not hyperactivity and learning Five-week-old DS mice received bilateral stereotaxic injections of the HCA-CAG-SCN1A vector (2 × 10 7 vg/injection) in basal ganglia, cerebellum, and prefrontal cortex (BG/Cb/pCtx). Control mice were left untreated (ut) or were injected with the reporter vector HCA-CAG-Luc (Luc). Wild-type littermates (WT) are included as a reference of normal score for each test. Mice were subjected to the following tests: NOR (A); MWM (B); OF (C); marble burying (D); nest building (E); rotarod (F); and clasping (G). Bars represent the average value of each group, and individual scores are indicated by symbols (WT, gray inverted triangles; ut, empty orange diamonds; Luc, red circles; SCN1A, green triangles). In the MWM (B), the visible and invisible platform phases of the test (VP and IP, respectively, mean ±SEM) and the 60-s probe tests (P60) are represented at left, center, and right, respectively. (C) In the OF the mean velocity, time spent in the center of the arena, and number of stereotypies/min are represented at left, center, and right, respectively. ∗p

    Techniques Used: High Content Screening, Mouse Assay, Plasmid Preparation, Injection

    Nav1.1 expression from HC-AdV vectors SH-SY5Y cells and primary mouse neurons were infected with the vectors at the indicated multiplicities of infection (MOIs), and Nav1.1 was detected by qRT-PCR (A) and IF (B). Scale bars: 50 μm. Bars indicate averages for each group, and individual values are represented by small circles. ITR, inverted terminal repeats; pA, polyadenylation signal; Ѱ, packaging signal. ∗p
    Figure Legend Snippet: Nav1.1 expression from HC-AdV vectors SH-SY5Y cells and primary mouse neurons were infected with the vectors at the indicated multiplicities of infection (MOIs), and Nav1.1 was detected by qRT-PCR (A) and IF (B). Scale bars: 50 μm. Bars indicate averages for each group, and individual values are represented by small circles. ITR, inverted terminal repeats; pA, polyadenylation signal; Ѱ, packaging signal. ∗p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    Plasmids encoding the codon-optimized SCN1A cDNA achieve efficient expression of Nav1.1. in HEK-293 cells (A) Schematic representation of the plasmids expressing SCN1A (not drawn to scale). The plasmids were transfected in HEK-293 cells. The pCDNA3 empty plasmid was used as a negative control. Transgene expression was analyzed 48 h after transfection. (B). Quantification of SCN1A mRNA was performed by qRT-PCR. The values correspond to copies of SCN1A mRNA per cell. (C). Detection of Nav1.1 protein by western blot in membrane-enriched protein extracts. GAPDH in total extracts is shown as a housekeeping control. (D). Detection of Nav1.1 by IF. Bars indicate averages for each group, and individual values are represented by small circles. IRES, internal ribosomal entry site; pA, polyadenylation signal. ∗∗p
    Figure Legend Snippet: Plasmids encoding the codon-optimized SCN1A cDNA achieve efficient expression of Nav1.1. in HEK-293 cells (A) Schematic representation of the plasmids expressing SCN1A (not drawn to scale). The plasmids were transfected in HEK-293 cells. The pCDNA3 empty plasmid was used as a negative control. Transgene expression was analyzed 48 h after transfection. (B). Quantification of SCN1A mRNA was performed by qRT-PCR. The values correspond to copies of SCN1A mRNA per cell. (C). Detection of Nav1.1 protein by western blot in membrane-enriched protein extracts. GAPDH in total extracts is shown as a housekeeping control. (D). Detection of Nav1.1 by IF. Bars indicate averages for each group, and individual values are represented by small circles. IRES, internal ribosomal entry site; pA, polyadenylation signal. ∗∗p

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Western Blot

    Intracerebral administration of HCA-CAG-SCN1A increases the expression of functional Nav1.1 in DS mice Two groups of 5-week-old DS mice received the vector by stereotaxic injection in basal ganglia at 4.6 × 10 6 or 2 × 10 7 vg/injection (DS+SCN1A (LD) and (HD), respectively). Control mice were injected with saline solution in the same location. Electrodes were placed close to the injection site and in the prefrontal area during the same surgical session. (A) One week later, electrophysiological signals were recorded from awake, freely moving animals. Representative signals from deep and superficial electrodes are shown at top and bottom, respectively. The number of IEDs per min was quantified and is represented at right. ∗p
    Figure Legend Snippet: Intracerebral administration of HCA-CAG-SCN1A increases the expression of functional Nav1.1 in DS mice Two groups of 5-week-old DS mice received the vector by stereotaxic injection in basal ganglia at 4.6 × 10 6 or 2 × 10 7 vg/injection (DS+SCN1A (LD) and (HD), respectively). Control mice were injected with saline solution in the same location. Electrodes were placed close to the injection site and in the prefrontal area during the same surgical session. (A) One week later, electrophysiological signals were recorded from awake, freely moving animals. Representative signals from deep and superficial electrodes are shown at top and bottom, respectively. The number of IEDs per min was quantified and is represented at right. ∗p

    Techniques Used: High Content Screening, Expressing, Functional Assay, Mouse Assay, Plasmid Preparation, Injection

    17) Product Images from "Inhibitory Interneuron Deficit Links Altered Network Activity and Cognitive Dysfunction in Alzheimer Model"

    Article Title: Inhibitory Interneuron Deficit Links Altered Network Activity and Cognitive Dysfunction in Alzheimer Model

    Journal: Cell

    doi: 10.1016/j.cell.2012.02.046

    Increasing Levels of Nav1.1 in hAPPJ20 Mice by Nav1.1-BAC Transgene Overexpression
    Figure Legend Snippet: Increasing Levels of Nav1.1 in hAPPJ20 Mice by Nav1.1-BAC Transgene Overexpression

    Techniques Used: Mouse Assay, BAC Assay, Over Expression

    Reduced Nav1.1 Levels in PV Cells of hAPPJ20 Mice and in AD Brains
    Figure Legend Snippet: Reduced Nav1.1 Levels in PV Cells of hAPPJ20 Mice and in AD Brains

    Techniques Used: Mouse Assay

    Enhancing Nav1.1 Levels Increases Inhibitory Synaptic Currents and Gamma Activity and Reduces Epileptiform Discharges in hAPPJ20 Mice
    Figure Legend Snippet: Enhancing Nav1.1 Levels Increases Inhibitory Synaptic Currents and Gamma Activity and Reduces Epileptiform Discharges in hAPPJ20 Mice

    Techniques Used: Activity Assay, Mouse Assay

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    Alomone Labs anti scn1a nav1 1 antibody
    Cultures from two isogenic pairs contain a similar proportion of GABAergic and glutamatergic neurons. (A) Representative micrographs from cultures of two isogenic pairs of iPSC-derived neurons stained with neuronal subtype markers GABA or CaMKII antibodies to identify GABAergic and glutamatergic neurons at D21-24 post plating. (B) The percentage of GABAergic and glutamatergic neurons (white arrows) in neuronal cultures at D21-24 post plating in each line. There was no significant difference between cell lines (p = 0.1, one-way ANOVA for GABAergic neurons and p = 0.6 Kruskal-Wallis test for glutamatergic neurons). (C) Identification of inhibitory (top) and excitatory (bottom) neurons at D21-24 post plating labeled with plasmids via transient transfection during live recording (white arrows). At least 3 coverslips of 2-3 individual platings were evaluated for each line. (D) Localization of <t>Nav1.1</t> in GABAergic and glutamatergic neurons of the control line (white arrows). Data represented as mean + s.e.m. Ctrl = control, mut ctrl = mutated control, corr pt = corrected patient, and pt = patient. Scale bar represents 50 µ m.
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    Cultures from two isogenic pairs contain a similar proportion of GABAergic and glutamatergic neurons. (A) Representative micrographs from cultures of two isogenic pairs of iPSC-derived neurons stained with neuronal subtype markers GABA or CaMKII antibodies to identify GABAergic and glutamatergic neurons at D21-24 post plating. (B) The percentage of GABAergic and glutamatergic neurons (white arrows) in neuronal cultures at D21-24 post plating in each line. There was no significant difference between cell lines (p = 0.1, one-way ANOVA for GABAergic neurons and p = 0.6 Kruskal-Wallis test for glutamatergic neurons). (C) Identification of inhibitory (top) and excitatory (bottom) neurons at D21-24 post plating labeled with plasmids via transient transfection during live recording (white arrows). At least 3 coverslips of 2-3 individual platings were evaluated for each line. (D) Localization of Nav1.1 in GABAergic and glutamatergic neurons of the control line (white arrows). Data represented as mean + s.e.m. Ctrl = control, mut ctrl = mutated control, corr pt = corrected patient, and pt = patient. Scale bar represents 50 µ m.

    Journal: bioRxiv

    Article Title: Comparisons of dual isogenic human iPSC pairs identify functional alterations directly caused by an epilepsy associated SCN1A mutation

    doi: 10.1101/524835

    Figure Lengend Snippet: Cultures from two isogenic pairs contain a similar proportion of GABAergic and glutamatergic neurons. (A) Representative micrographs from cultures of two isogenic pairs of iPSC-derived neurons stained with neuronal subtype markers GABA or CaMKII antibodies to identify GABAergic and glutamatergic neurons at D21-24 post plating. (B) The percentage of GABAergic and glutamatergic neurons (white arrows) in neuronal cultures at D21-24 post plating in each line. There was no significant difference between cell lines (p = 0.1, one-way ANOVA for GABAergic neurons and p = 0.6 Kruskal-Wallis test for glutamatergic neurons). (C) Identification of inhibitory (top) and excitatory (bottom) neurons at D21-24 post plating labeled with plasmids via transient transfection during live recording (white arrows). At least 3 coverslips of 2-3 individual platings were evaluated for each line. (D) Localization of Nav1.1 in GABAergic and glutamatergic neurons of the control line (white arrows). Data represented as mean + s.e.m. Ctrl = control, mut ctrl = mutated control, corr pt = corrected patient, and pt = patient. Scale bar represents 50 µ m.

    Article Snippet: Expression of Nav1.1 was examined in GABA+ (1:100; Aldrich-Sigma, A0310) and CaMKII-eGFP+ cells by staining with anti-Nav1.1 antibody (1:800; Alomone Labs, ASC-001).

    Techniques: Derivative Assay, Staining, Labeling, Transfection

    Isogenic pairs of iPSCs generated by CRISPR/Cas9 editing. (A) The K1270T mutation is located in the second transmembrane segment of the third homologous domain of Nav1.1 alpha subunit. (B) CRISPR/Cas9 editing was used to generate two isogenic pairs from iPSCs derived from two siblings in the GEFS+ family (control and patient) followed by differentiation into functional neurons. (C) Scheme of CRISPR/Cas9 editing design. Additional silent mutation introduced by ssODN1 resulted in an EcoRV restriction site for clone screening when knocking in the K1270T mutation. (D) Sequencing of two isogenic iPSC pairs confirmed the absence and the presence of the mutation. (E) All four lines had normal karyotypes (46, XY). (F) iPSCs of two isogenic pairs were stained with nuclei marker DAPI and pluripotency markers OCT-3/4, SOX2 and NANOG individually. The percentage of cells positive for each of the pluripotency markers was not significantly different between the 4 lines (p = 0.1, 0.06 and 0.2 for OCT-3/4, SOX2 and NANOG respectively, one-way ANOVA). Scale bar represents 50 µ m. Data represented as mean + s.e.m. Data represent counts from three fields in three coverslips from three platings for each genotype.

    Journal: bioRxiv

    Article Title: Comparisons of dual isogenic human iPSC pairs identify functional alterations directly caused by an epilepsy associated SCN1A mutation

    doi: 10.1101/524835

    Figure Lengend Snippet: Isogenic pairs of iPSCs generated by CRISPR/Cas9 editing. (A) The K1270T mutation is located in the second transmembrane segment of the third homologous domain of Nav1.1 alpha subunit. (B) CRISPR/Cas9 editing was used to generate two isogenic pairs from iPSCs derived from two siblings in the GEFS+ family (control and patient) followed by differentiation into functional neurons. (C) Scheme of CRISPR/Cas9 editing design. Additional silent mutation introduced by ssODN1 resulted in an EcoRV restriction site for clone screening when knocking in the K1270T mutation. (D) Sequencing of two isogenic iPSC pairs confirmed the absence and the presence of the mutation. (E) All four lines had normal karyotypes (46, XY). (F) iPSCs of two isogenic pairs were stained with nuclei marker DAPI and pluripotency markers OCT-3/4, SOX2 and NANOG individually. The percentage of cells positive for each of the pluripotency markers was not significantly different between the 4 lines (p = 0.1, 0.06 and 0.2 for OCT-3/4, SOX2 and NANOG respectively, one-way ANOVA). Scale bar represents 50 µ m. Data represented as mean + s.e.m. Data represent counts from three fields in three coverslips from three platings for each genotype.

    Article Snippet: Expression of Nav1.1 was examined in GABA+ (1:100; Aldrich-Sigma, A0310) and CaMKII-eGFP+ cells by staining with anti-Nav1.1 antibody (1:800; Alomone Labs, ASC-001).

    Techniques: Generated, CRISPR, Mutagenesis, Derivative Assay, Functional Assay, Sequencing, Staining, Marker

    MiR-9 induced the increased of total Nav1.1 and Nav1.2 in primary cultured neonatal rat neurons (NRNs). a , b Effects of miR-9 on total protein levels of endogenous Nav1.1 ( a ), Nav1.2 ( b ), in NRNs, using western blot analysis and . Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. * P

    Journal: Molecular Neurodegeneration

    Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

    doi: 10.1186/s13024-015-0032-9

    Figure Lengend Snippet: MiR-9 induced the increased of total Nav1.1 and Nav1.2 in primary cultured neonatal rat neurons (NRNs). a , b Effects of miR-9 on total protein levels of endogenous Nav1.1 ( a ), Nav1.2 ( b ), in NRNs, using western blot analysis and . Cells were transfected with miR-9, AMO-9, miR-9 + AMO-9, or NC. mean ± s.e.m from 3 batches of cells for each group. * P

    Article Snippet: After blocking, cultured neonatal rat neurons were incubated with the anti-β-Tubulin III (neuronal) antibody (Cat no. T8578; 1:5000; Sigma, Saint Louis, USA) or anti- Nav1.1, Nav1.2, Navβ2 antibodies (Alomone Labs, Jerusalem, Israel) overnight at 4 °C, and then the cultured neonatal rat neurons were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature.

    Techniques: Cell Culture, Western Blot, Transfection

    MiR-9 produces the disturbance of trafficking, cellular distribution of Nav1.1 and Nav1.2 in rats. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. Rats were transfected with lenti-pre- miR-9, lenti-pre-miR-9 + lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P

    Journal: Molecular Neurodegeneration

    Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

    doi: 10.1186/s13024-015-0032-9

    Figure Lengend Snippet: MiR-9 produces the disturbance of trafficking, cellular distribution of Nav1.1 and Nav1.2 in rats. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. Rats were transfected with lenti-pre- miR-9, lenti-pre-miR-9 + lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P

    Article Snippet: After blocking, cultured neonatal rat neurons were incubated with the anti-β-Tubulin III (neuronal) antibody (Cat no. T8578; 1:5000; Sigma, Saint Louis, USA) or anti- Nav1.1, Nav1.2, Navβ2 antibodies (Alomone Labs, Jerusalem, Israel) overnight at 4 °C, and then the cultured neonatal rat neurons were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature.

    Techniques: Injection, Quantitative RT-PCR, Transfection

    AMO-miR-9 prevented the disturbed trafficking of Nav1.1/Nav1.2 induced by 2VO. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. 2VO rats were transfected with lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P

    Journal: Molecular Neurodegeneration

    Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

    doi: 10.1186/s13024-015-0032-9

    Figure Lengend Snippet: AMO-miR-9 prevented the disturbed trafficking of Nav1.1/Nav1.2 induced by 2VO. a , Detection of miR-9 in hippocampi and cortices tissues after stereotaxic injection 8 weeks using qRT-PCR. 2VO rats were transfected with lenti-pre-AMO - miR-9, or NC. Data was shown by mean ± s.e.m from 6 rats for each group. * P

    Article Snippet: After blocking, cultured neonatal rat neurons were incubated with the anti-β-Tubulin III (neuronal) antibody (Cat no. T8578; 1:5000; Sigma, Saint Louis, USA) or anti- Nav1.1, Nav1.2, Navβ2 antibodies (Alomone Labs, Jerusalem, Israel) overnight at 4 °C, and then the cultured neonatal rat neurons were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature.

    Techniques: Injection, Quantitative RT-PCR, Transfection

    Nav1.1 and Nav1.2 trafficking were disturbed in hippocampi and cortices after chronic brain hypoperfusion (CBH). a , western-blot analysis of the surface protein levels of Nav1.1 and Nav1.2 in sham and 2VO rats, upper: representative immunoblots of Nav1.1 and Nav1.2; lower: the quantitative analysis data of the immunoblots. The optical density was evaluated for each band and values for 2VO rat tissue were normalized to sham group after correction for protein loading with TfR,** P

    Journal: Molecular Neurodegeneration

    Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

    doi: 10.1186/s13024-015-0032-9

    Figure Lengend Snippet: Nav1.1 and Nav1.2 trafficking were disturbed in hippocampi and cortices after chronic brain hypoperfusion (CBH). a , western-blot analysis of the surface protein levels of Nav1.1 and Nav1.2 in sham and 2VO rats, upper: representative immunoblots of Nav1.1 and Nav1.2; lower: the quantitative analysis data of the immunoblots. The optical density was evaluated for each band and values for 2VO rat tissue were normalized to sham group after correction for protein loading with TfR,** P

    Article Snippet: After blocking, cultured neonatal rat neurons were incubated with the anti-β-Tubulin III (neuronal) antibody (Cat no. T8578; 1:5000; Sigma, Saint Louis, USA) or anti- Nav1.1, Nav1.2, Navβ2 antibodies (Alomone Labs, Jerusalem, Israel) overnight at 4 °C, and then the cultured neonatal rat neurons were washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature.

    Techniques: Western Blot

    Inhibition of Na v currents in GABAergic interneurons by NRG1-ICD. a Increased rheobase and depolarized action potential threshold (APT) in the FS-GABAergic interneurons of gto Nrg1 ; Gfp mice, compared with gtoGfp mice. * P = 0.0321, ** P = 0.0015, two-sided t test, n = 17 cells from five gtoGfp mice, n = 21 cells from four gto Nrg1 ; Gfp mice. b Representative traces of a single AP evoked from a suprathreshold current injection (left) and corresponding phase plots (dV/dt vs voltage) (right) recorded in FS-GABAergic interneurons from gtoGfp and gto Nrg1 ; Gfp mice. c Reduced Na + current density in GABAergic interneurons from gto Nrg1 ; Gfp PFC. Representative current traces of Na v channels in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. Currents were elicited by step depolarizations from −80 to +30 mV in 5 mV increments from a holding potential of −80 mV. The tracts shown are for depolarizations from −80 to +20 mV. d I/V curves of Na v channel activation in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. **Genotype F (1, 28) = 8.264, P = 0.0076, two-way ANOVA, n = 13 cells from three gto Nrg1 ; Gfp mice, n = 17 cells from four gtoGfp mice. Data are presented as mean values + /− SEM. e Similar voltage-dependent activation curves of Na v channels in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. Genotype F (1, 28) = 0.023, P = 0.879, two-way ANOVA, n = 13 cells from three gto Nrg1 ; Gfp mice, n = 17 cells from four gtoGfp mice. Data are presented as mean values + /− SEM. f Transcriptional levels of Scn1a is higher than Scn2a1, Scn3a, and Scn8a in PV and SST-positive GABAergic interneurons ( n = 2688 cells). Shown are transcripts of Scn genes per 100 k total transcripts from single-cell RNA sequencing. g Transcriptional levels of Scn1a were lower than Scn2a1 and Scn8a in layer 2/3 PN ( n = 41827 cells). Shown are transcripts of Scn genes per 100 k total transcripts from single-cell RNA sequencing. h Diagram showing the structure of SCN1A. The SCN1A protein was composed of four transmembrane domains (I–IV) and two major cytoplasmic loops (CL1 and CL2). The NRG1-ICD could interact with the CL1 of SCN1A. i Coomassie blue staining of 30 μg GST and GST-NRG1-ICD proteins. Asterisks indicated degradation product of GST-NRG1-ICD proteins. Four independent experiments were repeated to get similar results. j Interaction of NRG1-ICD with His-CL1. The recombinant GST-NRG1-ICD and His-CL1, or His-CL2 proteins were used for GST pulldown experiments. Four independent experiments were repeated to get similar results. k Diagram showing delivery of GST-NRG1-ICD or GST proteins into GABAergic interneurons in gtoGfp slices. l Reduced Na + current density in GABAergic interneurons treated with GST-NRG1-ICD. Representative current traces of Na v channels in GABAergic interneurons treated with recombinant GST or GST-NRG1-ICD proteins. Currents were elicited by step depolarizations from −80 to +30 mV in 5 mV increments from a holding potential of −80 mV. The tracts shown are for depolarizations from −80 to +20 mV. m I/V curves of Na v channel activation in GABAergic interneurons treated with recombinant GST or GST-NRG1-ICD proteins. **Treatment F (1, 24) = 10.89, P = 0.003, two-way ANOVA, n = 14 cells treated with GST-NRG1-ICD, n = 12 cells treated with GST. Data are presented as mean values + /− SEM. n Similar voltage-dependent activation curves of Na v channels in GABAergic interneurons treated with GST or GST-NRG1-ICD. Genotype F (1, 24) = 0.059, P = 0.81, two-way ANOVA, n = 14 cells treated with GST-NRG1-ICD, n = 12 cells treated with GST. Data are presented as mean values + /− SEM.

    Journal: Nature Communications

    Article Title: Overexpression of neuregulin 1 in GABAergic interneurons results in reversible cortical disinhibition

    doi: 10.1038/s41467-020-20552-y

    Figure Lengend Snippet: Inhibition of Na v currents in GABAergic interneurons by NRG1-ICD. a Increased rheobase and depolarized action potential threshold (APT) in the FS-GABAergic interneurons of gto Nrg1 ; Gfp mice, compared with gtoGfp mice. * P = 0.0321, ** P = 0.0015, two-sided t test, n = 17 cells from five gtoGfp mice, n = 21 cells from four gto Nrg1 ; Gfp mice. b Representative traces of a single AP evoked from a suprathreshold current injection (left) and corresponding phase plots (dV/dt vs voltage) (right) recorded in FS-GABAergic interneurons from gtoGfp and gto Nrg1 ; Gfp mice. c Reduced Na + current density in GABAergic interneurons from gto Nrg1 ; Gfp PFC. Representative current traces of Na v channels in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. Currents were elicited by step depolarizations from −80 to +30 mV in 5 mV increments from a holding potential of −80 mV. The tracts shown are for depolarizations from −80 to +20 mV. d I/V curves of Na v channel activation in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. **Genotype F (1, 28) = 8.264, P = 0.0076, two-way ANOVA, n = 13 cells from three gto Nrg1 ; Gfp mice, n = 17 cells from four gtoGfp mice. Data are presented as mean values + /− SEM. e Similar voltage-dependent activation curves of Na v channels in GABAergic interneurons from gto Nrg1 ; Gfp and gtoGfp mice. Genotype F (1, 28) = 0.023, P = 0.879, two-way ANOVA, n = 13 cells from three gto Nrg1 ; Gfp mice, n = 17 cells from four gtoGfp mice. Data are presented as mean values + /− SEM. f Transcriptional levels of Scn1a is higher than Scn2a1, Scn3a, and Scn8a in PV and SST-positive GABAergic interneurons ( n = 2688 cells). Shown are transcripts of Scn genes per 100 k total transcripts from single-cell RNA sequencing. g Transcriptional levels of Scn1a were lower than Scn2a1 and Scn8a in layer 2/3 PN ( n = 41827 cells). Shown are transcripts of Scn genes per 100 k total transcripts from single-cell RNA sequencing. h Diagram showing the structure of SCN1A. The SCN1A protein was composed of four transmembrane domains (I–IV) and two major cytoplasmic loops (CL1 and CL2). The NRG1-ICD could interact with the CL1 of SCN1A. i Coomassie blue staining of 30 μg GST and GST-NRG1-ICD proteins. Asterisks indicated degradation product of GST-NRG1-ICD proteins. Four independent experiments were repeated to get similar results. j Interaction of NRG1-ICD with His-CL1. The recombinant GST-NRG1-ICD and His-CL1, or His-CL2 proteins were used for GST pulldown experiments. Four independent experiments were repeated to get similar results. k Diagram showing delivery of GST-NRG1-ICD or GST proteins into GABAergic interneurons in gtoGfp slices. l Reduced Na + current density in GABAergic interneurons treated with GST-NRG1-ICD. Representative current traces of Na v channels in GABAergic interneurons treated with recombinant GST or GST-NRG1-ICD proteins. Currents were elicited by step depolarizations from −80 to +30 mV in 5 mV increments from a holding potential of −80 mV. The tracts shown are for depolarizations from −80 to +20 mV. m I/V curves of Na v channel activation in GABAergic interneurons treated with recombinant GST or GST-NRG1-ICD proteins. **Treatment F (1, 24) = 10.89, P = 0.003, two-way ANOVA, n = 14 cells treated with GST-NRG1-ICD, n = 12 cells treated with GST. Data are presented as mean values + /− SEM. n Similar voltage-dependent activation curves of Na v channels in GABAergic interneurons treated with GST or GST-NRG1-ICD. Genotype F (1, 24) = 0.059, P = 0.81, two-way ANOVA, n = 14 cells treated with GST-NRG1-ICD, n = 12 cells treated with GST. Data are presented as mean values + /− SEM.

    Article Snippet: The following antibodies were used: rabbit anti-NRG1 (1:1000, Santa Cruz, sc-348), mouse anti-GAPDH (1:5000, Abways, ab0037), mouse anti-PSD95 (1:1000, Millipore, 2492127), rabbit anti-ErbB4 (1:1000, Cell Signaling, 111B2), rabbit anti-p-ErbB4 (1:200, Cell Signaling, Y1248) and rabbit anti-Nav1.1 (1:500, Alomone Labs, ASC-001), mouse anti-GST (1:5000, ImmunoWay, B2101) and mouse anti-His (1:2000, ImmunoWay, B0401).

    Techniques: Inhibition, Mouse Assay, Injection, Activation Assay, RNA Sequencing Assay, Staining, Recombinant

    Decreased VGNC expression and increased PKC and ERK activity in Tm4sf2 −/y mice. A) Western blot of crude membrane preparation from Tm4sf2+/y and Tm4sf2−/y mice habenulae detected using ChemiDoc XRS+ System (BioRad) and quantification of voltage-gated sodium and potassium channel protein levels normalized on tubulin. You can see a downward trend and a significant reduced expression for NaV1.1 and NaV1.6 respectively in Tm4sf2−/y mice, while no changes for Kv4.2 has been detected; B) Quantification of PKC brain activity obtained using a PKC Kinase Activity Assay Kit showing a hyperactivity in Tm4sf2−/y compared to Tm4sf2+/y mice; C) Western blot of habenulae homogenates from Tm4sf2+/y and Tm4sf2−/y mice and quantification of expression levels represented as pERK/ERK ratio. α-tubulin was used as loading control.

    Journal: Neurobiology of Disease

    Article Title: Lateral habenula dysfunctions in Tm4sf2−/y mice model for neurodevelopmental disorder

    doi: 10.1016/j.nbd.2020.105189

    Figure Lengend Snippet: Decreased VGNC expression and increased PKC and ERK activity in Tm4sf2 −/y mice. A) Western blot of crude membrane preparation from Tm4sf2+/y and Tm4sf2−/y mice habenulae detected using ChemiDoc XRS+ System (BioRad) and quantification of voltage-gated sodium and potassium channel protein levels normalized on tubulin. You can see a downward trend and a significant reduced expression for NaV1.1 and NaV1.6 respectively in Tm4sf2−/y mice, while no changes for Kv4.2 has been detected; B) Quantification of PKC brain activity obtained using a PKC Kinase Activity Assay Kit showing a hyperactivity in Tm4sf2−/y compared to Tm4sf2+/y mice; C) Western blot of habenulae homogenates from Tm4sf2+/y and Tm4sf2−/y mice and quantification of expression levels represented as pERK/ERK ratio. α-tubulin was used as loading control.

    Article Snippet: The following primary antibodies were used: NaV1.1 (Alomone Labs, 1:200), NaV1.6 (Sigma Aldrich, 1:200), Kv4.2 (Sigma Aldrich, 1:200), α-tubulin (Sigma Aldrich, 1:40000), α-ERK (Cell Signaling, 1:1000), α-pERK (Cell Signaling, 1:1000).

    Techniques: Expressing, Activity Assay, Mouse Assay, Western Blot, Kinase Assay