nav1 2 polyclonal  (Alomone Labs)


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    Structured Review

    Alomone Labs nav1 2 polyclonal
    Differential expression of Nav1.6 and <t>Nav1.2</t> at developing CG cell AISs
    Nav1 2 Polyclonal, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nav1 2 polyclonal/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nav1 2 polyclonal - by Bioz Stars, 2022-05
    94/100 stars

    Images

    1) Product Images from "Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells"

    Article Title: Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2010.183798

    Differential expression of Nav1.6 and Nav1.2 at developing CG cell AISs
    Figure Legend Snippet: Differential expression of Nav1.6 and Nav1.2 at developing CG cell AISs

    Techniques Used: Expressing

    Developmental regulation of Nav1.2 and Nav1.6 clustering at CG cell AISs in mice aged P10–P60
    Figure Legend Snippet: Developmental regulation of Nav1.2 and Nav1.6 clustering at CG cell AISs in mice aged P10–P60

    Techniques Used: Mouse Assay

    2) Product Images from "Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice"

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0099-2

    Altered action potentials of Scn2a KO excitatory neurons. Responses of neocortical pyramidal excitatory neurons and fast-spiking inhibitory neurons from Vgat -Venus/ Scn2a +/+ (WT) mice ( n = 19 pyramidal and 8 fast-spiking neurons, P7–8; n = 22 pyramidal and 11 fast-spiking neurons, P15–22) and Vgat -Venus/ Scn2a KO/+ (KO/ + ) mice ( n = 16 pyramidal and 11 fast-spiking neurons, P7–8; n = 22 pyramidal and 13 fast-spiking neurons, P15–22) to current injections. Representative action potential traces ( a , e , h , k ), peak amplitudes ( b , f , i , l ), half widths ( c , g , j , m ) and maximum rates of rise ( d ) were shown. b – g Peak amplitudes of P7–8 pyramidal cells are smaller in Scn2a KO/+ than in WT mice, while half widths of P7–8 and P15–22 pyramidal cells were broader in Scn2a KO/+ than in WT mice. Unpaired t -test, peak amplitudes, P7–8, t (33) = 6.0638, *** P = 0.0000008, P15–22, t (42) = 0.0791, P = 0.9373; half widths, P7–8, t (33) = −4.0342, *** P = 0.00031, P15–22, t (42) = −3.6442, * P = 0.0109. d Maximum rates of rise for action potential of P7–8 hippocampal pyramidal cells ( n = 19 WT neurons, n = 11 KO/ + neurons) were lower in Scn2a KO/+ than in WT mice. Unpaired t -test, −120mV, t (28) = 7.3005, *** P = 0.00000006, −110mV, t (28) = 7.3572, *** P = 0.00000005, −100mV, t (28) = 7.3237, *** P = 0.00000006, −90mV, t (28) = 7.4814, *** P = 0.00000004, −80mV, t (28) = 7.6586, *** P = 0.00000002, −70mV, t (28) = 7.2738, *** P = 0.00000006, −60mV, t (28) = 7.8954, *** P = 0.00000001, −50mV, t (27) = 7.1199, *** P = 0.00000012. There were no significant differences between WT and Scn2a KO/+ fast-spiking neurons. Unpaired t -test, peak amplitudes, P7–8, t (17) = 0.8793, P = 0.3915, P15–22, t (22) = 2.0676, P = 0.0506; half widths, P7–8, t (17) = 0.0435, P = 0.9658, P15–22, t (22) = −0.8992, P = 0.3783. Details of the results and statistical tests are reported in Supplementary Tables 1 and 2 . Black filled circles and gray filled circles represent WT and Scn2a KO/+ neurons, respectively. Data represent means ± SEM, * P
    Figure Legend Snippet: Altered action potentials of Scn2a KO excitatory neurons. Responses of neocortical pyramidal excitatory neurons and fast-spiking inhibitory neurons from Vgat -Venus/ Scn2a +/+ (WT) mice ( n = 19 pyramidal and 8 fast-spiking neurons, P7–8; n = 22 pyramidal and 11 fast-spiking neurons, P15–22) and Vgat -Venus/ Scn2a KO/+ (KO/ + ) mice ( n = 16 pyramidal and 11 fast-spiking neurons, P7–8; n = 22 pyramidal and 13 fast-spiking neurons, P15–22) to current injections. Representative action potential traces ( a , e , h , k ), peak amplitudes ( b , f , i , l ), half widths ( c , g , j , m ) and maximum rates of rise ( d ) were shown. b – g Peak amplitudes of P7–8 pyramidal cells are smaller in Scn2a KO/+ than in WT mice, while half widths of P7–8 and P15–22 pyramidal cells were broader in Scn2a KO/+ than in WT mice. Unpaired t -test, peak amplitudes, P7–8, t (33) = 6.0638, *** P = 0.0000008, P15–22, t (42) = 0.0791, P = 0.9373; half widths, P7–8, t (33) = −4.0342, *** P = 0.00031, P15–22, t (42) = −3.6442, * P = 0.0109. d Maximum rates of rise for action potential of P7–8 hippocampal pyramidal cells ( n = 19 WT neurons, n = 11 KO/ + neurons) were lower in Scn2a KO/+ than in WT mice. Unpaired t -test, −120mV, t (28) = 7.3005, *** P = 0.00000006, −110mV, t (28) = 7.3572, *** P = 0.00000005, −100mV, t (28) = 7.3237, *** P = 0.00000006, −90mV, t (28) = 7.4814, *** P = 0.00000004, −80mV, t (28) = 7.6586, *** P = 0.00000002, −70mV, t (28) = 7.2738, *** P = 0.00000006, −60mV, t (28) = 7.8954, *** P = 0.00000001, −50mV, t (27) = 7.1199, *** P = 0.00000012. There were no significant differences between WT and Scn2a KO/+ fast-spiking neurons. Unpaired t -test, peak amplitudes, P7–8, t (17) = 0.8793, P = 0.3915, P15–22, t (22) = 2.0676, P = 0.0506; half widths, P7–8, t (17) = 0.0435, P = 0.9658, P15–22, t (22) = −0.8992, P = 0.3783. Details of the results and statistical tests are reported in Supplementary Tables 1 and 2 . Black filled circles and gray filled circles represent WT and Scn2a KO/+ neurons, respectively. Data represent means ± SEM, * P

    Techniques Used: Mouse Assay

    Scn2a deletion in dorsal telencephalic excitatory but not global inhibitory neurons triggered SWDs in mice. a Survival rates at P2.5 of Scn2a fl/fl ( N = 7), Scn2a fl/fl / Emx1 -Cre ( N = 6) and Scn2a fl/fl / Vgat -Cre mice ( N = 5). All Scn2a fl/fl / Emx1 -Cre and all but one Scn2a fl/fl / Vgat -Cre mice died before P2.5. One Scn2a fl/fl / Vgat -Cre survivor died at P8.5. b Survival curves during P3–30 of Scn2a fl/+ / Emx1 -Cre ( N = 39), Scn2a fl/+ / Vgat -Cre ( N = 30) and Scn2a fl/+ mice ( N = 95). About 30% of Scn2a fl/+ / Vgat -Cre mice suffered premature death between P16 and P25. c Representative ECoG/EMG traces in Scn2a fl/+ / Emx1 -Cre mice. SWDs during waking were often associated with behavioral arrest (right). Black arrowheads indicate the onsets of SWDs. Gray arrowheads indicate the onset and end of behavioral arrest. d , e Frequencies of SWDs during 24 h ECoG recordings in Scn2a fl/+ / Emx1 -Cre ( N = 5) and littermate controls (Cntl) (2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 5), Scn2a fl/+ / Vgat -Cre ( N = 4) and littermate Cntl (2 Scn2a fl/+ , 1 Scn2a +/+ / Vgat -Cre, N = 3), and Scn2a RX/+ mice ( N = 7). All recorded mice were over 8 weeks of age. f Ethosuximide (33.3 mg mL −1 in saline, 200 mg per kg, i.p .) efficiently suppressed SWDs in Scn2a fl/+ / Emx1 -Cre mice ( N = 6). g , h LFP recordings from 3–6-month-old Scn2a fl/+ / Emx1 -Cre ( N = 4) and littermate Cntl (2 Scn2a +/+ , 2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 7). g Representative ECoG/EMG/LFPs traces in Scn2a fl/+ / Emx1 -Cre mice. h Epileptiform discharges were predominantly detected in medial prefrontal cortex and caudate putamen of Scn2a fl/+ / Emx1 -Cre mice (Mann–Whitney test, ECoG, somatosensory cortex: U = 0, * P = 0.0286; LFP, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 6, P > 0.999; basolateral amygdala: U = 0, * P = 0.0268; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0268; ventroposterior thalamus: U = 0, * P = 0.0286). Data represent means ± SEM, * P
    Figure Legend Snippet: Scn2a deletion in dorsal telencephalic excitatory but not global inhibitory neurons triggered SWDs in mice. a Survival rates at P2.5 of Scn2a fl/fl ( N = 7), Scn2a fl/fl / Emx1 -Cre ( N = 6) and Scn2a fl/fl / Vgat -Cre mice ( N = 5). All Scn2a fl/fl / Emx1 -Cre and all but one Scn2a fl/fl / Vgat -Cre mice died before P2.5. One Scn2a fl/fl / Vgat -Cre survivor died at P8.5. b Survival curves during P3–30 of Scn2a fl/+ / Emx1 -Cre ( N = 39), Scn2a fl/+ / Vgat -Cre ( N = 30) and Scn2a fl/+ mice ( N = 95). About 30% of Scn2a fl/+ / Vgat -Cre mice suffered premature death between P16 and P25. c Representative ECoG/EMG traces in Scn2a fl/+ / Emx1 -Cre mice. SWDs during waking were often associated with behavioral arrest (right). Black arrowheads indicate the onsets of SWDs. Gray arrowheads indicate the onset and end of behavioral arrest. d , e Frequencies of SWDs during 24 h ECoG recordings in Scn2a fl/+ / Emx1 -Cre ( N = 5) and littermate controls (Cntl) (2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 5), Scn2a fl/+ / Vgat -Cre ( N = 4) and littermate Cntl (2 Scn2a fl/+ , 1 Scn2a +/+ / Vgat -Cre, N = 3), and Scn2a RX/+ mice ( N = 7). All recorded mice were over 8 weeks of age. f Ethosuximide (33.3 mg mL −1 in saline, 200 mg per kg, i.p .) efficiently suppressed SWDs in Scn2a fl/+ / Emx1 -Cre mice ( N = 6). g , h LFP recordings from 3–6-month-old Scn2a fl/+ / Emx1 -Cre ( N = 4) and littermate Cntl (2 Scn2a +/+ , 2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 7). g Representative ECoG/EMG/LFPs traces in Scn2a fl/+ / Emx1 -Cre mice. h Epileptiform discharges were predominantly detected in medial prefrontal cortex and caudate putamen of Scn2a fl/+ / Emx1 -Cre mice (Mann–Whitney test, ECoG, somatosensory cortex: U = 0, * P = 0.0286; LFP, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 6, P > 0.999; basolateral amygdala: U = 0, * P = 0.0268; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0268; ventroposterior thalamus: U = 0, * P = 0.0286). Data represent means ± SEM, * P

    Techniques Used: Mouse Assay, MANN-WHITNEY

    Nav1.2 expressions at AISs of excitatory neurons in neocortex and hippocampus. Immunofluorescence histochemistry of P15.5 wild-type neocortices and hippocampi stained with anti-Nav1.2 (G-20, magenta), anti-ankyrin G (green), and anti-Tbr1 (cyan) antibodies, and counterstained with 4′-6-diamidino-2-phenylindole (DAPI, gray) and their merged images. Arrows indicate Nav1.2 and ankyrin G-double immunoreactive AISs of Tbr1-expressing neocortical pyramidal cells. Arrowheads indicate Nav1.2 and ankyrin G-double immunoreactive AISs of hippocampal pyramidal cells. Representative images of four or more slices are shown. o stratum oriens, p stratum pyramidale. Scale bars: 20 µm
    Figure Legend Snippet: Nav1.2 expressions at AISs of excitatory neurons in neocortex and hippocampus. Immunofluorescence histochemistry of P15.5 wild-type neocortices and hippocampi stained with anti-Nav1.2 (G-20, magenta), anti-ankyrin G (green), and anti-Tbr1 (cyan) antibodies, and counterstained with 4′-6-diamidino-2-phenylindole (DAPI, gray) and their merged images. Arrows indicate Nav1.2 and ankyrin G-double immunoreactive AISs of Tbr1-expressing neocortical pyramidal cells. Arrowheads indicate Nav1.2 and ankyrin G-double immunoreactive AISs of hippocampal pyramidal cells. Representative images of four or more slices are shown. o stratum oriens, p stratum pyramidale. Scale bars: 20 µm

    Techniques Used: Immunofluorescence, Staining, Expressing

    Scn2a haplodeficiency in dorsal telencephalic excitatory but in those in global inhibitory neurons reduced neocortical and hippocampal Nav1.2 expression levels. Western blot analyses of 6-weeks neocortex or hippocampus for Scn2a fl/+ / Emx1 -Cre, Scn2a fl/+ / Vgat -Cre and Scn2a fl/+ controls ( N = 3, each genotype). Unpaired t -test, Scn2a fl/+ vs. Scn2a fl/+ / Emx1 -Cre, neocortex: t (4) = 5.91, ** P = 0.0041; hippocampus: t (4) = 5.74, ** P = 0.0046; Scn2a fl/+ vs. Scn2a fl/+ / Vgat -Cre, neocortex: t (4) = 1.413, P = 0.2305; hippocampus: t (4) = 0.489, P = 0.6503. Nav1.2 protein was normalized by β-tubulin. Mean Nav1.2 expression levels are represented as percentages relative to the level of Scn2a fl/+ control littermates (100%). Data represent means ± SEM, ** P
    Figure Legend Snippet: Scn2a haplodeficiency in dorsal telencephalic excitatory but in those in global inhibitory neurons reduced neocortical and hippocampal Nav1.2 expression levels. Western blot analyses of 6-weeks neocortex or hippocampus for Scn2a fl/+ / Emx1 -Cre, Scn2a fl/+ / Vgat -Cre and Scn2a fl/+ controls ( N = 3, each genotype). Unpaired t -test, Scn2a fl/+ vs. Scn2a fl/+ / Emx1 -Cre, neocortex: t (4) = 5.91, ** P = 0.0041; hippocampus: t (4) = 5.74, ** P = 0.0046; Scn2a fl/+ vs. Scn2a fl/+ / Vgat -Cre, neocortex: t (4) = 1.413, P = 0.2305; hippocampus: t (4) = 0.489, P = 0.6503. Nav1.2 protein was normalized by β-tubulin. Mean Nav1.2 expression levels are represented as percentages relative to the level of Scn2a fl/+ control littermates (100%). Data represent means ± SEM, ** P

    Techniques Used: Expressing, Western Blot

    Developmental changes of Nav1.2 distribution in mouse brain. Brain sections of wild-type mice at P0.5 ( a , f , k , p ), P2.5 ( b , g , l , q ), P7.5 ( c , h , m , r ), P15.5 ( d , i , n , s ), and 8-week-old ( e , j , o , t ) were stained with anti-Nav1.2 (G-20, red). Higher-magnified images outlined in a – e are shown in f – t . Nav1.2 immunoreactivities were observed at AISs of neocortical neurons (single black arrowheads), nodes of Ranvier within white matter (double black arrowheads), AISs of hippocampal pyramidal neurons (white arrowheads), mossy fibers of dentate granule cells (black arrows), etc. Note that, while Nav1.2 at AISs and nodes of Ranvier peaked at P15.5 and became less at 8-weeks, diffused Nav1.2 signals in neocortex continued to become dense until 8-weeks-old. The brain slices were processed in parallel. Representative images of four or more slices per stage are shown. MZ marginal zone, UCP upper cortical plate, LCP lower cortical plate, DG dentate gyrus, WM white matter, IZ intermediate zone, o stratum oriens, p stratum pyramidale, l stratum lucidum, r stratum radiatum. Scale bars: a – e 500 µm; f – o 50 µm; p – t 100 µm
    Figure Legend Snippet: Developmental changes of Nav1.2 distribution in mouse brain. Brain sections of wild-type mice at P0.5 ( a , f , k , p ), P2.5 ( b , g , l , q ), P7.5 ( c , h , m , r ), P15.5 ( d , i , n , s ), and 8-week-old ( e , j , o , t ) were stained with anti-Nav1.2 (G-20, red). Higher-magnified images outlined in a – e are shown in f – t . Nav1.2 immunoreactivities were observed at AISs of neocortical neurons (single black arrowheads), nodes of Ranvier within white matter (double black arrowheads), AISs of hippocampal pyramidal neurons (white arrowheads), mossy fibers of dentate granule cells (black arrows), etc. Note that, while Nav1.2 at AISs and nodes of Ranvier peaked at P15.5 and became less at 8-weeks, diffused Nav1.2 signals in neocortex continued to become dense until 8-weeks-old. The brain slices were processed in parallel. Representative images of four or more slices per stage are shown. MZ marginal zone, UCP upper cortical plate, LCP lower cortical plate, DG dentate gyrus, WM white matter, IZ intermediate zone, o stratum oriens, p stratum pyramidale, l stratum lucidum, r stratum radiatum. Scale bars: a – e 500 µm; f – o 50 µm; p – t 100 µm

    Techniques Used: Mouse Assay, Staining

    A pathogenic Scn2a nonsense mutation inactivated the mutated allele. a Schematic of the voltage-gated sodium channel Nav1.2, showing the location of R102* (RX) nonsense mutation. Full-length wild type Nav1.2 is composed of 2006-amino acid (aa) residues with the predicted molecular weight of ~228 kD. The RX mutation can cause a truncated peptides, Nav1.2-RX, consisting of the first 101-aa residues of Nav1.2 with the theoretical molecular weight of ~12 kD. Epitope locations for the anti-Nav1.2 (EM-1, ASC-002) and anti-pan Nav1 (SP19) antibodies are indicated. b , c The RX allele was effectively inactivated in Scn2a knock-in mice. Western blot analyses of P0.5 whole brain membrane ( b ) and cytosolic ( c ) fractions were performed using EM-1. b Full-length Nav1.2 was moderate and negligible in Scn2a RX/+ (RX/+, N = 4) and Scn2a RX/RX (RX/RX, N = 3) mice, respectively, compared with that in Scn2a +/+ mice (+/+, N = 3) [one-way analysis of variance; genotype: F (2, 7) = 1737, *** P = 0.00000000036, Tukey’s test; Scn2a +/+ vs . Scn2a RX/+ *** P
    Figure Legend Snippet: A pathogenic Scn2a nonsense mutation inactivated the mutated allele. a Schematic of the voltage-gated sodium channel Nav1.2, showing the location of R102* (RX) nonsense mutation. Full-length wild type Nav1.2 is composed of 2006-amino acid (aa) residues with the predicted molecular weight of ~228 kD. The RX mutation can cause a truncated peptides, Nav1.2-RX, consisting of the first 101-aa residues of Nav1.2 with the theoretical molecular weight of ~12 kD. Epitope locations for the anti-Nav1.2 (EM-1, ASC-002) and anti-pan Nav1 (SP19) antibodies are indicated. b , c The RX allele was effectively inactivated in Scn2a knock-in mice. Western blot analyses of P0.5 whole brain membrane ( b ) and cytosolic ( c ) fractions were performed using EM-1. b Full-length Nav1.2 was moderate and negligible in Scn2a RX/+ (RX/+, N = 4) and Scn2a RX/RX (RX/RX, N = 3) mice, respectively, compared with that in Scn2a +/+ mice (+/+, N = 3) [one-way analysis of variance; genotype: F (2, 7) = 1737, *** P = 0.00000000036, Tukey’s test; Scn2a +/+ vs . Scn2a RX/+ *** P

    Techniques Used: Mutagenesis, Molecular Weight, Knock-In, Mouse Assay, Western Blot

    The pathogenic Scn2a nonsense mutation caused absence-like seizures with SWDs in mice. a Representative traces of somatosensory ECoG/EMG recordings in 6–11 weeks-old Scn2a RX/+ mice ( N = 7). Behavioral arrest during waking state associated with ECoG epileptiform SWDs (left). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. Positivity was plotted up. b Frequencies of SWDs during a 24 h ECoG recording period in Scn2a +/+ ( N = 5) and Scn2a RX/+ ( N = 7) mice. c An episode of non-convulsive epileptiform discharges detected in 1 out of 7 Scn2a RX/+ mice. An arrowhead indicates the onset of epileptiform discharge. Positivity was plotted up. d , e Seizure susceptibility to PTZ in Scn2a RX/+ and Scn2a +/+ mice (10 weeks of age). The latencies to the first appearance of absence seizure-like sudden immobility, myoclonus, clonic convulsion and tonic-clonic convulsion after intraperitoneal administration of PTZ at dose of 50 ( d , N = 20, each genotype) and 25 ( e , N = 14, each genotype) mg per kg body weight. Scn2a RX/+ mice had significantly shorter latencies to absence-like sudden immobility (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 106, * P = 0.0100, myoclonus, U = 116.5, * P = 0.0231, clonic convulsion, U = 162, P = 0.3098, tonic-clonic convulsion, U = 175, P = 0.5023; 25 mg per kg, sudden immobility, U = 53, * P = 0.0383). Data represent means ± SEM, * P
    Figure Legend Snippet: The pathogenic Scn2a nonsense mutation caused absence-like seizures with SWDs in mice. a Representative traces of somatosensory ECoG/EMG recordings in 6–11 weeks-old Scn2a RX/+ mice ( N = 7). Behavioral arrest during waking state associated with ECoG epileptiform SWDs (left). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. Positivity was plotted up. b Frequencies of SWDs during a 24 h ECoG recording period in Scn2a +/+ ( N = 5) and Scn2a RX/+ ( N = 7) mice. c An episode of non-convulsive epileptiform discharges detected in 1 out of 7 Scn2a RX/+ mice. An arrowhead indicates the onset of epileptiform discharge. Positivity was plotted up. d , e Seizure susceptibility to PTZ in Scn2a RX/+ and Scn2a +/+ mice (10 weeks of age). The latencies to the first appearance of absence seizure-like sudden immobility, myoclonus, clonic convulsion and tonic-clonic convulsion after intraperitoneal administration of PTZ at dose of 50 ( d , N = 20, each genotype) and 25 ( e , N = 14, each genotype) mg per kg body weight. Scn2a RX/+ mice had significantly shorter latencies to absence-like sudden immobility (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 106, * P = 0.0100, myoclonus, U = 116.5, * P = 0.0231, clonic convulsion, U = 162, P = 0.3098, tonic-clonic convulsion, U = 175, P = 0.5023; 25 mg per kg, sudden immobility, U = 53, * P = 0.0383). Data represent means ± SEM, * P

    Techniques Used: Mutagenesis, Mouse Assay, MANN-WHITNEY

    Scn2a haplodeficiency did not alter expression levels of other sodium channel subunits. Quantitative RT-PCR analyses of brain mRNAs prepared from P14.5 Scn2a +/+ and Scn2a KO/+ mice ( N = 11, each genotype). Scn2a mRNA expression in Scn2a KO/+ whole brain was reduced to about 50% level of that in Scn2a +/+ mice while there were no significant changes in Scn1a , Scn3a , Scn5a , Scn8a , Scn1b , Scn2b , Scn3b , and Scn4b mRNA expression levels in Scn2a KO/+ , compared with those in Scn2a +/+ mice [unpaired t-test, Scn1a ; t (20) = 1.461, P = 0.1595, Scn2a ; t (20) = 5.250, *** P = 0.000039, Scn3a ; t (20) = 0.5066, P = 0.6180, Scn5a ; t (20) = 0.4223, P = 0.6773, Scn8a ; t (20) = 0.3952, P = 0.6969, Scn1b ; t (20) = 0.7407, P = 0.4675, Scn2b ; t (20) = 0.4259, P = 0.6747, Scn3b ; t (20) = 0.8664, P = 0.3965, Scn4b ; t (20) = 0.5273, P = 0.6038]. White and gray bars represent Scn2a +/+ and Scn2a KO/+ mice, respectively. Data represent means ± SEM, *** P
    Figure Legend Snippet: Scn2a haplodeficiency did not alter expression levels of other sodium channel subunits. Quantitative RT-PCR analyses of brain mRNAs prepared from P14.5 Scn2a +/+ and Scn2a KO/+ mice ( N = 11, each genotype). Scn2a mRNA expression in Scn2a KO/+ whole brain was reduced to about 50% level of that in Scn2a +/+ mice while there were no significant changes in Scn1a , Scn3a , Scn5a , Scn8a , Scn1b , Scn2b , Scn3b , and Scn4b mRNA expression levels in Scn2a KO/+ , compared with those in Scn2a +/+ mice [unpaired t-test, Scn1a ; t (20) = 1.461, P = 0.1595, Scn2a ; t (20) = 5.250, *** P = 0.000039, Scn3a ; t (20) = 0.5066, P = 0.6180, Scn5a ; t (20) = 0.4223, P = 0.6773, Scn8a ; t (20) = 0.3952, P = 0.6969, Scn1b ; t (20) = 0.7407, P = 0.4675, Scn2b ; t (20) = 0.4259, P = 0.6747, Scn3b ; t (20) = 0.8664, P = 0.3965, Scn4b ; t (20) = 0.5273, P = 0.6038]. White and gray bars represent Scn2a +/+ and Scn2a KO/+ mice, respectively. Data represent means ± SEM, *** P

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

    Heterozygous Scn2a knockout mice showed absence-like seizures with SWDs. a Representative traces of ECoG/EMG recordings from 10–27 weeks-old Scn2a KO/+ (KO/+) mice ( N = 6). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. b A representative trace of prolonged non-convulsive seizure. ECoG recordings detected 2 episodes of prolonged non-convulsive seizure with duration of 30–45 s in 2 out of 6 Scn2a KO/+ mice, which were neither accompanied by convulsions, nor followed by post-ictal depression. c Representative ECoG/EMG/LFP recordings during an SWD episode in Scn2a KO/+ mice. Epileptiform discharges with large amplitudes are seen in mPFC and CPu. Positivity was plotted up ( a , b , c ). d Quantification of ECoG SWDs and LFP epileptiform discharges [3-hour recording, light period, Scn2a +/+ , Scn2a KO/+ ( N = 4, each genotype)]. Mann–Whitney test, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 8, P > 0.9999; basolateral amygdala: U = 3, P = 0.2571; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0286; ventroposterior thalamus: U = 7.5, P > 0.9999. e Thresholds of body temperature for hyperthermia-induced seizures did not differ between Scn2a KO/+ and Scn2a +/+ mice (4-week-old, N = 10, each genotype) [unpaired t -test, t (18) = 1.149, P = 0.2658]. f , g Increased seizure susceptibility to PTZ in 10-week-old Scn2a KO/+ mice. Latencies to the first appearance of sudden immobility, myoclonus, clonic convulsion, and tonic-clonic convulsion after administrating of PTZ at doses of 50 ( f , N = 20, each genotype) or 25 ( g , N = 12, each genotype) mg per kg body weight. The latencies to the appearance of sudden immobility, myoclonus and clonic convulsion were shorter in Scn2a KO/+ than in Scn2a +/+ mice (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 69.5, *** P = 0.0002, myoclonus, U = 110.5, * P = 0.0145, clonic convulsion, U = 119, * P = 0.0278, tonic-clonic convulsion, U = 188.5, P = 0.7624; 25 mg per kg, sudden immobility, U = 26.5, ** P = 0.0071). Data represent means ± SEM, * P
    Figure Legend Snippet: Heterozygous Scn2a knockout mice showed absence-like seizures with SWDs. a Representative traces of ECoG/EMG recordings from 10–27 weeks-old Scn2a KO/+ (KO/+) mice ( N = 6). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. b A representative trace of prolonged non-convulsive seizure. ECoG recordings detected 2 episodes of prolonged non-convulsive seizure with duration of 30–45 s in 2 out of 6 Scn2a KO/+ mice, which were neither accompanied by convulsions, nor followed by post-ictal depression. c Representative ECoG/EMG/LFP recordings during an SWD episode in Scn2a KO/+ mice. Epileptiform discharges with large amplitudes are seen in mPFC and CPu. Positivity was plotted up ( a , b , c ). d Quantification of ECoG SWDs and LFP epileptiform discharges [3-hour recording, light period, Scn2a +/+ , Scn2a KO/+ ( N = 4, each genotype)]. Mann–Whitney test, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 8, P > 0.9999; basolateral amygdala: U = 3, P = 0.2571; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0286; ventroposterior thalamus: U = 7.5, P > 0.9999. e Thresholds of body temperature for hyperthermia-induced seizures did not differ between Scn2a KO/+ and Scn2a +/+ mice (4-week-old, N = 10, each genotype) [unpaired t -test, t (18) = 1.149, P = 0.2658]. f , g Increased seizure susceptibility to PTZ in 10-week-old Scn2a KO/+ mice. Latencies to the first appearance of sudden immobility, myoclonus, clonic convulsion, and tonic-clonic convulsion after administrating of PTZ at doses of 50 ( f , N = 20, each genotype) or 25 ( g , N = 12, each genotype) mg per kg body weight. The latencies to the appearance of sudden immobility, myoclonus and clonic convulsion were shorter in Scn2a KO/+ than in Scn2a +/+ mice (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 69.5, *** P = 0.0002, myoclonus, U = 110.5, * P = 0.0145, clonic convulsion, U = 119, * P = 0.0278, tonic-clonic convulsion, U = 188.5, P = 0.7624; 25 mg per kg, sudden immobility, U = 26.5, ** P = 0.0071). Data represent means ± SEM, * P

    Techniques Used: Knock-Out, Mouse Assay, MANN-WHITNEY

    3) Product Images from "Severe deficiency of the voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice"

    Article Title: Severe deficiency of the voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.109495

    Elevated neuronal firing is reversible by FlpO-mediated restoration of Na V 1.2 expression in adult Na V 1.2-deficient mice (A) Cartoon illustration of mice systemically administered PHP.eB.AAV-control or PHP.eB.AAV-FlpO via tail vein injection. (B) Coronal views of LacZ staining of the striatum from WT and Scn2a gt/gt (HOM) mice injected with AAV-control or AAV-FlpO. Blue staining of HOM mice largely disappeared in the AAV-FlpO group. CPu, caudate nucleus and the putamen (dorsal striatum). Scale bar, 1 mm. (C) Western blot analysis showing Na V 1.2 protein levels in whole-brain homogenates from HOM mice in the AAV-control or AAV-FlpO group. One-way ANOVA with multiple comparisons. (D) Representative current-clamp recordings of MSNs from WT mice transduced with AAV-FlpO (red), HOM mice transduced with AAV-Control (blue), and HOM mice transduced with AAV-Control (magenta) obtained at the RMP. Inset: representative trace in response to +350 pA injection. (E) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired Mann-Whitney U test for each current pulse. (F) Typical spikes of MSNs were obtained at the normal RMP. (G) Associated phase-plane plots. (H) Individuals and average spike rheobase. Unpaired t test. (I) Typical spikes of MSNs at a fixed MP of −80 mV. (J) Associated phase-plane plots at −80 mV. Data were shown as mean ± SEM.
    Figure Legend Snippet: Elevated neuronal firing is reversible by FlpO-mediated restoration of Na V 1.2 expression in adult Na V 1.2-deficient mice (A) Cartoon illustration of mice systemically administered PHP.eB.AAV-control or PHP.eB.AAV-FlpO via tail vein injection. (B) Coronal views of LacZ staining of the striatum from WT and Scn2a gt/gt (HOM) mice injected with AAV-control or AAV-FlpO. Blue staining of HOM mice largely disappeared in the AAV-FlpO group. CPu, caudate nucleus and the putamen (dorsal striatum). Scale bar, 1 mm. (C) Western blot analysis showing Na V 1.2 protein levels in whole-brain homogenates from HOM mice in the AAV-control or AAV-FlpO group. One-way ANOVA with multiple comparisons. (D) Representative current-clamp recordings of MSNs from WT mice transduced with AAV-FlpO (red), HOM mice transduced with AAV-Control (blue), and HOM mice transduced with AAV-Control (magenta) obtained at the RMP. Inset: representative trace in response to +350 pA injection. (E) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired Mann-Whitney U test for each current pulse. (F) Typical spikes of MSNs were obtained at the normal RMP. (G) Associated phase-plane plots. (H) Individuals and average spike rheobase. Unpaired t test. (I) Typical spikes of MSNs at a fixed MP of −80 mV. (J) Associated phase-plane plots at −80 mV. Data were shown as mean ± SEM.

    Techniques Used: Expressing, Mouse Assay, Injection, Staining, Western Blot, Transduction, Generated, MANN-WHITNEY

    Elevated neuronal firing of striatal medium spiny neurons (MSNs) in adult Na V 1.2-deficient mice (A) A typical MSN labeled by neurobiotin. Scale bar, 40 μm. (B) Representative current-clamp recordings of MSNs from wild-type (WT, black) and homozygous (HOM) Scn2a gt/gt (blue) mice were obtained at the RMP. Inset: representative trace in response to +350 pA injection. (C) The average number of action potentials (APs) generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U test for each current pulse. (D) Individuals and mean RMP values. Unpaired t test. (Ei) Representative traces in response to −100 pA injection. V steady-state (V ss ) is the voltage recorded 0–10 ms before the end of the stimulus. (Eii) Individuals and mean input resistance values at the RMP. Unpaired t test. (F) Typical spikes of MSNs from WT (black) and HOM (blue) mice were obtained at the normal RMP. (G) Associated phase-plane plots. (H–L) Individual and mean spike rheobase, voltage threshold, amplitude, fast after-hyperpolarization (AHP), and half-width values. Unpaired t test. Data are shown as mean ± SEM.
    Figure Legend Snippet: Elevated neuronal firing of striatal medium spiny neurons (MSNs) in adult Na V 1.2-deficient mice (A) A typical MSN labeled by neurobiotin. Scale bar, 40 μm. (B) Representative current-clamp recordings of MSNs from wild-type (WT, black) and homozygous (HOM) Scn2a gt/gt (blue) mice were obtained at the RMP. Inset: representative trace in response to +350 pA injection. (C) The average number of action potentials (APs) generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U test for each current pulse. (D) Individuals and mean RMP values. Unpaired t test. (Ei) Representative traces in response to −100 pA injection. V steady-state (V ss ) is the voltage recorded 0–10 ms before the end of the stimulus. (Eii) Individuals and mean input resistance values at the RMP. Unpaired t test. (F) Typical spikes of MSNs from WT (black) and HOM (blue) mice were obtained at the normal RMP. (G) Associated phase-plane plots. (H–L) Individual and mean spike rheobase, voltage threshold, amplitude, fast after-hyperpolarization (AHP), and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Techniques Used: Mouse Assay, Labeling, Injection, Generated, Two Tailed Test, MANN-WHITNEY

    Elevated neuronal excitability is autonomous in adult Na V 1.2-deficient mice (A) Scn2a gt/gt (HOM) mice were injected with a dilute FlpO virus, sparsely transducing a subset of neurons in the striatum. Dashed circles highlight two neighboring AAV-negative (blue circle) and AAV-FlpO-positive (magenta circle) neurons. Scale bar, 10 μm. (B) Representative current-clamp recordings of AAV-negative (blue) and AAV-FlpO-positive (magenta) MSNs in the CPu of HOM mice were obtained at the RMP. Inset: representative trace in response to +350 pA injection. (C) The average number of APs generated in response to depolarizing current pulses. Unpaired Mann-Whitney U test for each current pulse. (D) Individuals and average RMP values. Unpaired t test. (E) Individuals and average input resistance values at the RMP. Unpaired t test. (F) Typical spikes were obtained at the RMP. (G) Associated phase-plane plots. (H–L) Individual and average spike rheobase, voltage threshold, amplitude, AHP, and half-width values. Unpaired t test. Data are shown as mean ± SEM.
    Figure Legend Snippet: Elevated neuronal excitability is autonomous in adult Na V 1.2-deficient mice (A) Scn2a gt/gt (HOM) mice were injected with a dilute FlpO virus, sparsely transducing a subset of neurons in the striatum. Dashed circles highlight two neighboring AAV-negative (blue circle) and AAV-FlpO-positive (magenta circle) neurons. Scale bar, 10 μm. (B) Representative current-clamp recordings of AAV-negative (blue) and AAV-FlpO-positive (magenta) MSNs in the CPu of HOM mice were obtained at the RMP. Inset: representative trace in response to +350 pA injection. (C) The average number of APs generated in response to depolarizing current pulses. Unpaired Mann-Whitney U test for each current pulse. (D) Individuals and average RMP values. Unpaired t test. (E) Individuals and average input resistance values at the RMP. Unpaired t test. (F) Typical spikes were obtained at the RMP. (G) Associated phase-plane plots. (H–L) Individual and average spike rheobase, voltage threshold, amplitude, AHP, and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Techniques Used: Mouse Assay, Injection, Generated, MANN-WHITNEY

    Activation of K V channels reverses elevated neuronal firing in adult Na V 1.2-deficient mice (A) Volcano plot displaying Scn2a and Scn8a as well as potassium channels that are statistically downregulated in Scn2a gt/gt (HOM) mice compared with WT mice identified by RNA-seq. Significantly upregulated genes are shown in yellow, and downregulated genes are shown in blue. (B) List of potassium channels that are significantly downregulated in HOM mice compared with the WT. Hits were identified from both DESeq2 and edgeR differential expression analysis with a false discovery rate of less than 0.05. “% expression” of WT level (100%). n = 4 mice for each group. (C) Representative whole-cell voltage-clamp recordings of MSNs in brain slices from WT and HOM mice, showing potassium currents at voltage steps from −120 mV to +50 mV. Voltage-gated Ca 2+ channels were not blocked to allow activation of Ca 2+ -dependent K + channels. (D) Summary of the total sustained current (measured at the end of steps). Two-way ANOVA with repeated measures. (E) Representative current-clamp recordings of MSNs from WT slices perfused with 0.1% DMSO in artificial cerebrospinal fluid (aCSF) (WT control, black), HOM slices perfused with 0.1% DMSO in aCSF (HOM control, blue), and HOM slices perfused with 0.1% DMSO in aCSF containing PiMA (HOM 10 μM PiMA, magenta) at the RMP. Inset: representative trace in response to +400 pA injection. (F) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired Mann-Whitney U test for each current pulse. (G) Individuals and average RMP values. Unpaired t test. (H) Individuals and average input resistance values at the RMP. Unpaired t test. (I) Typical spikes of MSNs were obtained at the RMP. (J) Associated phase-plane plots. (K–O) Individuals and average spike rheobase, voltage threshold, amplitude, AHP, and half-width values. Unpaired t test. Data are shown as mean ± SEM.
    Figure Legend Snippet: Activation of K V channels reverses elevated neuronal firing in adult Na V 1.2-deficient mice (A) Volcano plot displaying Scn2a and Scn8a as well as potassium channels that are statistically downregulated in Scn2a gt/gt (HOM) mice compared with WT mice identified by RNA-seq. Significantly upregulated genes are shown in yellow, and downregulated genes are shown in blue. (B) List of potassium channels that are significantly downregulated in HOM mice compared with the WT. Hits were identified from both DESeq2 and edgeR differential expression analysis with a false discovery rate of less than 0.05. “% expression” of WT level (100%). n = 4 mice for each group. (C) Representative whole-cell voltage-clamp recordings of MSNs in brain slices from WT and HOM mice, showing potassium currents at voltage steps from −120 mV to +50 mV. Voltage-gated Ca 2+ channels were not blocked to allow activation of Ca 2+ -dependent K + channels. (D) Summary of the total sustained current (measured at the end of steps). Two-way ANOVA with repeated measures. (E) Representative current-clamp recordings of MSNs from WT slices perfused with 0.1% DMSO in artificial cerebrospinal fluid (aCSF) (WT control, black), HOM slices perfused with 0.1% DMSO in aCSF (HOM control, blue), and HOM slices perfused with 0.1% DMSO in aCSF containing PiMA (HOM 10 μM PiMA, magenta) at the RMP. Inset: representative trace in response to +400 pA injection. (F) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired Mann-Whitney U test for each current pulse. (G) Individuals and average RMP values. Unpaired t test. (H) Individuals and average input resistance values at the RMP. Unpaired t test. (I) Typical spikes of MSNs were obtained at the RMP. (J) Associated phase-plane plots. (K–O) Individuals and average spike rheobase, voltage threshold, amplitude, AHP, and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Techniques Used: Activation Assay, Mouse Assay, RNA Sequencing Assay, Expressing, Injection, Generated, MANN-WHITNEY

    4) Product Images from "Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice"

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    Journal: bioRxiv

    doi: 10.1101/2021.07.19.452930

    Altered anxiety-related behavior and rotarod performance in Scn2a E/+ mice. A) Percent time spent in the open arms of a zero-maze apparatus in Scn2a E/+ mice compared to WT at 6 weeks of age. Scn2a E/+ males spent significantly more time in the open arms compared to WT (WT: 15.3 ± 2.2%, Scn2a E/+ : 30.5 ± 4.1%, **p=0.0023; Student’s t-test). Scn2a E/+ females spent significantly more time in the open arms compared to WT (WT: 22.0 ± 2.0%, Scn2a E/+ : 34.8 ± 4.9%, *p=0.0297; Welch’s t test). B) Percent time spent in the light zone of a light/dark box in Scn2a E/+ mice compared to WT at 7 weeks of age. Scn2a E/+ males spent significantly more time in the light zone compared to WT (WT: 20.9 ± 2.0%, Scn2a E/+ : 29.2 ± 3.2%, *p=0.0367; Student’s t-test). Scn2a E/+ females also spent significantly more time in the light zone compared to WT (WT: 21.1 ± 1.7%, Scn2a E/+ : 32.1 ± 3.5%, *p=0.0367; Mann-Whitney test). C) Percent time spent in the center zone of an open field apparatus in Scn2a E/+ mice compared to WT at 8 weeks of age. There was not a significant difference in the amount of time spent in the center zone between Scn2a E/+ and WT males (p=0.0556, Mann-Whitney test). However, Scn2a E/+ females spent significantly more time in the center zone compared to WT (WT: 6.27 ± 0.9%, Scn2a E/+ : 10.1 ± 1.2%, *p=0.0167; Mann-Whitney test). D) Number of marbles buried during a 30-minute trial by Scn2a E/+ mice compared to WT at 6 weeks of age is displayed. Scn2a E/+ males buried significantly fewer marbles compared to WT (WT: 12 ± 1, Scn2a E/+ : 5 ± 2, **p=0.0019; Mann-Whitney test). Scn2a E/+ females also buried significantly fewer marbles compared to WT (WT: 13 ± 2, Scn2a E/+ : 2 ± 1, ****p=0.0001; Mann-Whitney test). E) Average latency to fall during an accelerating rotarod task measured on three consecutive days in Scn2a E/+ mice compared to WT at 9 weeks of age. Daily performance for each animal was assessed by averaging across three trials. Two-way repeated measures ANOVA comparing average latency to fall between Scn2a E/+ and WT males showed significant main effects of test day [ F (1.511,34.76) = 8.450, **p=0.0022] and genotype [ F (1,23) = 10.18, *p=0.0041]. Scn2a E/+ males took significantly longer to fall compared to WT on day 3 (WT: 121 ± 6 sec, Scn2a E/+ : 152 ± 7 sec, p=0.0106; Sidak’s post-hoc test). Two-way repeated measures ANOVA comparing average latency to fall between Scn2a E/+ and WT females showed a significant main effect of test day only [ F (1.594,36.65) = 6.646, **p=0.0059]. For panels A-D symbols represent individual mice, horizontal lines represent mean, and error bars represent SEM. For panel E symbols and error bars represent mean ± SEM. Males and females were analyzed separately, with n=12-14 per genotype for males and n=11-15 per genotype for females.
    Figure Legend Snippet: Altered anxiety-related behavior and rotarod performance in Scn2a E/+ mice. A) Percent time spent in the open arms of a zero-maze apparatus in Scn2a E/+ mice compared to WT at 6 weeks of age. Scn2a E/+ males spent significantly more time in the open arms compared to WT (WT: 15.3 ± 2.2%, Scn2a E/+ : 30.5 ± 4.1%, **p=0.0023; Student’s t-test). Scn2a E/+ females spent significantly more time in the open arms compared to WT (WT: 22.0 ± 2.0%, Scn2a E/+ : 34.8 ± 4.9%, *p=0.0297; Welch’s t test). B) Percent time spent in the light zone of a light/dark box in Scn2a E/+ mice compared to WT at 7 weeks of age. Scn2a E/+ males spent significantly more time in the light zone compared to WT (WT: 20.9 ± 2.0%, Scn2a E/+ : 29.2 ± 3.2%, *p=0.0367; Student’s t-test). Scn2a E/+ females also spent significantly more time in the light zone compared to WT (WT: 21.1 ± 1.7%, Scn2a E/+ : 32.1 ± 3.5%, *p=0.0367; Mann-Whitney test). C) Percent time spent in the center zone of an open field apparatus in Scn2a E/+ mice compared to WT at 8 weeks of age. There was not a significant difference in the amount of time spent in the center zone between Scn2a E/+ and WT males (p=0.0556, Mann-Whitney test). However, Scn2a E/+ females spent significantly more time in the center zone compared to WT (WT: 6.27 ± 0.9%, Scn2a E/+ : 10.1 ± 1.2%, *p=0.0167; Mann-Whitney test). D) Number of marbles buried during a 30-minute trial by Scn2a E/+ mice compared to WT at 6 weeks of age is displayed. Scn2a E/+ males buried significantly fewer marbles compared to WT (WT: 12 ± 1, Scn2a E/+ : 5 ± 2, **p=0.0019; Mann-Whitney test). Scn2a E/+ females also buried significantly fewer marbles compared to WT (WT: 13 ± 2, Scn2a E/+ : 2 ± 1, ****p=0.0001; Mann-Whitney test). E) Average latency to fall during an accelerating rotarod task measured on three consecutive days in Scn2a E/+ mice compared to WT at 9 weeks of age. Daily performance for each animal was assessed by averaging across three trials. Two-way repeated measures ANOVA comparing average latency to fall between Scn2a E/+ and WT males showed significant main effects of test day [ F (1.511,34.76) = 8.450, **p=0.0022] and genotype [ F (1,23) = 10.18, *p=0.0041]. Scn2a E/+ males took significantly longer to fall compared to WT on day 3 (WT: 121 ± 6 sec, Scn2a E/+ : 152 ± 7 sec, p=0.0106; Sidak’s post-hoc test). Two-way repeated measures ANOVA comparing average latency to fall between Scn2a E/+ and WT females showed a significant main effect of test day only [ F (1.594,36.65) = 6.646, **p=0.0059]. For panels A-D symbols represent individual mice, horizontal lines represent mean, and error bars represent SEM. For panel E symbols and error bars represent mean ± SEM. Males and females were analyzed separately, with n=12-14 per genotype for males and n=11-15 per genotype for females.

    Techniques Used: Mouse Assay, MANN-WHITNEY

    Altered social behavior in Scn2a E/+ mice. A) Sociability phase of three-chamber assay. Amount of time spent sniffing either an empty cup (EC) or unfamiliar mouse (S1) in Scn2a E/+ mice compared to WT at 10 weeks of age. Two-way ANOVA (using target as a within-subject variable) comparing average sniffing time between Scn2a E/+ and WT males showed a significant main effect of target [ F (1,24) = 57.28, p
    Figure Legend Snippet: Altered social behavior in Scn2a E/+ mice. A) Sociability phase of three-chamber assay. Amount of time spent sniffing either an empty cup (EC) or unfamiliar mouse (S1) in Scn2a E/+ mice compared to WT at 10 weeks of age. Two-way ANOVA (using target as a within-subject variable) comparing average sniffing time between Scn2a E/+ and WT males showed a significant main effect of target [ F (1,24) = 57.28, p

    Techniques Used: Mouse Assay, Boyden Chamber Assay

    EEG abnormalities and altered susceptibility to induced seizures in Scn2a E/+ mice. A) Representative 5-minute epoch of EEG from Scn2a E/+ mice. A localized seizure occurred as an abrupt onset of rhythmic 2 Hz sharp waves with overriding fast activity that evolve in amplitude and frequency for ∼45 seconds before abruptly terminating with return to typical sleep background. B) 30-second epoch corresponding to the purple bar segment from A . The top line in both A and B corresponds to channel 1 (right posterior-left posterior) and second line is channel 2 (right anterior-left posterior). C) Example of an isolated high amplitude sharp wave with overriding fast activity corresponding to the blue bar segment in B . D) Power spectrum for the sharp wave in C showing elevated power in decibels across the 1-170 Hz frequency range at the time of discharge. E) Latency to flurothyl-induced GTCS in Scn2a E/+ mice compared to WT at 6-9 weeks of age. Scn2a E/+ males had an elevated threshold for flurothyl-induced seizures compared to WT (WT: 175 ± 8 sec, Scn2a E/+ : 247 ± 13 sec, ****p
    Figure Legend Snippet: EEG abnormalities and altered susceptibility to induced seizures in Scn2a E/+ mice. A) Representative 5-minute epoch of EEG from Scn2a E/+ mice. A localized seizure occurred as an abrupt onset of rhythmic 2 Hz sharp waves with overriding fast activity that evolve in amplitude and frequency for ∼45 seconds before abruptly terminating with return to typical sleep background. B) 30-second epoch corresponding to the purple bar segment from A . The top line in both A and B corresponds to channel 1 (right posterior-left posterior) and second line is channel 2 (right anterior-left posterior). C) Example of an isolated high amplitude sharp wave with overriding fast activity corresponding to the blue bar segment in B . D) Power spectrum for the sharp wave in C showing elevated power in decibels across the 1-170 Hz frequency range at the time of discharge. E) Latency to flurothyl-induced GTCS in Scn2a E/+ mice compared to WT at 6-9 weeks of age. Scn2a E/+ males had an elevated threshold for flurothyl-induced seizures compared to WT (WT: 175 ± 8 sec, Scn2a E/+ : 247 ± 13 sec, ****p

    Techniques Used: Mouse Assay, Activity Assay, Isolation

    Scn2a K1422E prefrontal pyramidal cell AP waveform has loss-of-function characteristics. A) Example whole-cell voltage response to somatic current injection in WT (black) and Scn2a E/+ (K1422E, cyan) neurons. Numbers between examples correspond to current injection amplitude. B) AP number per 300 ms current injection, color coded as in A. Bars are mean ± SEM. n = 13 cells each. C) Rheobase AP as dV/dt vs. voltage (phase-plane plot). Note reduction of peak dV/dt for Scn2a E/+ cells compared to WT, indicative of loss-of-function in Na V 1.2-mediated somatic depolarization. D) Summaries of AP waveform and intrinsic excitability characteristics. Circles are single cells, bars are mean ± SEM. Peak dV/dt is reduced in Scn2a E/+ cells, unpaired t-test. n = 14 WT, 13 Scn2a E/+ cells.
    Figure Legend Snippet: Scn2a K1422E prefrontal pyramidal cell AP waveform has loss-of-function characteristics. A) Example whole-cell voltage response to somatic current injection in WT (black) and Scn2a E/+ (K1422E, cyan) neurons. Numbers between examples correspond to current injection amplitude. B) AP number per 300 ms current injection, color coded as in A. Bars are mean ± SEM. n = 13 cells each. C) Rheobase AP as dV/dt vs. voltage (phase-plane plot). Note reduction of peak dV/dt for Scn2a E/+ cells compared to WT, indicative of loss-of-function in Na V 1.2-mediated somatic depolarization. D) Summaries of AP waveform and intrinsic excitability characteristics. Circles are single cells, bars are mean ± SEM. Peak dV/dt is reduced in Scn2a E/+ cells, unpaired t-test. n = 14 WT, 13 Scn2a E/+ cells.

    Techniques Used: Injection

    Generation and molecular characterization of Scn2a K1422E mice. A) Sequencing chromatograms of Scn2a genomic PCR products with the first nucleotide of the K1422E codon highlighted in black. Top chromatogram from a WT littermate control mouse shows homozygosity for the WT nucleotide at the K1422E codon. Bottom chromatogram from a heterozygous Scn2a E/+ mouse shows heterozygosity for the single nucleotide change introduced by CRISPR/Cas9 genome editing and homology directed repair. B) Relative expression of whole brain Scn2a transcript in WT and Scn2a E/+ mice assayed by RT-ddPCR. Relative transcript levels are expressed as a ratio of Scn2a concentration to Tbp concentration (normalized to WT average). There was no difference in transcript expression between genotypes (p=0.6295; n=8 mice per genotype). C) Relative expression of whole brain Na V 1.2 protein in WT and Scn2a E/+ mice assayed by immunoblotting. Quantification is expressed as a ratio of Na V 1.2 immunofluorescence relative to GRP75/Mortalin (normalized to WT average). There was no difference in protein expression between genotypes (p=0.2937; n=8 mice per genotype). For both B and C , circles represent samples from individual mice, horizontal lines rep resent mean, and error bars represent SEM. D) Representative immunoblot. Bands corresponding to Na V 1.2 (MW: 260 kDa) are visualized in green (Alexa Fluor 790) while bands corresponding to GRP75 (MW: 73 kDa) are visualized in red (Alexa Fluor 680).
    Figure Legend Snippet: Generation and molecular characterization of Scn2a K1422E mice. A) Sequencing chromatograms of Scn2a genomic PCR products with the first nucleotide of the K1422E codon highlighted in black. Top chromatogram from a WT littermate control mouse shows homozygosity for the WT nucleotide at the K1422E codon. Bottom chromatogram from a heterozygous Scn2a E/+ mouse shows heterozygosity for the single nucleotide change introduced by CRISPR/Cas9 genome editing and homology directed repair. B) Relative expression of whole brain Scn2a transcript in WT and Scn2a E/+ mice assayed by RT-ddPCR. Relative transcript levels are expressed as a ratio of Scn2a concentration to Tbp concentration (normalized to WT average). There was no difference in transcript expression between genotypes (p=0.6295; n=8 mice per genotype). C) Relative expression of whole brain Na V 1.2 protein in WT and Scn2a E/+ mice assayed by immunoblotting. Quantification is expressed as a ratio of Na V 1.2 immunofluorescence relative to GRP75/Mortalin (normalized to WT average). There was no difference in protein expression between genotypes (p=0.2937; n=8 mice per genotype). For both B and C , circles represent samples from individual mice, horizontal lines rep resent mean, and error bars represent SEM. D) Representative immunoblot. Bands corresponding to Na V 1.2 (MW: 260 kDa) are visualized in green (Alexa Fluor 790) while bands corresponding to GRP75 (MW: 73 kDa) are visualized in red (Alexa Fluor 680).

    Techniques Used: Mouse Assay, Sequencing, Polymerase Chain Reaction, CRISPR, Expressing, Concentration Assay, Immunofluorescence

    Whole-cell sodium currents of acutely isolated neuron. Representative whole-cell sodium currents and current voltage relationships of A) total sodium current, B) TTX-resistant currents, and C) TTX-sensitive currents from acutely dissociated hippocampal pyramidal neurons from WT and Scn2a E/+ mice, D) Sodium reversal potential of total sodium current (left, p=0.0364), TTX-resistant current (middle, p=0.0006), and TTX-sensitive currents (right, p=0.6964). All data are plotted as mean ± SEM of n=8-11 cells.
    Figure Legend Snippet: Whole-cell sodium currents of acutely isolated neuron. Representative whole-cell sodium currents and current voltage relationships of A) total sodium current, B) TTX-resistant currents, and C) TTX-sensitive currents from acutely dissociated hippocampal pyramidal neurons from WT and Scn2a E/+ mice, D) Sodium reversal potential of total sodium current (left, p=0.0364), TTX-resistant current (middle, p=0.0006), and TTX-sensitive currents (right, p=0.6964). All data are plotted as mean ± SEM of n=8-11 cells.

    Techniques Used: Isolation, Mouse Assay

    Heterologous expression of K1422E in HEK293T cells reveals altered ion selectivity. A) Homology model of voltage gated sodium channel alpha subunit (PDB 6MWA Na V Ab, residues1-239). Four internally homologous domains coalesce with four-fold symmetry around an ion conducting pore denoted by an asterisk. The Cα carbons from each residue (DEKA) of the highly conserved ion selectivity filter are represented by colored ellipses. The red ellipse denotes the Cα carbon from the K1422 residue that is substituted for glutamate (E) in the Scn2a K1422E model. B) Representative whole-cell sodium currents (top) and calcium currents (bottom) from WT (left) and K1422E (right) expressing cells. C) Summary current-voltage relationship of whole cells sodium current showing reduced sodium current density of K1422E at -10 mV (p=0.0033). D) Sodium reversal potential of WT and K1422E expressing cells (p
    Figure Legend Snippet: Heterologous expression of K1422E in HEK293T cells reveals altered ion selectivity. A) Homology model of voltage gated sodium channel alpha subunit (PDB 6MWA Na V Ab, residues1-239). Four internally homologous domains coalesce with four-fold symmetry around an ion conducting pore denoted by an asterisk. The Cα carbons from each residue (DEKA) of the highly conserved ion selectivity filter are represented by colored ellipses. The red ellipse denotes the Cα carbon from the K1422 residue that is substituted for glutamate (E) in the Scn2a K1422E model. B) Representative whole-cell sodium currents (top) and calcium currents (bottom) from WT (left) and K1422E (right) expressing cells. C) Summary current-voltage relationship of whole cells sodium current showing reduced sodium current density of K1422E at -10 mV (p=0.0033). D) Sodium reversal potential of WT and K1422E expressing cells (p

    Techniques Used: Expressing

    Lower olfactory dishabituation to social odors and intact olfactory-guided behavior in Scn2a E/+ mice. A) Average sniffing times during an odor habituation/dishabituation assay in Scn2a E/+ mice compared to WT at 11 weeks of age. Overall, olfactory discrimination in Scn2a E/+ males was not significantly different from WT males. However, Scn2a E/+ males spent significantly less time sniffing same sex urine during the first two presentations compared to WT males. During the first presentation, average sniffing time was 42.3 ± 3.3 sec for WT males and 18.8 ± 3.4 sec for Scn2a E/+ males (***p=0.0005, multiple t-tests). During the second presentation, average sniffing time was 26.3 ± 8.9 sec for WT males and 8.9 ± 2.1 sec for Scn2a E/+ males (*p=0.0307, multiple t-tests). Olfactory discrimination in Scn2a E/+ females was also not significantly different from WT females. However, Scn2a E/+ females spent significantly less time sniffing same sex urine during the first presentation compared to WT females (WT: 46.3 ± 4.6 sec, Scn2a E/+ : 19.9 ± 4.2 sec, **p=0.0045, multiple t-tests). Symbols and error bars represent mean ± SEM. Males and females were analyzed separately, with n=14 per genotype for males and n=12-13 per genotype for females. B) Latency to find a buried food item in Scn2a E/+ mice compared to WT at 8 weeks of age. Latency to find a buried food item was not significantly different between Scn2a E/+ and WT males (WT: 44.86 ± 11.58 sec, Scn2a E/+ : 32.46 ± 9.95 sec, p = 0.7471; Mann-Whitney test). Latency to find a buried food item was not significantly different between Scn2a E/+ and WT females (WT: 89.00 ± 44.31 sec, Scn2a E/+ : 161.7 ± 70.02, p = 0.2927; Mann-Whitney test). Symbols represent measured values from individual mice, horizontal lines represent mean, and error bars represent SEM. Males and females were analyzed separately, with n=13-14 per genotype for males and n=13 per genotype for females.
    Figure Legend Snippet: Lower olfactory dishabituation to social odors and intact olfactory-guided behavior in Scn2a E/+ mice. A) Average sniffing times during an odor habituation/dishabituation assay in Scn2a E/+ mice compared to WT at 11 weeks of age. Overall, olfactory discrimination in Scn2a E/+ males was not significantly different from WT males. However, Scn2a E/+ males spent significantly less time sniffing same sex urine during the first two presentations compared to WT males. During the first presentation, average sniffing time was 42.3 ± 3.3 sec for WT males and 18.8 ± 3.4 sec for Scn2a E/+ males (***p=0.0005, multiple t-tests). During the second presentation, average sniffing time was 26.3 ± 8.9 sec for WT males and 8.9 ± 2.1 sec for Scn2a E/+ males (*p=0.0307, multiple t-tests). Olfactory discrimination in Scn2a E/+ females was also not significantly different from WT females. However, Scn2a E/+ females spent significantly less time sniffing same sex urine during the first presentation compared to WT females (WT: 46.3 ± 4.6 sec, Scn2a E/+ : 19.9 ± 4.2 sec, **p=0.0045, multiple t-tests). Symbols and error bars represent mean ± SEM. Males and females were analyzed separately, with n=14 per genotype for males and n=12-13 per genotype for females. B) Latency to find a buried food item in Scn2a E/+ mice compared to WT at 8 weeks of age. Latency to find a buried food item was not significantly different between Scn2a E/+ and WT males (WT: 44.86 ± 11.58 sec, Scn2a E/+ : 32.46 ± 9.95 sec, p = 0.7471; Mann-Whitney test). Latency to find a buried food item was not significantly different between Scn2a E/+ and WT females (WT: 89.00 ± 44.31 sec, Scn2a E/+ : 161.7 ± 70.02, p = 0.2927; Mann-Whitney test). Symbols represent measured values from individual mice, horizontal lines represent mean, and error bars represent SEM. Males and females were analyzed separately, with n=13-14 per genotype for males and n=13 per genotype for females.

    Techniques Used: Mouse Assay, MANN-WHITNEY

    AP-evoked Ca 2+ influx during the rising phase of the AP in the proximal AIS of Scn2a E/+ cells. A) Pyramidal cell initial segments are enriched with Na V 1.2 proximal to the soma and Na V 1.6 more distal to the soma. Pointscan imaging was performed 5 and 30 µm from the axon hillock, corresponding to Na V 1.2 and Na V 1.6-enriched regions, respectively. B) Examples of AP-evoked (2 nA, 2 ms stimulus; black, top) calcium transients imaged in pointscan mode in the proximal (green, middle) and distal (violet, bottom) AIS in WT (left) and Scn2a E/+ cells (right). Vertical line is aligned to peak AP voltage. Grey shaded area encompasses imaging signal root-mean-squared error (RMSE) during baseline, before AP. Consistent deviation above this error value defines onset of Ca 2+ transient. Note Ca 2+ influx before AP peak in proximal AIS of K1422E condition, only. C) Amplitude of Ca 2+ transient is higher in proximal AIS of Scn2a E/+ cells, consistent with influx from both local voltage-gated calcium channels and additional influx through K1422E Na V 1.2 channels. Circles are single cells and bars are mean ± SEM. p values from unpaired t-tests. D) Ca 2+ transient onset occurs earlier in the proximal AIS of Scn2a E/+ cells, consistent with Ca 2+ influx through K1422E Na V 1.2 channels. Display as in C .
    Figure Legend Snippet: AP-evoked Ca 2+ influx during the rising phase of the AP in the proximal AIS of Scn2a E/+ cells. A) Pyramidal cell initial segments are enriched with Na V 1.2 proximal to the soma and Na V 1.6 more distal to the soma. Pointscan imaging was performed 5 and 30 µm from the axon hillock, corresponding to Na V 1.2 and Na V 1.6-enriched regions, respectively. B) Examples of AP-evoked (2 nA, 2 ms stimulus; black, top) calcium transients imaged in pointscan mode in the proximal (green, middle) and distal (violet, bottom) AIS in WT (left) and Scn2a E/+ cells (right). Vertical line is aligned to peak AP voltage. Grey shaded area encompasses imaging signal root-mean-squared error (RMSE) during baseline, before AP. Consistent deviation above this error value defines onset of Ca 2+ transient. Note Ca 2+ influx before AP peak in proximal AIS of K1422E condition, only. C) Amplitude of Ca 2+ transient is higher in proximal AIS of Scn2a E/+ cells, consistent with influx from both local voltage-gated calcium channels and additional influx through K1422E Na V 1.2 channels. Circles are single cells and bars are mean ± SEM. p values from unpaired t-tests. D) Ca 2+ transient onset occurs earlier in the proximal AIS of Scn2a E/+ cells, consistent with Ca 2+ influx through K1422E Na V 1.2 channels. Display as in C .

    Techniques Used: Imaging

    5) 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

    6) Product Images from "Scn2a Haploinsufficiency in Mice Suppresses Hippocampal Neuronal Excitability, Excitatory Synaptic Drive, and Long-Term Potentiation, and Spatial Learning and Memory"

    Article Title: Scn2a Haploinsufficiency in Mice Suppresses Hippocampal Neuronal Excitability, Excitatory Synaptic Drive, and Long-Term Potentiation, and Spatial Learning and Memory

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2019.00145

    Decreased neuronal excitability and enhanced excitatory synaptic transmission in the presence of network activity in the Scn2a +/- hippocampus. (A,B) Suppressed intrinsic excitability in hippocampal CA1 pyramidal neurons of Scn2a +/- mice (3 weeks), as shown by the decrease in input resistance. Note that the sag ratio and current-spike relationship were normal, despite a decreasing trend. Data are presented as means ± SEM. n = 14 cells from 3 mice for WT and HT, ∗ P
    Figure Legend Snippet: Decreased neuronal excitability and enhanced excitatory synaptic transmission in the presence of network activity in the Scn2a +/- hippocampus. (A,B) Suppressed intrinsic excitability in hippocampal CA1 pyramidal neurons of Scn2a +/- mice (3 weeks), as shown by the decrease in input resistance. Note that the sag ratio and current-spike relationship were normal, despite a decreasing trend. Data are presented as means ± SEM. n = 14 cells from 3 mice for WT and HT, ∗ P

    Techniques Used: Transmission Assay, Activity Assay, Mouse Assay

    Newborn and juvenile Scn2a +/- mice show modestly increased direct social interaction and modestly decreased locomotion but normal social communication and mother-attachment behavior. (A) Normal mother-seeking USVs in newborn Scn2a +/- mice (P4–10), as shown by the number of USV calls, duration of each call, and latency to the first call. Data are presented as means ± SEM. n = 20 mice for WT and 27 for HT, ns, not significant, Mann–Whitney test. (B) Moderately increased direct social interaction (or juvenile play) in juvenile Scn2a +/- mice (3 weeks), as indicated by nose-to-nose sniffing. Note that the total social interaction is unaltered in the mutant mice n = 8 mice for WT and 14 for HT, ∗ P
    Figure Legend Snippet: Newborn and juvenile Scn2a +/- mice show modestly increased direct social interaction and modestly decreased locomotion but normal social communication and mother-attachment behavior. (A) Normal mother-seeking USVs in newborn Scn2a +/- mice (P4–10), as shown by the number of USV calls, duration of each call, and latency to the first call. Data are presented as means ± SEM. n = 20 mice for WT and 27 for HT, ns, not significant, Mann–Whitney test. (B) Moderately increased direct social interaction (or juvenile play) in juvenile Scn2a +/- mice (3 weeks), as indicated by nose-to-nose sniffing. Note that the total social interaction is unaltered in the mutant mice n = 8 mice for WT and 14 for HT, ∗ P

    Techniques Used: Mouse Assay, MANN-WHITNEY, Mutagenesis

    Expression of Scn2a mRNA in both glutamatergic and GABAergic neurons. (A,B) Expression of Scn2a mRNAs in Vglut1/2-positive glutamatergic neurons (A) and Gad1/2-positive GABAergic neurons (B) in the neocortex and hippocampus in the mouse brain (P56), as detected by fluorescence in situ hybridization. Coronal brain sections were triply stained for Scn2a, Vglut1/2 or Gad1/2, and DAPI (nuclear stain; blue). Images at right show enlarged views of white boxes in the images at left. Arrowheads indicate neurons that express both Scn2a and neuronal markers; cells indicated by yellow arrowheads were further enlarged to highlight coexpression of the markers. Scale bar, 20 and 10 μm for left and right scale bars, respectively, in each row. (C–E) Expression of Scn2a mRNA in PV-, SST-, or VIP-expressing GABAergic neurons in the neocortex and hippocampus in the mouse brain (P56), as detected by fluorescence in situ hybridization. Scale bar, 20 and 10 μm for left and right scale bars, respectively, in each row.
    Figure Legend Snippet: Expression of Scn2a mRNA in both glutamatergic and GABAergic neurons. (A,B) Expression of Scn2a mRNAs in Vglut1/2-positive glutamatergic neurons (A) and Gad1/2-positive GABAergic neurons (B) in the neocortex and hippocampus in the mouse brain (P56), as detected by fluorescence in situ hybridization. Coronal brain sections were triply stained for Scn2a, Vglut1/2 or Gad1/2, and DAPI (nuclear stain; blue). Images at right show enlarged views of white boxes in the images at left. Arrowheads indicate neurons that express both Scn2a and neuronal markers; cells indicated by yellow arrowheads were further enlarged to highlight coexpression of the markers. Scale bar, 20 and 10 μm for left and right scale bars, respectively, in each row. (C–E) Expression of Scn2a mRNA in PV-, SST-, or VIP-expressing GABAergic neurons in the neocortex and hippocampus in the mouse brain (P56), as detected by fluorescence in situ hybridization. Scale bar, 20 and 10 μm for left and right scale bars, respectively, in each row.

    Techniques Used: Expressing, Fluorescence, In Situ Hybridization, Staining

    Scn2a +/- mice show impaired spatial learning and memory and enhanced long-term fear memory. (A–C) Impaired spatial learning and memory in both the initial and reversal phases of the Morris water-maze test in Scn2a +/- mice (3–4 months), as shown by escape latency, time spent in quadrant, and number of exact platform crossings in the learning phase (day 1–7), reversal phase (day 9–11), and respective probe tests (days 8 and 12). Data are presented as means ± SEM. n = 23 mice for WT and 28 for HT, ∗ P
    Figure Legend Snippet: Scn2a +/- mice show impaired spatial learning and memory and enhanced long-term fear memory. (A–C) Impaired spatial learning and memory in both the initial and reversal phases of the Morris water-maze test in Scn2a +/- mice (3–4 months), as shown by escape latency, time spent in quadrant, and number of exact platform crossings in the learning phase (day 1–7), reversal phase (day 9–11), and respective probe tests (days 8 and 12). Data are presented as means ± SEM. n = 23 mice for WT and 28 for HT, ∗ P

    Techniques Used: Mouse Assay

    Scn2a +/- mice display suppressed LTP, but normal LTD. (A) Normal basal excitatory synaptic transmission at hippocampal SC-CA1 synapses in Scn2a +/- mice (P20–22), as shown by the input–output ratio of fEPSP slopes plotted against fiber volley (FV) amplitudes. Data are presented as means ± SEM. n = 10 slices from 3 mice for WT and HT, two-way ANOVA with Sidak’s multiple comparison test. (B) Normal paired-pulse facilitation at hippocampal SC-CA1 synapses of Scn2a +/- mice (P20–22), as shown by fEPSP slopes plotted against inter-stimulus intervals. n = 10 slices from 3 mice for WT and HT, two-way ANOVA with Sidak’s multiple comparison test. (C) Decreased LTP induced by high-frequency stimulation (100 Hz, 1 s) at hippocampal SC-CA1 synapses of Scn2a +/- mice (3 months). n = 12 slices from 6 mice for WT and 14 slices from 7 mice for HT, ∗ P
    Figure Legend Snippet: Scn2a +/- mice display suppressed LTP, but normal LTD. (A) Normal basal excitatory synaptic transmission at hippocampal SC-CA1 synapses in Scn2a +/- mice (P20–22), as shown by the input–output ratio of fEPSP slopes plotted against fiber volley (FV) amplitudes. Data are presented as means ± SEM. n = 10 slices from 3 mice for WT and HT, two-way ANOVA with Sidak’s multiple comparison test. (B) Normal paired-pulse facilitation at hippocampal SC-CA1 synapses of Scn2a +/- mice (P20–22), as shown by fEPSP slopes plotted against inter-stimulus intervals. n = 10 slices from 3 mice for WT and HT, two-way ANOVA with Sidak’s multiple comparison test. (C) Decreased LTP induced by high-frequency stimulation (100 Hz, 1 s) at hippocampal SC-CA1 synapses of Scn2a +/- mice (3 months). n = 12 slices from 6 mice for WT and 14 slices from 7 mice for HT, ∗ P

    Techniques Used: Mouse Assay, Transmission Assay

    Scn2a +/- mice show abnormally enhanced direct social interaction but normal social approach, social communication, and repetitive behavior. (A–D) Normal social approach in Scn2a +/- mice (3–4 months) in the three-chamber test, as shown by time spent sniffing or in the chamber. Ob, object; S1, familiar stranger; S2, novel stranger. Data are presented as means ± SEM. n = 15 mice for WT and 21 for HT, ∗ P
    Figure Legend Snippet: Scn2a +/- mice show abnormally enhanced direct social interaction but normal social approach, social communication, and repetitive behavior. (A–D) Normal social approach in Scn2a +/- mice (3–4 months) in the three-chamber test, as shown by time spent sniffing or in the chamber. Ob, object; S1, familiar stranger; S2, novel stranger. Data are presented as means ± SEM. n = 15 mice for WT and 21 for HT, ∗ P

    Techniques Used: Mouse Assay

    Generation of Scn2a +/- mice and characterization of Scn2a mRNA expression. (A) Schematic diagram of the strategy for targeting exons 4–6 of the Scn2a gene. The two sets of primers used for PCR genotyping are indicated. (B) PCR genotyping of Scn2a +/+ , Scn2a +/- , and Scn2a -/- mice (2 months for WT and Scn2a +/- /HT mice; E20.5 for Scn2a -/- /KO mice). (C) Nav1.2 protein levels in whole-brain lysates from WT, Scn2a -/- , and Scn2a +/- mice (2 months for WT and Scn2a +/- mice; E20 for Scn2a -/- mice). Data are presented as means ± SEM. n = 4 mice, ∗∗ P
    Figure Legend Snippet: Generation of Scn2a +/- mice and characterization of Scn2a mRNA expression. (A) Schematic diagram of the strategy for targeting exons 4–6 of the Scn2a gene. The two sets of primers used for PCR genotyping are indicated. (B) PCR genotyping of Scn2a +/+ , Scn2a +/- , and Scn2a -/- mice (2 months for WT and Scn2a +/- /HT mice; E20.5 for Scn2a -/- /KO mice). (C) Nav1.2 protein levels in whole-brain lysates from WT, Scn2a -/- , and Scn2a +/- mice (2 months for WT and Scn2a +/- mice; E20 for Scn2a -/- mice). Data are presented as means ± SEM. n = 4 mice, ∗∗ P

    Techniques Used: Mouse Assay, Expressing, Polymerase Chain Reaction

    Scn2a +/- mice show suppressed locomotion in a familiar environment, but normal anxiety-like behavior and susceptibility to induced seizure. (A,B) Normal locomotor activity of Scn2a +/- mice (2–3 months) in the open-field test. Data are presented as means ± SEM. n = 8 mice for WT and 11 for HT, ns, not significant, Two-way ANOVA with Sidak’s multiple comparison test, Student’s t -test and Mann–Whitney test. (C,D) Decreased distance moved and rearing time, but normal self-grooming and climbing, in Scn2a +/- mice (2–3 months) in the LABORAS test, in which mouse movements are continuously monitored for four consecutive days. Note that the extent of the decrease in distance moved became more evident as the time in LABORAS cages increased. The shaded regions indicate light-off periods. n = 8 mice for WT and 12 for HT, ∗ P
    Figure Legend Snippet: Scn2a +/- mice show suppressed locomotion in a familiar environment, but normal anxiety-like behavior and susceptibility to induced seizure. (A,B) Normal locomotor activity of Scn2a +/- mice (2–3 months) in the open-field test. Data are presented as means ± SEM. n = 8 mice for WT and 11 for HT, ns, not significant, Two-way ANOVA with Sidak’s multiple comparison test, Student’s t -test and Mann–Whitney test. (C,D) Decreased distance moved and rearing time, but normal self-grooming and climbing, in Scn2a +/- mice (2–3 months) in the LABORAS test, in which mouse movements are continuously monitored for four consecutive days. Note that the extent of the decrease in distance moved became more evident as the time in LABORAS cages increased. The shaded regions indicate light-off periods. n = 8 mice for WT and 12 for HT, ∗ P

    Techniques Used: Mouse Assay, Activity Assay, MANN-WHITNEY

    7) Product Images from "Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice"

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0099-2

    Altered action potentials of Scn2a KO excitatory neurons. Responses of neocortical pyramidal excitatory neurons and fast-spiking inhibitory neurons from Vgat -Venus/ Scn2a +/+ (WT) mice ( n = 19 pyramidal and 8 fast-spiking neurons, P7–8; n = 22 pyramidal and 11 fast-spiking neurons, P15–22) and Vgat -Venus/ Scn2a KO/+ (KO/ + ) mice ( n = 16 pyramidal and 11 fast-spiking neurons, P7–8; n = 22 pyramidal and 13 fast-spiking neurons, P15–22) to current injections. Representative action potential traces ( a , e , h , k ), peak amplitudes ( b , f , i , l ), half widths ( c , g , j , m ) and maximum rates of rise ( d ) were shown. b – g Peak amplitudes of P7–8 pyramidal cells are smaller in Scn2a KO/+ than in WT mice, while half widths of P7–8 and P15–22 pyramidal cells were broader in Scn2a KO/+ than in WT mice. Unpaired t -test, peak amplitudes, P7–8, t (33) = 6.0638, *** P = 0.0000008, P15–22, t (42) = 0.0791, P = 0.9373; half widths, P7–8, t (33) = −4.0342, *** P = 0.00031, P15–22, t (42) = −3.6442, * P = 0.0109. d Maximum rates of rise for action potential of P7–8 hippocampal pyramidal cells ( n = 19 WT neurons, n = 11 KO/ + neurons) were lower in Scn2a KO/+ than in WT mice. Unpaired t -test, −120mV, t (28) = 7.3005, *** P = 0.00000006, −110mV, t (28) = 7.3572, *** P = 0.00000005, −100mV, t (28) = 7.3237, *** P = 0.00000006, −90mV, t (28) = 7.4814, *** P = 0.00000004, −80mV, t (28) = 7.6586, *** P = 0.00000002, −70mV, t (28) = 7.2738, *** P = 0.00000006, −60mV, t (28) = 7.8954, *** P = 0.00000001, −50mV, t (27) = 7.1199, *** P = 0.00000012. There were no significant differences between WT and Scn2a KO/+ fast-spiking neurons. Unpaired t -test, peak amplitudes, P7–8, t (17) = 0.8793, P = 0.3915, P15–22, t (22) = 2.0676, P = 0.0506; half widths, P7–8, t (17) = 0.0435, P = 0.9658, P15–22, t (22) = −0.8992, P = 0.3783. Details of the results and statistical tests are reported in Supplementary Tables 1 and 2 . Black filled circles and gray filled circles represent WT and Scn2a KO/+ neurons, respectively. Data represent means ± SEM, * P
    Figure Legend Snippet: Altered action potentials of Scn2a KO excitatory neurons. Responses of neocortical pyramidal excitatory neurons and fast-spiking inhibitory neurons from Vgat -Venus/ Scn2a +/+ (WT) mice ( n = 19 pyramidal and 8 fast-spiking neurons, P7–8; n = 22 pyramidal and 11 fast-spiking neurons, P15–22) and Vgat -Venus/ Scn2a KO/+ (KO/ + ) mice ( n = 16 pyramidal and 11 fast-spiking neurons, P7–8; n = 22 pyramidal and 13 fast-spiking neurons, P15–22) to current injections. Representative action potential traces ( a , e , h , k ), peak amplitudes ( b , f , i , l ), half widths ( c , g , j , m ) and maximum rates of rise ( d ) were shown. b – g Peak amplitudes of P7–8 pyramidal cells are smaller in Scn2a KO/+ than in WT mice, while half widths of P7–8 and P15–22 pyramidal cells were broader in Scn2a KO/+ than in WT mice. Unpaired t -test, peak amplitudes, P7–8, t (33) = 6.0638, *** P = 0.0000008, P15–22, t (42) = 0.0791, P = 0.9373; half widths, P7–8, t (33) = −4.0342, *** P = 0.00031, P15–22, t (42) = −3.6442, * P = 0.0109. d Maximum rates of rise for action potential of P7–8 hippocampal pyramidal cells ( n = 19 WT neurons, n = 11 KO/ + neurons) were lower in Scn2a KO/+ than in WT mice. Unpaired t -test, −120mV, t (28) = 7.3005, *** P = 0.00000006, −110mV, t (28) = 7.3572, *** P = 0.00000005, −100mV, t (28) = 7.3237, *** P = 0.00000006, −90mV, t (28) = 7.4814, *** P = 0.00000004, −80mV, t (28) = 7.6586, *** P = 0.00000002, −70mV, t (28) = 7.2738, *** P = 0.00000006, −60mV, t (28) = 7.8954, *** P = 0.00000001, −50mV, t (27) = 7.1199, *** P = 0.00000012. There were no significant differences between WT and Scn2a KO/+ fast-spiking neurons. Unpaired t -test, peak amplitudes, P7–8, t (17) = 0.8793, P = 0.3915, P15–22, t (22) = 2.0676, P = 0.0506; half widths, P7–8, t (17) = 0.0435, P = 0.9658, P15–22, t (22) = −0.8992, P = 0.3783. Details of the results and statistical tests are reported in Supplementary Tables 1 and 2 . Black filled circles and gray filled circles represent WT and Scn2a KO/+ neurons, respectively. Data represent means ± SEM, * P

    Techniques Used: Mouse Assay

    Scn2a deletion in dorsal telencephalic excitatory but not global inhibitory neurons triggered SWDs in mice. a Survival rates at P2.5 of Scn2a fl/fl ( N = 7), Scn2a fl/fl / Emx1 -Cre ( N = 6) and Scn2a fl/fl / Vgat -Cre mice ( N = 5). All Scn2a fl/fl / Emx1 -Cre and all but one Scn2a fl/fl / Vgat -Cre mice died before P2.5. One Scn2a fl/fl / Vgat -Cre survivor died at P8.5. b Survival curves during P3–30 of Scn2a fl/+ / Emx1 -Cre ( N = 39), Scn2a fl/+ / Vgat -Cre ( N = 30) and Scn2a fl/+ mice ( N = 95). About 30% of Scn2a fl/+ / Vgat -Cre mice suffered premature death between P16 and P25. c Representative ECoG/EMG traces in Scn2a fl/+ / Emx1 -Cre mice. SWDs during waking were often associated with behavioral arrest (right). Black arrowheads indicate the onsets of SWDs. Gray arrowheads indicate the onset and end of behavioral arrest. d , e Frequencies of SWDs during 24 h ECoG recordings in Scn2a fl/+ / Emx1 -Cre ( N = 5) and littermate controls (Cntl) (2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 5), Scn2a fl/+ / Vgat -Cre ( N = 4) and littermate Cntl (2 Scn2a fl/+ , 1 Scn2a +/+ / Vgat -Cre, N = 3), and Scn2a RX/+ mice ( N = 7). All recorded mice were over 8 weeks of age. f Ethosuximide (33.3 mg mL −1 in saline, 200 mg per kg, i.p .) efficiently suppressed SWDs in Scn2a fl/+ / Emx1 -Cre mice ( N = 6). g , h LFP recordings from 3–6-month-old Scn2a fl/+ / Emx1 -Cre ( N = 4) and littermate Cntl (2 Scn2a +/+ , 2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 7). g Representative ECoG/EMG/LFPs traces in Scn2a fl/+ / Emx1 -Cre mice. h Epileptiform discharges were predominantly detected in medial prefrontal cortex and caudate putamen of Scn2a fl/+ / Emx1 -Cre mice (Mann–Whitney test, ECoG, somatosensory cortex: U = 0, * P = 0.0286; LFP, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 6, P > 0.999; basolateral amygdala: U = 0, * P = 0.0268; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0268; ventroposterior thalamus: U = 0, * P = 0.0286). Data represent means ± SEM, * P
    Figure Legend Snippet: Scn2a deletion in dorsal telencephalic excitatory but not global inhibitory neurons triggered SWDs in mice. a Survival rates at P2.5 of Scn2a fl/fl ( N = 7), Scn2a fl/fl / Emx1 -Cre ( N = 6) and Scn2a fl/fl / Vgat -Cre mice ( N = 5). All Scn2a fl/fl / Emx1 -Cre and all but one Scn2a fl/fl / Vgat -Cre mice died before P2.5. One Scn2a fl/fl / Vgat -Cre survivor died at P8.5. b Survival curves during P3–30 of Scn2a fl/+ / Emx1 -Cre ( N = 39), Scn2a fl/+ / Vgat -Cre ( N = 30) and Scn2a fl/+ mice ( N = 95). About 30% of Scn2a fl/+ / Vgat -Cre mice suffered premature death between P16 and P25. c Representative ECoG/EMG traces in Scn2a fl/+ / Emx1 -Cre mice. SWDs during waking were often associated with behavioral arrest (right). Black arrowheads indicate the onsets of SWDs. Gray arrowheads indicate the onset and end of behavioral arrest. d , e Frequencies of SWDs during 24 h ECoG recordings in Scn2a fl/+ / Emx1 -Cre ( N = 5) and littermate controls (Cntl) (2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 5), Scn2a fl/+ / Vgat -Cre ( N = 4) and littermate Cntl (2 Scn2a fl/+ , 1 Scn2a +/+ / Vgat -Cre, N = 3), and Scn2a RX/+ mice ( N = 7). All recorded mice were over 8 weeks of age. f Ethosuximide (33.3 mg mL −1 in saline, 200 mg per kg, i.p .) efficiently suppressed SWDs in Scn2a fl/+ / Emx1 -Cre mice ( N = 6). g , h LFP recordings from 3–6-month-old Scn2a fl/+ / Emx1 -Cre ( N = 4) and littermate Cntl (2 Scn2a +/+ , 2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 7). g Representative ECoG/EMG/LFPs traces in Scn2a fl/+ / Emx1 -Cre mice. h Epileptiform discharges were predominantly detected in medial prefrontal cortex and caudate putamen of Scn2a fl/+ / Emx1 -Cre mice (Mann–Whitney test, ECoG, somatosensory cortex: U = 0, * P = 0.0286; LFP, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 6, P > 0.999; basolateral amygdala: U = 0, * P = 0.0268; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0268; ventroposterior thalamus: U = 0, * P = 0.0286). Data represent means ± SEM, * P

    Techniques Used: Mouse Assay, MANN-WHITNEY

    Nav1.2 expressions at AISs of excitatory neurons in neocortex and hippocampus. Immunofluorescence histochemistry of P15.5 wild-type neocortices and hippocampi stained with anti-Nav1.2 (G-20, magenta), anti-ankyrin G (green), and anti-Tbr1 (cyan) antibodies, and counterstained with 4′-6-diamidino-2-phenylindole (DAPI, gray) and their merged images. Arrows indicate Nav1.2 and ankyrin G-double immunoreactive AISs of Tbr1-expressing neocortical pyramidal cells. Arrowheads indicate Nav1.2 and ankyrin G-double immunoreactive AISs of hippocampal pyramidal cells. Representative images of four or more slices are shown. o stratum oriens, p stratum pyramidale. Scale bars: 20 µm
    Figure Legend Snippet: Nav1.2 expressions at AISs of excitatory neurons in neocortex and hippocampus. Immunofluorescence histochemistry of P15.5 wild-type neocortices and hippocampi stained with anti-Nav1.2 (G-20, magenta), anti-ankyrin G (green), and anti-Tbr1 (cyan) antibodies, and counterstained with 4′-6-diamidino-2-phenylindole (DAPI, gray) and their merged images. Arrows indicate Nav1.2 and ankyrin G-double immunoreactive AISs of Tbr1-expressing neocortical pyramidal cells. Arrowheads indicate Nav1.2 and ankyrin G-double immunoreactive AISs of hippocampal pyramidal cells. Representative images of four or more slices are shown. o stratum oriens, p stratum pyramidale. Scale bars: 20 µm

    Techniques Used: Immunofluorescence, Staining, Expressing

    Scn2a haplodeficiency in dorsal telencephalic excitatory but in those in global inhibitory neurons reduced neocortical and hippocampal Nav1.2 expression levels. Western blot analyses of 6-weeks neocortex or hippocampus for Scn2a fl/+ / Emx1 -Cre, Scn2a fl/+ / Vgat -Cre and Scn2a fl/+ controls ( N = 3, each genotype). Unpaired t -test, Scn2a fl/+ vs. Scn2a fl/+ / Emx1 -Cre, neocortex: t (4) = 5.91, ** P = 0.0041; hippocampus: t (4) = 5.74, ** P = 0.0046; Scn2a fl/+ vs. Scn2a fl/+ / Vgat -Cre, neocortex: t (4) = 1.413, P = 0.2305; hippocampus: t (4) = 0.489, P = 0.6503. Nav1.2 protein was normalized by β-tubulin. Mean Nav1.2 expression levels are represented as percentages relative to the level of Scn2a fl/+ control littermates (100%). Data represent means ± SEM, ** P
    Figure Legend Snippet: Scn2a haplodeficiency in dorsal telencephalic excitatory but in those in global inhibitory neurons reduced neocortical and hippocampal Nav1.2 expression levels. Western blot analyses of 6-weeks neocortex or hippocampus for Scn2a fl/+ / Emx1 -Cre, Scn2a fl/+ / Vgat -Cre and Scn2a fl/+ controls ( N = 3, each genotype). Unpaired t -test, Scn2a fl/+ vs. Scn2a fl/+ / Emx1 -Cre, neocortex: t (4) = 5.91, ** P = 0.0041; hippocampus: t (4) = 5.74, ** P = 0.0046; Scn2a fl/+ vs. Scn2a fl/+ / Vgat -Cre, neocortex: t (4) = 1.413, P = 0.2305; hippocampus: t (4) = 0.489, P = 0.6503. Nav1.2 protein was normalized by β-tubulin. Mean Nav1.2 expression levels are represented as percentages relative to the level of Scn2a fl/+ control littermates (100%). Data represent means ± SEM, ** P

    Techniques Used: Expressing, Western Blot

    Developmental changes of Nav1.2 distribution in mouse brain. Brain sections of wild-type mice at P0.5 ( a , f , k , p ), P2.5 ( b , g , l , q ), P7.5 ( c , h , m , r ), P15.5 ( d , i , n , s ), and 8-week-old ( e , j , o , t ) were stained with anti-Nav1.2 (G-20, red). Higher-magnified images outlined in a – e are shown in f – t . Nav1.2 immunoreactivities were observed at AISs of neocortical neurons (single black arrowheads), nodes of Ranvier within white matter (double black arrowheads), AISs of hippocampal pyramidal neurons (white arrowheads), mossy fibers of dentate granule cells (black arrows), etc. Note that, while Nav1.2 at AISs and nodes of Ranvier peaked at P15.5 and became less at 8-weeks, diffused Nav1.2 signals in neocortex continued to become dense until 8-weeks-old. The brain slices were processed in parallel. Representative images of four or more slices per stage are shown. MZ marginal zone, UCP upper cortical plate, LCP lower cortical plate, DG dentate gyrus, WM white matter, IZ intermediate zone, o stratum oriens, p stratum pyramidale, l stratum lucidum, r stratum radiatum. Scale bars: a – e 500 µm; f – o 50 µm; p – t 100 µm
    Figure Legend Snippet: Developmental changes of Nav1.2 distribution in mouse brain. Brain sections of wild-type mice at P0.5 ( a , f , k , p ), P2.5 ( b , g , l , q ), P7.5 ( c , h , m , r ), P15.5 ( d , i , n , s ), and 8-week-old ( e , j , o , t ) were stained with anti-Nav1.2 (G-20, red). Higher-magnified images outlined in a – e are shown in f – t . Nav1.2 immunoreactivities were observed at AISs of neocortical neurons (single black arrowheads), nodes of Ranvier within white matter (double black arrowheads), AISs of hippocampal pyramidal neurons (white arrowheads), mossy fibers of dentate granule cells (black arrows), etc. Note that, while Nav1.2 at AISs and nodes of Ranvier peaked at P15.5 and became less at 8-weeks, diffused Nav1.2 signals in neocortex continued to become dense until 8-weeks-old. The brain slices were processed in parallel. Representative images of four or more slices per stage are shown. MZ marginal zone, UCP upper cortical plate, LCP lower cortical plate, DG dentate gyrus, WM white matter, IZ intermediate zone, o stratum oriens, p stratum pyramidale, l stratum lucidum, r stratum radiatum. Scale bars: a – e 500 µm; f – o 50 µm; p – t 100 µm

    Techniques Used: Mouse Assay, Staining

    A pathogenic Scn2a nonsense mutation inactivated the mutated allele. a Schematic of the voltage-gated sodium channel Nav1.2, showing the location of R102* (RX) nonsense mutation. Full-length wild type Nav1.2 is composed of 2006-amino acid (aa) residues with the predicted molecular weight of ~228 kD. The RX mutation can cause a truncated peptides, Nav1.2-RX, consisting of the first 101-aa residues of Nav1.2 with the theoretical molecular weight of ~12 kD. Epitope locations for the anti-Nav1.2 (EM-1, ASC-002) and anti-pan Nav1 (SP19) antibodies are indicated. b , c The RX allele was effectively inactivated in Scn2a knock-in mice. Western blot analyses of P0.5 whole brain membrane ( b ) and cytosolic ( c ) fractions were performed using EM-1. b Full-length Nav1.2 was moderate and negligible in Scn2a RX/+ (RX/+, N = 4) and Scn2a RX/RX (RX/RX, N = 3) mice, respectively, compared with that in Scn2a +/+ mice (+/+, N = 3) [one-way analysis of variance; genotype: F (2, 7) = 1737, *** P = 0.00000000036, Tukey’s test; Scn2a +/+ vs . Scn2a RX/+ *** P
    Figure Legend Snippet: A pathogenic Scn2a nonsense mutation inactivated the mutated allele. a Schematic of the voltage-gated sodium channel Nav1.2, showing the location of R102* (RX) nonsense mutation. Full-length wild type Nav1.2 is composed of 2006-amino acid (aa) residues with the predicted molecular weight of ~228 kD. The RX mutation can cause a truncated peptides, Nav1.2-RX, consisting of the first 101-aa residues of Nav1.2 with the theoretical molecular weight of ~12 kD. Epitope locations for the anti-Nav1.2 (EM-1, ASC-002) and anti-pan Nav1 (SP19) antibodies are indicated. b , c The RX allele was effectively inactivated in Scn2a knock-in mice. Western blot analyses of P0.5 whole brain membrane ( b ) and cytosolic ( c ) fractions were performed using EM-1. b Full-length Nav1.2 was moderate and negligible in Scn2a RX/+ (RX/+, N = 4) and Scn2a RX/RX (RX/RX, N = 3) mice, respectively, compared with that in Scn2a +/+ mice (+/+, N = 3) [one-way analysis of variance; genotype: F (2, 7) = 1737, *** P = 0.00000000036, Tukey’s test; Scn2a +/+ vs . Scn2a RX/+ *** P

    Techniques Used: Mutagenesis, Molecular Weight, Knock-In, Mouse Assay, Western Blot

    The pathogenic Scn2a nonsense mutation caused absence-like seizures with SWDs in mice. a Representative traces of somatosensory ECoG/EMG recordings in 6–11 weeks-old Scn2a RX/+ mice ( N = 7). Behavioral arrest during waking state associated with ECoG epileptiform SWDs (left). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. Positivity was plotted up. b Frequencies of SWDs during a 24 h ECoG recording period in Scn2a +/+ ( N = 5) and Scn2a RX/+ ( N = 7) mice. c An episode of non-convulsive epileptiform discharges detected in 1 out of 7 Scn2a RX/+ mice. An arrowhead indicates the onset of epileptiform discharge. Positivity was plotted up. d , e Seizure susceptibility to PTZ in Scn2a RX/+ and Scn2a +/+ mice (10 weeks of age). The latencies to the first appearance of absence seizure-like sudden immobility, myoclonus, clonic convulsion and tonic-clonic convulsion after intraperitoneal administration of PTZ at dose of 50 ( d , N = 20, each genotype) and 25 ( e , N = 14, each genotype) mg per kg body weight. Scn2a RX/+ mice had significantly shorter latencies to absence-like sudden immobility (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 106, * P = 0.0100, myoclonus, U = 116.5, * P = 0.0231, clonic convulsion, U = 162, P = 0.3098, tonic-clonic convulsion, U = 175, P = 0.5023; 25 mg per kg, sudden immobility, U = 53, * P = 0.0383). Data represent means ± SEM, * P
    Figure Legend Snippet: The pathogenic Scn2a nonsense mutation caused absence-like seizures with SWDs in mice. a Representative traces of somatosensory ECoG/EMG recordings in 6–11 weeks-old Scn2a RX/+ mice ( N = 7). Behavioral arrest during waking state associated with ECoG epileptiform SWDs (left). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. Positivity was plotted up. b Frequencies of SWDs during a 24 h ECoG recording period in Scn2a +/+ ( N = 5) and Scn2a RX/+ ( N = 7) mice. c An episode of non-convulsive epileptiform discharges detected in 1 out of 7 Scn2a RX/+ mice. An arrowhead indicates the onset of epileptiform discharge. Positivity was plotted up. d , e Seizure susceptibility to PTZ in Scn2a RX/+ and Scn2a +/+ mice (10 weeks of age). The latencies to the first appearance of absence seizure-like sudden immobility, myoclonus, clonic convulsion and tonic-clonic convulsion after intraperitoneal administration of PTZ at dose of 50 ( d , N = 20, each genotype) and 25 ( e , N = 14, each genotype) mg per kg body weight. Scn2a RX/+ mice had significantly shorter latencies to absence-like sudden immobility (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 106, * P = 0.0100, myoclonus, U = 116.5, * P = 0.0231, clonic convulsion, U = 162, P = 0.3098, tonic-clonic convulsion, U = 175, P = 0.5023; 25 mg per kg, sudden immobility, U = 53, * P = 0.0383). Data represent means ± SEM, * P

    Techniques Used: Mutagenesis, Mouse Assay, MANN-WHITNEY

    Scn2a haplodeficiency did not alter expression levels of other sodium channel subunits. Quantitative RT-PCR analyses of brain mRNAs prepared from P14.5 Scn2a +/+ and Scn2a KO/+ mice ( N = 11, each genotype). Scn2a mRNA expression in Scn2a KO/+ whole brain was reduced to about 50% level of that in Scn2a +/+ mice while there were no significant changes in Scn1a , Scn3a , Scn5a , Scn8a , Scn1b , Scn2b , Scn3b , and Scn4b mRNA expression levels in Scn2a KO/+ , compared with those in Scn2a +/+ mice [unpaired t-test, Scn1a ; t (20) = 1.461, P = 0.1595, Scn2a ; t (20) = 5.250, *** P = 0.000039, Scn3a ; t (20) = 0.5066, P = 0.6180, Scn5a ; t (20) = 0.4223, P = 0.6773, Scn8a ; t (20) = 0.3952, P = 0.6969, Scn1b ; t (20) = 0.7407, P = 0.4675, Scn2b ; t (20) = 0.4259, P = 0.6747, Scn3b ; t (20) = 0.8664, P = 0.3965, Scn4b ; t (20) = 0.5273, P = 0.6038]. White and gray bars represent Scn2a +/+ and Scn2a KO/+ mice, respectively. Data represent means ± SEM, *** P
    Figure Legend Snippet: Scn2a haplodeficiency did not alter expression levels of other sodium channel subunits. Quantitative RT-PCR analyses of brain mRNAs prepared from P14.5 Scn2a +/+ and Scn2a KO/+ mice ( N = 11, each genotype). Scn2a mRNA expression in Scn2a KO/+ whole brain was reduced to about 50% level of that in Scn2a +/+ mice while there were no significant changes in Scn1a , Scn3a , Scn5a , Scn8a , Scn1b , Scn2b , Scn3b , and Scn4b mRNA expression levels in Scn2a KO/+ , compared with those in Scn2a +/+ mice [unpaired t-test, Scn1a ; t (20) = 1.461, P = 0.1595, Scn2a ; t (20) = 5.250, *** P = 0.000039, Scn3a ; t (20) = 0.5066, P = 0.6180, Scn5a ; t (20) = 0.4223, P = 0.6773, Scn8a ; t (20) = 0.3952, P = 0.6969, Scn1b ; t (20) = 0.7407, P = 0.4675, Scn2b ; t (20) = 0.4259, P = 0.6747, Scn3b ; t (20) = 0.8664, P = 0.3965, Scn4b ; t (20) = 0.5273, P = 0.6038]. White and gray bars represent Scn2a +/+ and Scn2a KO/+ mice, respectively. Data represent means ± SEM, *** P

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

    Heterozygous Scn2a knockout mice showed absence-like seizures with SWDs. a Representative traces of ECoG/EMG recordings from 10–27 weeks-old Scn2a KO/+ (KO/+) mice ( N = 6). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. b A representative trace of prolonged non-convulsive seizure. ECoG recordings detected 2 episodes of prolonged non-convulsive seizure with duration of 30–45 s in 2 out of 6 Scn2a KO/+ mice, which were neither accompanied by convulsions, nor followed by post-ictal depression. c Representative ECoG/EMG/LFP recordings during an SWD episode in Scn2a KO/+ mice. Epileptiform discharges with large amplitudes are seen in mPFC and CPu. Positivity was plotted up ( a , b , c ). d Quantification of ECoG SWDs and LFP epileptiform discharges [3-hour recording, light period, Scn2a +/+ , Scn2a KO/+ ( N = 4, each genotype)]. Mann–Whitney test, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 8, P > 0.9999; basolateral amygdala: U = 3, P = 0.2571; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0286; ventroposterior thalamus: U = 7.5, P > 0.9999. e Thresholds of body temperature for hyperthermia-induced seizures did not differ between Scn2a KO/+ and Scn2a +/+ mice (4-week-old, N = 10, each genotype) [unpaired t -test, t (18) = 1.149, P = 0.2658]. f , g Increased seizure susceptibility to PTZ in 10-week-old Scn2a KO/+ mice. Latencies to the first appearance of sudden immobility, myoclonus, clonic convulsion, and tonic-clonic convulsion after administrating of PTZ at doses of 50 ( f , N = 20, each genotype) or 25 ( g , N = 12, each genotype) mg per kg body weight. The latencies to the appearance of sudden immobility, myoclonus and clonic convulsion were shorter in Scn2a KO/+ than in Scn2a +/+ mice (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 69.5, *** P = 0.0002, myoclonus, U = 110.5, * P = 0.0145, clonic convulsion, U = 119, * P = 0.0278, tonic-clonic convulsion, U = 188.5, P = 0.7624; 25 mg per kg, sudden immobility, U = 26.5, ** P = 0.0071). Data represent means ± SEM, * P
    Figure Legend Snippet: Heterozygous Scn2a knockout mice showed absence-like seizures with SWDs. a Representative traces of ECoG/EMG recordings from 10–27 weeks-old Scn2a KO/+ (KO/+) mice ( N = 6). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. b A representative trace of prolonged non-convulsive seizure. ECoG recordings detected 2 episodes of prolonged non-convulsive seizure with duration of 30–45 s in 2 out of 6 Scn2a KO/+ mice, which were neither accompanied by convulsions, nor followed by post-ictal depression. c Representative ECoG/EMG/LFP recordings during an SWD episode in Scn2a KO/+ mice. Epileptiform discharges with large amplitudes are seen in mPFC and CPu. Positivity was plotted up ( a , b , c ). d Quantification of ECoG SWDs and LFP epileptiform discharges [3-hour recording, light period, Scn2a +/+ , Scn2a KO/+ ( N = 4, each genotype)]. Mann–Whitney test, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 8, P > 0.9999; basolateral amygdala: U = 3, P = 0.2571; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0286; ventroposterior thalamus: U = 7.5, P > 0.9999. e Thresholds of body temperature for hyperthermia-induced seizures did not differ between Scn2a KO/+ and Scn2a +/+ mice (4-week-old, N = 10, each genotype) [unpaired t -test, t (18) = 1.149, P = 0.2658]. f , g Increased seizure susceptibility to PTZ in 10-week-old Scn2a KO/+ mice. Latencies to the first appearance of sudden immobility, myoclonus, clonic convulsion, and tonic-clonic convulsion after administrating of PTZ at doses of 50 ( f , N = 20, each genotype) or 25 ( g , N = 12, each genotype) mg per kg body weight. The latencies to the appearance of sudden immobility, myoclonus and clonic convulsion were shorter in Scn2a KO/+ than in Scn2a +/+ mice (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 69.5, *** P = 0.0002, myoclonus, U = 110.5, * P = 0.0145, clonic convulsion, U = 119, * P = 0.0278, tonic-clonic convulsion, U = 188.5, P = 0.7624; 25 mg per kg, sudden immobility, U = 26.5, ** P = 0.0071). Data represent means ± SEM, * P

    Techniques Used: Knock-Out, Mouse Assay, MANN-WHITNEY

    8) Product Images from "Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons"

    Article Title: Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons

    Journal: bioRxiv

    doi: 10.1101/2021.03.31.437794

    AISs of GFP-positive cell are Nav1.2-negative in Scn1a -GFP mouse neocortex. Immunofluorescence images of neocortical L2/3 and L5 of Scn1a -GFP mice (#233 line) at P15 stained by mouse anti-GFP (green), rabbit anti-Nav1.2 (magenta), and goat anti-ankyrin G (cyan) antibodies. Note that AISs of GFP-positive cell are Nav1.2-negative (arrowheads). Scale bars: 50µm.
    Figure Legend Snippet: AISs of GFP-positive cell are Nav1.2-negative in Scn1a -GFP mouse neocortex. Immunofluorescence images of neocortical L2/3 and L5 of Scn1a -GFP mice (#233 line) at P15 stained by mouse anti-GFP (green), rabbit anti-Nav1.2 (magenta), and goat anti-ankyrin G (cyan) antibodies. Note that AISs of GFP-positive cell are Nav1.2-negative (arrowheads). Scale bars: 50µm.

    Techniques Used: Immunofluorescence, Mouse Assay, Staining

    A major population of AISs of TBR1-positive cells are Nav1.2-positive at mouse neocortical layer V/VI Immunofluorescence images of neocortical L5/6 of C57BL/6J at P15 stained with rabbit anti-Nav1.2 (green), alexa 647-conjugated anti-TBR1 (magenta) antibodies, and DAPI (blue). Arrowheads indicate TBR1-positive cells that have Nav1.2-positive AIS. Scale bars: Scale bars: 20µm.
    Figure Legend Snippet: A major population of AISs of TBR1-positive cells are Nav1.2-positive at mouse neocortical layer V/VI Immunofluorescence images of neocortical L5/6 of C57BL/6J at P15 stained with rabbit anti-Nav1.2 (green), alexa 647-conjugated anti-TBR1 (magenta) antibodies, and DAPI (blue). Arrowheads indicate TBR1-positive cells that have Nav1.2-positive AIS. Scale bars: Scale bars: 20µm.

    Techniques Used: Immunofluorescence, Staining

    Nav1.1 and Nav1.2 distributions among neocortical projection neurons. Nav1.1 is expressed in PT, CCa, CCb, while Nav1.2 is expressed in CT, iCS and CCc neocortical projection neurons. A subpopulation of CCc neurons are TBR1-positive, and most of those are assumed to be FEZF2-negative because of the paucity of cells with FEZF2 signals in L2/3. PT: pyramidal tract projection neurons. CCa, CCb, CCc: cortico-cortical projection neuron subpopulations. iCS: intratelencephalic cortico-striatal projection neurons. CT: cortico-thalamic projection neurons.
    Figure Legend Snippet: Nav1.1 and Nav1.2 distributions among neocortical projection neurons. Nav1.1 is expressed in PT, CCa, CCb, while Nav1.2 is expressed in CT, iCS and CCc neocortical projection neurons. A subpopulation of CCc neurons are TBR1-positive, and most of those are assumed to be FEZF2-negative because of the paucity of cells with FEZF2 signals in L2/3. PT: pyramidal tract projection neurons. CCa, CCb, CCc: cortico-cortical projection neuron subpopulations. iCS: intratelencephalic cortico-striatal projection neurons. CT: cortico-thalamic projection neurons.

    Techniques Used: Countercurrent Chromatography

    9) Product Images from "Poly-dipeptides produced from C9orf72 hexanucleotide repeats cause selective motor neuron hyperexcitability in ALS"

    Article Title: Poly-dipeptides produced from C9orf72 hexanucleotide repeats cause selective motor neuron hyperexcitability in ALS

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.2113813119

    β4 is critical for the PR 20 -mediated modulation of Nav1.2 channels. ( A–J ) Effects of PR 20 peptide on I-V relationships of the hNav1.2 channel coexpressed with β1 and β2 ( A ), the hNav1.2 channel coexpressed with β1 and β4 ( B ), the hNav1.6 channel coexpressed with β1 and β2 ( C ), the hNav1.6 channel coexpressed with β1 and β4 ( D ), the hNav1.2 channel coexpressed with β1 ( E ), the hNav1.2 channel coexpressed with β2 ( F ), the hNav1.2 channel coexpressed with β4 ( G ), the hNav1.2 channel coexpressed with the β4 C58A mutant ( H ), the hNav1.2 channel coexpressed with the β1 and β4 C58A mutant ( I ), and the hNav1.5 channel coexpressed with β4 ( J ) in HEK-293T cells. Currents were elicited from a V h of −120 mV to test potentials ranging from −60 to +20 mV in 5-mV increments. Black circles indicate control; red circles indicate 150 s after PR 20 application. All data are mean ± SEM. * P
    Figure Legend Snippet: β4 is critical for the PR 20 -mediated modulation of Nav1.2 channels. ( A–J ) Effects of PR 20 peptide on I-V relationships of the hNav1.2 channel coexpressed with β1 and β2 ( A ), the hNav1.2 channel coexpressed with β1 and β4 ( B ), the hNav1.6 channel coexpressed with β1 and β2 ( C ), the hNav1.6 channel coexpressed with β1 and β4 ( D ), the hNav1.2 channel coexpressed with β1 ( E ), the hNav1.2 channel coexpressed with β2 ( F ), the hNav1.2 channel coexpressed with β4 ( G ), the hNav1.2 channel coexpressed with the β4 C58A mutant ( H ), the hNav1.2 channel coexpressed with the β1 and β4 C58A mutant ( I ), and the hNav1.5 channel coexpressed with β4 ( J ) in HEK-293T cells. Currents were elicited from a V h of −120 mV to test potentials ranging from −60 to +20 mV in 5-mV increments. Black circles indicate control; red circles indicate 150 s after PR 20 application. All data are mean ± SEM. * P

    Techniques Used: Mutagenesis

    Both I NaP and hyperexcitability induced by PR 20 are blocked by antibodies targeting Nav1.2, β1, and β4. ( A ) A percentage increase graph for changes in I NaP at −20 mV of cortical motor neurons by PR 20 treatment (100 nM, 20 min) in the presence of the control IgG or Nav antibodies as indicated. I NaP was evoked by a depolarizing voltage ramp (−60 to 0 mV, 60 mV/s). All data are mean ± SEM. IgG: n = 5 and 2 mice. Nav1.2: n = 7 and 4 mice. Nav1.6: n = 5 and 2 mice. β1: n = 5 and 2 mice. β2: n = 3 and 2 mice. β4: n = 6 and 3 mice. Numbers of cells/mice analyzed are shown in parentheses. NS indicates not significantly different. * P
    Figure Legend Snippet: Both I NaP and hyperexcitability induced by PR 20 are blocked by antibodies targeting Nav1.2, β1, and β4. ( A ) A percentage increase graph for changes in I NaP at −20 mV of cortical motor neurons by PR 20 treatment (100 nM, 20 min) in the presence of the control IgG or Nav antibodies as indicated. I NaP was evoked by a depolarizing voltage ramp (−60 to 0 mV, 60 mV/s). All data are mean ± SEM. IgG: n = 5 and 2 mice. Nav1.2: n = 7 and 4 mice. Nav1.6: n = 5 and 2 mice. β1: n = 5 and 2 mice. β2: n = 3 and 2 mice. β4: n = 6 and 3 mice. Numbers of cells/mice analyzed are shown in parentheses. NS indicates not significantly different. * P

    Techniques Used: Mouse Assay

    10) Product Images from "Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons"

    Article Title: Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons

    Journal: bioRxiv

    doi: 10.1101/2021.03.31.437794

    AISs of GFP-positive cell are Nav1.2-negative in Scn1a -GFP mouse neocortex. Immunofluorescence images of neocortical L2/3 and L5 of Scn1a -GFP mice (#233 line) at P15 stained by mouse anti-GFP (green), rabbit anti-Nav1.2 (magenta), and goat anti-ankyrin G (cyan) antibodies. Note that AISs of GFP-positive cell are Nav1.2-negative (arrowheads). Scale bars: 50µm.
    Figure Legend Snippet: AISs of GFP-positive cell are Nav1.2-negative in Scn1a -GFP mouse neocortex. Immunofluorescence images of neocortical L2/3 and L5 of Scn1a -GFP mice (#233 line) at P15 stained by mouse anti-GFP (green), rabbit anti-Nav1.2 (magenta), and goat anti-ankyrin G (cyan) antibodies. Note that AISs of GFP-positive cell are Nav1.2-negative (arrowheads). Scale bars: 50µm.

    Techniques Used: Immunofluorescence, Mouse Assay, Staining

    A major population of AISs of TBR1-positive cells are Nav1.2-positive at mouse neocortical layer V/VI Immunofluorescence images of neocortical L5/6 of C57BL/6J at P15 stained with rabbit anti-Nav1.2 (green), alexa 647-conjugated anti-TBR1 (magenta) antibodies, and DAPI (blue). Arrowheads indicate TBR1-positive cells that have Nav1.2-positive AIS. Scale bars: Scale bars: 20µm.
    Figure Legend Snippet: A major population of AISs of TBR1-positive cells are Nav1.2-positive at mouse neocortical layer V/VI Immunofluorescence images of neocortical L5/6 of C57BL/6J at P15 stained with rabbit anti-Nav1.2 (green), alexa 647-conjugated anti-TBR1 (magenta) antibodies, and DAPI (blue). Arrowheads indicate TBR1-positive cells that have Nav1.2-positive AIS. Scale bars: Scale bars: 20µm.

    Techniques Used: Immunofluorescence, Staining

    Nav1.1 and Nav1.2 dis l projection neurons. Nav1.1 is expressed in PT, CCa, CCb, while Nav1.2 is expressed in CT, iCS and CCc neocortical projection neurons. A subpopulation of CCc neurons are TBR1-positive, and most of those are assumed to be FEZF2-negative because of the paucity of cells with FEZF2 signals in L2/3. PT: pyramidal tract projection neurons. CCa, CCb, CCc: cortico-cortical projection neuron subpopulations. iCS: intratelencephalic cortico-striatal projection neurons. CT: cortico-thalamic projection neurons.
    Figure Legend Snippet: Nav1.1 and Nav1.2 dis l projection neurons. Nav1.1 is expressed in PT, CCa, CCb, while Nav1.2 is expressed in CT, iCS and CCc neocortical projection neurons. A subpopulation of CCc neurons are TBR1-positive, and most of those are assumed to be FEZF2-negative because of the paucity of cells with FEZF2 signals in L2/3. PT: pyramidal tract projection neurons. CCa, CCb, CCc: cortico-cortical projection neuron subpopulations. iCS: intratelencephalic cortico-striatal projection neurons. CT: cortico-thalamic projection neurons.

    Techniques Used: Countercurrent Chromatography

    11) Product Images from "Severe deficiency of voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice"

    Article Title: Severe deficiency of voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice

    Journal: bioRxiv

    doi: 10.1101/2021.02.02.429384

    Elevated neuronal excitability is autonomous. ( A ) Scn2a gtKO/ gtKO (HOM) mice were injected with a dilute FlpO virus, transducing a subset of neurons in the striatum sparsely. Dashed circles highlight two neighboring AAV-negative (blue circle) and AAV-FlpO-positive (magenta circle) neurons. The images were taken in the cell-attached configuration, and after that, the target neurons were used for whole-cell recordings. ( B ) Representative current-clamp recordings of AAV-negative (blue) and AAV-FlpO-positive (magenta) MSNs in CPu of Scn2a gtKO/ gtKO mice were obtained at the RMP. A series of 400-ms hyperpolarizing and depolarizing steps in 50-pA increments were applied to produce the traces. Inset: representative trace in response to 350 pA positive current injection. ( C ) The average number of APs generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U -test for each current pulse: ***p
    Figure Legend Snippet: Elevated neuronal excitability is autonomous. ( A ) Scn2a gtKO/ gtKO (HOM) mice were injected with a dilute FlpO virus, transducing a subset of neurons in the striatum sparsely. Dashed circles highlight two neighboring AAV-negative (blue circle) and AAV-FlpO-positive (magenta circle) neurons. The images were taken in the cell-attached configuration, and after that, the target neurons were used for whole-cell recordings. ( B ) Representative current-clamp recordings of AAV-negative (blue) and AAV-FlpO-positive (magenta) MSNs in CPu of Scn2a gtKO/ gtKO mice were obtained at the RMP. A series of 400-ms hyperpolarizing and depolarizing steps in 50-pA increments were applied to produce the traces. Inset: representative trace in response to 350 pA positive current injection. ( C ) The average number of APs generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U -test for each current pulse: ***p

    Techniques Used: Mouse Assay, Injection, Generated, Two Tailed Test, MANN-WHITNEY

    Activation of KV channels reverses elevated neuronal firings in adult Na v 1.2-deficient mice. ( A ) Volcano plot displays Scn2a and Scn8a , as well all potassium channels that are statistically down-regulated in Scn2a gtKO/ gtKO (HOM) mice compared to WT mice identified by RNA-seq. Statistically significantly upregulated genes are shown in yellow and downregulated genes are shown in blue. ( B ) List of potassium channels that are significantly down-regulated in HOM mice compared to WT. (Hits that are identified from both DESeq2 and edgeR differential expression analysis with False Discovery Rate
    Figure Legend Snippet: Activation of KV channels reverses elevated neuronal firings in adult Na v 1.2-deficient mice. ( A ) Volcano plot displays Scn2a and Scn8a , as well all potassium channels that are statistically down-regulated in Scn2a gtKO/ gtKO (HOM) mice compared to WT mice identified by RNA-seq. Statistically significantly upregulated genes are shown in yellow and downregulated genes are shown in blue. ( B ) List of potassium channels that are significantly down-regulated in HOM mice compared to WT. (Hits that are identified from both DESeq2 and edgeR differential expression analysis with False Discovery Rate

    Techniques Used: Activation Assay, Mouse Assay, RNA Sequencing Assay, Expressing

    Elevated neuronal firings of striatal MSNs at a fixed membrane potential of −80 mV in adult Na v 1.2-deficient mice. Related to Figure 1 . ( A ) gtKO allele has an inserted tm1a trapping cassette between the Exon 1 and Exon 2 of Scn2a gene in the genome, which traps the transcription from Exon 1 to tm1a cassette, resulting in “gene-trap” knockout of Scn2a . In the presence of Flp recombinase, frt sites flanked trapping cassette will be removed, producing conditional (“rescue”) allele that allows the expression of Scn2a at the WT level. frt , Flp recognition target (purple); En2 , engrailed-2 splice acceptor (red); LacZ , lacZ β-galactosidase (light blue); LoxP , locus of X-over P1 (dark blue); and Neo , neomycin (green). ( B ) gtKO cassette contains a LacZ element and is driven by the native Scn2a promoter. Thus, the LacZ expression can be used as a surrogate of Scn2a expression. Representative LacZ staining of a sagittal slice from a Scn2a gtKO/gtKO (HOM) mouse showing a strong blue signal across the brain including the prefrontal cortex (PFC) and dorsal striatum (CPu, caudate nucleus and the putamen). ( C ) Upper: Representative Western blots of striatal tissues from WT (black circle), HET (magenta diamond), and HOM (blue square) mice. Lower: associated quantification of Na v 1.2 protein. One-way ANOVA followed by Tukey’s multiple-comparison test: ***p
    Figure Legend Snippet: Elevated neuronal firings of striatal MSNs at a fixed membrane potential of −80 mV in adult Na v 1.2-deficient mice. Related to Figure 1 . ( A ) gtKO allele has an inserted tm1a trapping cassette between the Exon 1 and Exon 2 of Scn2a gene in the genome, which traps the transcription from Exon 1 to tm1a cassette, resulting in “gene-trap” knockout of Scn2a . In the presence of Flp recombinase, frt sites flanked trapping cassette will be removed, producing conditional (“rescue”) allele that allows the expression of Scn2a at the WT level. frt , Flp recognition target (purple); En2 , engrailed-2 splice acceptor (red); LacZ , lacZ β-galactosidase (light blue); LoxP , locus of X-over P1 (dark blue); and Neo , neomycin (green). ( B ) gtKO cassette contains a LacZ element and is driven by the native Scn2a promoter. Thus, the LacZ expression can be used as a surrogate of Scn2a expression. Representative LacZ staining of a sagittal slice from a Scn2a gtKO/gtKO (HOM) mouse showing a strong blue signal across the brain including the prefrontal cortex (PFC) and dorsal striatum (CPu, caudate nucleus and the putamen). ( C ) Upper: Representative Western blots of striatal tissues from WT (black circle), HET (magenta diamond), and HOM (blue square) mice. Lower: associated quantification of Na v 1.2 protein. One-way ANOVA followed by Tukey’s multiple-comparison test: ***p

    Techniques Used: Mouse Assay, Knock-Out, Expressing, Staining, Western Blot

    Elevated neuronal firings of layer V pyramidal cells in the mPFC are reversible by FlpO-mediated rescue in adult Na v 1.2-deficient mice. Related to Figure 2 . ( A-B ) LacZ staining of coronal brain slices containing mPFC from WT and Scn2a gtKO/gtKO (HOM) mice, which were systemically administered with AAV-Control or AAV-FlpO. PrL, prelimbic cortex; IL, infralimbic cortex. ( C ) A typical layer V pyramidal neuron in the mPFC was labeled by neurobiotin. Scale bar, 50 μm. ( D ) Representative current-clamp recordings of pyramidal cells from WT mice transduced with AAV-FlpO (red), HOM mice transduced with AAV-Control (blue), and HOM mice transduced with AAV-Control (magenta) at the RMP. A series of 400-ms hyperpolarizing and depolarizing steps in 50-pA increments were applied to produce the traces. Inset: representative trace in response to 250 pA positive current injection. ( E ) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired two-tailed non-parametric Mann-Whitney U -test for each current pulse: ns, no significance, p > 0.05; *p
    Figure Legend Snippet: Elevated neuronal firings of layer V pyramidal cells in the mPFC are reversible by FlpO-mediated rescue in adult Na v 1.2-deficient mice. Related to Figure 2 . ( A-B ) LacZ staining of coronal brain slices containing mPFC from WT and Scn2a gtKO/gtKO (HOM) mice, which were systemically administered with AAV-Control or AAV-FlpO. PrL, prelimbic cortex; IL, infralimbic cortex. ( C ) A typical layer V pyramidal neuron in the mPFC was labeled by neurobiotin. Scale bar, 50 μm. ( D ) Representative current-clamp recordings of pyramidal cells from WT mice transduced with AAV-FlpO (red), HOM mice transduced with AAV-Control (blue), and HOM mice transduced with AAV-Control (magenta) at the RMP. A series of 400-ms hyperpolarizing and depolarizing steps in 50-pA increments were applied to produce the traces. Inset: representative trace in response to 250 pA positive current injection. ( E ) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired two-tailed non-parametric Mann-Whitney U -test for each current pulse: ns, no significance, p > 0.05; *p

    Techniques Used: Mouse Assay, Staining, Labeling, Transduction, Injection, Generated, Two Tailed Test, MANN-WHITNEY

    Elevated neuronal firing is reversible by FlpO-mediated restoration of Na v 1.2 expression in adult Na v 1.2-deficient mice. ( A ) Cartoon illustration of mice systemically administrated with PHP.eB.AAV-control or AAV-FlpO via tail vein injection. ( B ) Coronal views of LacZ staining of striatum from WT and Scn2a gtKO/gtKO (HOM) mice injected with AAV-control or AAV-FlpO. Blue staining of HOM mice largely disappeared in the AAV-FlpO group. CPu, caudate nucleus and the putamen (dorsal striatum). ( C ) The Western blot analysis showed Na v 1.2 protein levels in whole-brain tissues from Scn2a gtKO/ gtKO (HOM) mice in AAV-Control or AAV-FlpO group. One-way ANOVA with Bonferroni’s multiple-comparison test: ns, no significance, p > 0.05; *p
    Figure Legend Snippet: Elevated neuronal firing is reversible by FlpO-mediated restoration of Na v 1.2 expression in adult Na v 1.2-deficient mice. ( A ) Cartoon illustration of mice systemically administrated with PHP.eB.AAV-control or AAV-FlpO via tail vein injection. ( B ) Coronal views of LacZ staining of striatum from WT and Scn2a gtKO/gtKO (HOM) mice injected with AAV-control or AAV-FlpO. Blue staining of HOM mice largely disappeared in the AAV-FlpO group. CPu, caudate nucleus and the putamen (dorsal striatum). ( C ) The Western blot analysis showed Na v 1.2 protein levels in whole-brain tissues from Scn2a gtKO/ gtKO (HOM) mice in AAV-Control or AAV-FlpO group. One-way ANOVA with Bonferroni’s multiple-comparison test: ns, no significance, p > 0.05; *p

    Techniques Used: Expressing, Mouse Assay, Injection, Staining, Western Blot

    Ex vivo recordings of MSNs at a fixed membrane potential of −80 mV in adult Na v 1.2-deficient mice with a dilute AAV-FlpO-mCherry injection. Related to Figure 3 . ( A ) Representative current-clamp recordings of MSNs with AAV-negative (blue) and with AAV-FlpO-positive (magenta) in Scn2a gtKO/gtKO (HOM) mice were obtained at a fixed membrane potential of −80 mV. A series of 400-ms hyperpolarizing and depolarizing steps in 50-pA increments were applied to produce the traces. Inset: representative trace in response to 350 pA positive current injection. ( B ) The average number of APs generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U -test for each current pulse: *p
    Figure Legend Snippet: Ex vivo recordings of MSNs at a fixed membrane potential of −80 mV in adult Na v 1.2-deficient mice with a dilute AAV-FlpO-mCherry injection. Related to Figure 3 . ( A ) Representative current-clamp recordings of MSNs with AAV-negative (blue) and with AAV-FlpO-positive (magenta) in Scn2a gtKO/gtKO (HOM) mice were obtained at a fixed membrane potential of −80 mV. A series of 400-ms hyperpolarizing and depolarizing steps in 50-pA increments were applied to produce the traces. Inset: representative trace in response to 350 pA positive current injection. ( B ) The average number of APs generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U -test for each current pulse: *p

    Techniques Used: Ex Vivo, Mouse Assay, Injection, Generated, Two Tailed Test, MANN-WHITNEY

    Elevated neuronal firings of striatal medium spiny neurons (MSNs) in adult Na V 1.2-deficient mice. ( A ) A typical MSN labeled by neurobiotin. Scale bar, 40 μm. ( B ) Representative current-clamp recordings of MSNs from WT (black) and homozygous (HOM), Scn2a gtKO/gtKO (blue) mice were obtained at the resting membrane potential (RMP). A series of 400-ms hyperpolarizing and depolarizing steps in 50-pA increments were applied to produce the traces. Inset: representative trace in response to 350 pA positive current injection. ( C ) The average number of action potentials (APs) generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U -test for each current pulse: ***p
    Figure Legend Snippet: Elevated neuronal firings of striatal medium spiny neurons (MSNs) in adult Na V 1.2-deficient mice. ( A ) A typical MSN labeled by neurobiotin. Scale bar, 40 μm. ( B ) Representative current-clamp recordings of MSNs from WT (black) and homozygous (HOM), Scn2a gtKO/gtKO (blue) mice were obtained at the resting membrane potential (RMP). A series of 400-ms hyperpolarizing and depolarizing steps in 50-pA increments were applied to produce the traces. Inset: representative trace in response to 350 pA positive current injection. ( C ) The average number of action potentials (APs) generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U -test for each current pulse: ***p

    Techniques Used: Mouse Assay, Labeling, Injection, Generated, Two Tailed Test, MANN-WHITNEY

    Specific activation of KV1.1 channel by 4TFMPG reverses the elevated neuronal firings in adult Na v 1.2-deficient mice. Related to Figure 4 . ( A ) Quantitative (q)PCR analysis of Scn2a and Scn8a mRNA in the striatum samples from WT and Scn2a gtKO/ gtKO mice. Unpaired two-tailed Student’s t -test for each group: ns, no significance, *p > 0.05; **p
    Figure Legend Snippet: Specific activation of KV1.1 channel by 4TFMPG reverses the elevated neuronal firings in adult Na v 1.2-deficient mice. Related to Figure 4 . ( A ) Quantitative (q)PCR analysis of Scn2a and Scn8a mRNA in the striatum samples from WT and Scn2a gtKO/ gtKO mice. Unpaired two-tailed Student’s t -test for each group: ns, no significance, *p > 0.05; **p

    Techniques Used: Activation Assay, Mouse Assay, Two Tailed Test

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    Alomone Labs nav1 2 polyclonal
    Differential expression of Nav1.6 and <t>Nav1.2</t> at developing CG cell AISs
    Nav1 2 Polyclonal, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Differential expression of Nav1.6 and Nav1.2 at developing CG cell AISs

    Journal: The Journal of Physiology

    Article Title: Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells

    doi: 10.1113/jphysiol.2010.183798

    Figure Lengend Snippet: Differential expression of Nav1.6 and Nav1.2 at developing CG cell AISs

    Article Snippet: Primary antibodies used and dilutions were as follows: serine-rich domain of ankyrin-G node (polyclonal, ), 1/500; ankyrin-G (monoclonal 4G3F8, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1/50; Nav1.1 (polyclonal AB5204, Chemicon), 1/150; Nav1.2 polyclonal (ASC-002, Alomone Laboratories, Jerusalem, Israel), 1/100; Nav1.2 monoclonal (K69/3, Upstate Biotechnology, Lake Placid, NY, USA), 1/100; Nav1.6 polyclonal (ASC-009, Alomone), 1/100; Nav1.6 monoclonal (K87A/10, Neuromab, Davis, CA, USA), 1/200.

    Techniques: Expressing

    Developmental regulation of Nav1.2 and Nav1.6 clustering at CG cell AISs in mice aged P10–P60

    Journal: The Journal of Physiology

    Article Title: Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells

    doi: 10.1113/jphysiol.2010.183798

    Figure Lengend Snippet: Developmental regulation of Nav1.2 and Nav1.6 clustering at CG cell AISs in mice aged P10–P60

    Article Snippet: Primary antibodies used and dilutions were as follows: serine-rich domain of ankyrin-G node (polyclonal, ), 1/500; ankyrin-G (monoclonal 4G3F8, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1/50; Nav1.1 (polyclonal AB5204, Chemicon), 1/150; Nav1.2 polyclonal (ASC-002, Alomone Laboratories, Jerusalem, Israel), 1/100; Nav1.2 monoclonal (K69/3, Upstate Biotechnology, Lake Placid, NY, USA), 1/100; Nav1.6 polyclonal (ASC-009, Alomone), 1/100; Nav1.6 monoclonal (K87A/10, Neuromab, Davis, CA, USA), 1/200.

    Techniques: Mouse Assay

    Altered action potentials of Scn2a KO excitatory neurons. Responses of neocortical pyramidal excitatory neurons and fast-spiking inhibitory neurons from Vgat -Venus/ Scn2a +/+ (WT) mice ( n = 19 pyramidal and 8 fast-spiking neurons, P7–8; n = 22 pyramidal and 11 fast-spiking neurons, P15–22) and Vgat -Venus/ Scn2a KO/+ (KO/ + ) mice ( n = 16 pyramidal and 11 fast-spiking neurons, P7–8; n = 22 pyramidal and 13 fast-spiking neurons, P15–22) to current injections. Representative action potential traces ( a , e , h , k ), peak amplitudes ( b , f , i , l ), half widths ( c , g , j , m ) and maximum rates of rise ( d ) were shown. b – g Peak amplitudes of P7–8 pyramidal cells are smaller in Scn2a KO/+ than in WT mice, while half widths of P7–8 and P15–22 pyramidal cells were broader in Scn2a KO/+ than in WT mice. Unpaired t -test, peak amplitudes, P7–8, t (33) = 6.0638, *** P = 0.0000008, P15–22, t (42) = 0.0791, P = 0.9373; half widths, P7–8, t (33) = −4.0342, *** P = 0.00031, P15–22, t (42) = −3.6442, * P = 0.0109. d Maximum rates of rise for action potential of P7–8 hippocampal pyramidal cells ( n = 19 WT neurons, n = 11 KO/ + neurons) were lower in Scn2a KO/+ than in WT mice. Unpaired t -test, −120mV, t (28) = 7.3005, *** P = 0.00000006, −110mV, t (28) = 7.3572, *** P = 0.00000005, −100mV, t (28) = 7.3237, *** P = 0.00000006, −90mV, t (28) = 7.4814, *** P = 0.00000004, −80mV, t (28) = 7.6586, *** P = 0.00000002, −70mV, t (28) = 7.2738, *** P = 0.00000006, −60mV, t (28) = 7.8954, *** P = 0.00000001, −50mV, t (27) = 7.1199, *** P = 0.00000012. There were no significant differences between WT and Scn2a KO/+ fast-spiking neurons. Unpaired t -test, peak amplitudes, P7–8, t (17) = 0.8793, P = 0.3915, P15–22, t (22) = 2.0676, P = 0.0506; half widths, P7–8, t (17) = 0.0435, P = 0.9658, P15–22, t (22) = −0.8992, P = 0.3783. Details of the results and statistical tests are reported in Supplementary Tables 1 and 2 . Black filled circles and gray filled circles represent WT and Scn2a KO/+ neurons, respectively. Data represent means ± SEM, * P

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: Altered action potentials of Scn2a KO excitatory neurons. Responses of neocortical pyramidal excitatory neurons and fast-spiking inhibitory neurons from Vgat -Venus/ Scn2a +/+ (WT) mice ( n = 19 pyramidal and 8 fast-spiking neurons, P7–8; n = 22 pyramidal and 11 fast-spiking neurons, P15–22) and Vgat -Venus/ Scn2a KO/+ (KO/ + ) mice ( n = 16 pyramidal and 11 fast-spiking neurons, P7–8; n = 22 pyramidal and 13 fast-spiking neurons, P15–22) to current injections. Representative action potential traces ( a , e , h , k ), peak amplitudes ( b , f , i , l ), half widths ( c , g , j , m ) and maximum rates of rise ( d ) were shown. b – g Peak amplitudes of P7–8 pyramidal cells are smaller in Scn2a KO/+ than in WT mice, while half widths of P7–8 and P15–22 pyramidal cells were broader in Scn2a KO/+ than in WT mice. Unpaired t -test, peak amplitudes, P7–8, t (33) = 6.0638, *** P = 0.0000008, P15–22, t (42) = 0.0791, P = 0.9373; half widths, P7–8, t (33) = −4.0342, *** P = 0.00031, P15–22, t (42) = −3.6442, * P = 0.0109. d Maximum rates of rise for action potential of P7–8 hippocampal pyramidal cells ( n = 19 WT neurons, n = 11 KO/ + neurons) were lower in Scn2a KO/+ than in WT mice. Unpaired t -test, −120mV, t (28) = 7.3005, *** P = 0.00000006, −110mV, t (28) = 7.3572, *** P = 0.00000005, −100mV, t (28) = 7.3237, *** P = 0.00000006, −90mV, t (28) = 7.4814, *** P = 0.00000004, −80mV, t (28) = 7.6586, *** P = 0.00000002, −70mV, t (28) = 7.2738, *** P = 0.00000006, −60mV, t (28) = 7.8954, *** P = 0.00000001, −50mV, t (27) = 7.1199, *** P = 0.00000012. There were no significant differences between WT and Scn2a KO/+ fast-spiking neurons. Unpaired t -test, peak amplitudes, P7–8, t (17) = 0.8793, P = 0.3915, P15–22, t (22) = 2.0676, P = 0.0506; half widths, P7–8, t (17) = 0.0435, P = 0.9658, P15–22, t (22) = −0.8992, P = 0.3783. Details of the results and statistical tests are reported in Supplementary Tables 1 and 2 . Black filled circles and gray filled circles represent WT and Scn2a KO/+ neurons, respectively. Data represent means ± SEM, * P

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Mouse Assay

    Scn2a deletion in dorsal telencephalic excitatory but not global inhibitory neurons triggered SWDs in mice. a Survival rates at P2.5 of Scn2a fl/fl ( N = 7), Scn2a fl/fl / Emx1 -Cre ( N = 6) and Scn2a fl/fl / Vgat -Cre mice ( N = 5). All Scn2a fl/fl / Emx1 -Cre and all but one Scn2a fl/fl / Vgat -Cre mice died before P2.5. One Scn2a fl/fl / Vgat -Cre survivor died at P8.5. b Survival curves during P3–30 of Scn2a fl/+ / Emx1 -Cre ( N = 39), Scn2a fl/+ / Vgat -Cre ( N = 30) and Scn2a fl/+ mice ( N = 95). About 30% of Scn2a fl/+ / Vgat -Cre mice suffered premature death between P16 and P25. c Representative ECoG/EMG traces in Scn2a fl/+ / Emx1 -Cre mice. SWDs during waking were often associated with behavioral arrest (right). Black arrowheads indicate the onsets of SWDs. Gray arrowheads indicate the onset and end of behavioral arrest. d , e Frequencies of SWDs during 24 h ECoG recordings in Scn2a fl/+ / Emx1 -Cre ( N = 5) and littermate controls (Cntl) (2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 5), Scn2a fl/+ / Vgat -Cre ( N = 4) and littermate Cntl (2 Scn2a fl/+ , 1 Scn2a +/+ / Vgat -Cre, N = 3), and Scn2a RX/+ mice ( N = 7). All recorded mice were over 8 weeks of age. f Ethosuximide (33.3 mg mL −1 in saline, 200 mg per kg, i.p .) efficiently suppressed SWDs in Scn2a fl/+ / Emx1 -Cre mice ( N = 6). g , h LFP recordings from 3–6-month-old Scn2a fl/+ / Emx1 -Cre ( N = 4) and littermate Cntl (2 Scn2a +/+ , 2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 7). g Representative ECoG/EMG/LFPs traces in Scn2a fl/+ / Emx1 -Cre mice. h Epileptiform discharges were predominantly detected in medial prefrontal cortex and caudate putamen of Scn2a fl/+ / Emx1 -Cre mice (Mann–Whitney test, ECoG, somatosensory cortex: U = 0, * P = 0.0286; LFP, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 6, P > 0.999; basolateral amygdala: U = 0, * P = 0.0268; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0268; ventroposterior thalamus: U = 0, * P = 0.0286). Data represent means ± SEM, * P

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: Scn2a deletion in dorsal telencephalic excitatory but not global inhibitory neurons triggered SWDs in mice. a Survival rates at P2.5 of Scn2a fl/fl ( N = 7), Scn2a fl/fl / Emx1 -Cre ( N = 6) and Scn2a fl/fl / Vgat -Cre mice ( N = 5). All Scn2a fl/fl / Emx1 -Cre and all but one Scn2a fl/fl / Vgat -Cre mice died before P2.5. One Scn2a fl/fl / Vgat -Cre survivor died at P8.5. b Survival curves during P3–30 of Scn2a fl/+ / Emx1 -Cre ( N = 39), Scn2a fl/+ / Vgat -Cre ( N = 30) and Scn2a fl/+ mice ( N = 95). About 30% of Scn2a fl/+ / Vgat -Cre mice suffered premature death between P16 and P25. c Representative ECoG/EMG traces in Scn2a fl/+ / Emx1 -Cre mice. SWDs during waking were often associated with behavioral arrest (right). Black arrowheads indicate the onsets of SWDs. Gray arrowheads indicate the onset and end of behavioral arrest. d , e Frequencies of SWDs during 24 h ECoG recordings in Scn2a fl/+ / Emx1 -Cre ( N = 5) and littermate controls (Cntl) (2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 5), Scn2a fl/+ / Vgat -Cre ( N = 4) and littermate Cntl (2 Scn2a fl/+ , 1 Scn2a +/+ / Vgat -Cre, N = 3), and Scn2a RX/+ mice ( N = 7). All recorded mice were over 8 weeks of age. f Ethosuximide (33.3 mg mL −1 in saline, 200 mg per kg, i.p .) efficiently suppressed SWDs in Scn2a fl/+ / Emx1 -Cre mice ( N = 6). g , h LFP recordings from 3–6-month-old Scn2a fl/+ / Emx1 -Cre ( N = 4) and littermate Cntl (2 Scn2a +/+ , 2 Scn2a fl/+ , 3 Scn2a +/+ / Emx1 -Cre, N = 7). g Representative ECoG/EMG/LFPs traces in Scn2a fl/+ / Emx1 -Cre mice. h Epileptiform discharges were predominantly detected in medial prefrontal cortex and caudate putamen of Scn2a fl/+ / Emx1 -Cre mice (Mann–Whitney test, ECoG, somatosensory cortex: U = 0, * P = 0.0286; LFP, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 6, P > 0.999; basolateral amygdala: U = 0, * P = 0.0268; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0268; ventroposterior thalamus: U = 0, * P = 0.0286). Data represent means ± SEM, * P

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Mouse Assay, MANN-WHITNEY

    Nav1.2 expressions at AISs of excitatory neurons in neocortex and hippocampus. Immunofluorescence histochemistry of P15.5 wild-type neocortices and hippocampi stained with anti-Nav1.2 (G-20, magenta), anti-ankyrin G (green), and anti-Tbr1 (cyan) antibodies, and counterstained with 4′-6-diamidino-2-phenylindole (DAPI, gray) and their merged images. Arrows indicate Nav1.2 and ankyrin G-double immunoreactive AISs of Tbr1-expressing neocortical pyramidal cells. Arrowheads indicate Nav1.2 and ankyrin G-double immunoreactive AISs of hippocampal pyramidal cells. Representative images of four or more slices are shown. o stratum oriens, p stratum pyramidale. Scale bars: 20 µm

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: Nav1.2 expressions at AISs of excitatory neurons in neocortex and hippocampus. Immunofluorescence histochemistry of P15.5 wild-type neocortices and hippocampi stained with anti-Nav1.2 (G-20, magenta), anti-ankyrin G (green), and anti-Tbr1 (cyan) antibodies, and counterstained with 4′-6-diamidino-2-phenylindole (DAPI, gray) and their merged images. Arrows indicate Nav1.2 and ankyrin G-double immunoreactive AISs of Tbr1-expressing neocortical pyramidal cells. Arrowheads indicate Nav1.2 and ankyrin G-double immunoreactive AISs of hippocampal pyramidal cells. Representative images of four or more slices are shown. o stratum oriens, p stratum pyramidale. Scale bars: 20 µm

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Immunofluorescence, Staining, Expressing

    Scn2a haplodeficiency in dorsal telencephalic excitatory but in those in global inhibitory neurons reduced neocortical and hippocampal Nav1.2 expression levels. Western blot analyses of 6-weeks neocortex or hippocampus for Scn2a fl/+ / Emx1 -Cre, Scn2a fl/+ / Vgat -Cre and Scn2a fl/+ controls ( N = 3, each genotype). Unpaired t -test, Scn2a fl/+ vs. Scn2a fl/+ / Emx1 -Cre, neocortex: t (4) = 5.91, ** P = 0.0041; hippocampus: t (4) = 5.74, ** P = 0.0046; Scn2a fl/+ vs. Scn2a fl/+ / Vgat -Cre, neocortex: t (4) = 1.413, P = 0.2305; hippocampus: t (4) = 0.489, P = 0.6503. Nav1.2 protein was normalized by β-tubulin. Mean Nav1.2 expression levels are represented as percentages relative to the level of Scn2a fl/+ control littermates (100%). Data represent means ± SEM, ** P

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: Scn2a haplodeficiency in dorsal telencephalic excitatory but in those in global inhibitory neurons reduced neocortical and hippocampal Nav1.2 expression levels. Western blot analyses of 6-weeks neocortex or hippocampus for Scn2a fl/+ / Emx1 -Cre, Scn2a fl/+ / Vgat -Cre and Scn2a fl/+ controls ( N = 3, each genotype). Unpaired t -test, Scn2a fl/+ vs. Scn2a fl/+ / Emx1 -Cre, neocortex: t (4) = 5.91, ** P = 0.0041; hippocampus: t (4) = 5.74, ** P = 0.0046; Scn2a fl/+ vs. Scn2a fl/+ / Vgat -Cre, neocortex: t (4) = 1.413, P = 0.2305; hippocampus: t (4) = 0.489, P = 0.6503. Nav1.2 protein was normalized by β-tubulin. Mean Nav1.2 expression levels are represented as percentages relative to the level of Scn2a fl/+ control littermates (100%). Data represent means ± SEM, ** P

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Expressing, Western Blot

    Developmental changes of Nav1.2 distribution in mouse brain. Brain sections of wild-type mice at P0.5 ( a , f , k , p ), P2.5 ( b , g , l , q ), P7.5 ( c , h , m , r ), P15.5 ( d , i , n , s ), and 8-week-old ( e , j , o , t ) were stained with anti-Nav1.2 (G-20, red). Higher-magnified images outlined in a – e are shown in f – t . Nav1.2 immunoreactivities were observed at AISs of neocortical neurons (single black arrowheads), nodes of Ranvier within white matter (double black arrowheads), AISs of hippocampal pyramidal neurons (white arrowheads), mossy fibers of dentate granule cells (black arrows), etc. Note that, while Nav1.2 at AISs and nodes of Ranvier peaked at P15.5 and became less at 8-weeks, diffused Nav1.2 signals in neocortex continued to become dense until 8-weeks-old. The brain slices were processed in parallel. Representative images of four or more slices per stage are shown. MZ marginal zone, UCP upper cortical plate, LCP lower cortical plate, DG dentate gyrus, WM white matter, IZ intermediate zone, o stratum oriens, p stratum pyramidale, l stratum lucidum, r stratum radiatum. Scale bars: a – e 500 µm; f – o 50 µm; p – t 100 µm

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: Developmental changes of Nav1.2 distribution in mouse brain. Brain sections of wild-type mice at P0.5 ( a , f , k , p ), P2.5 ( b , g , l , q ), P7.5 ( c , h , m , r ), P15.5 ( d , i , n , s ), and 8-week-old ( e , j , o , t ) were stained with anti-Nav1.2 (G-20, red). Higher-magnified images outlined in a – e are shown in f – t . Nav1.2 immunoreactivities were observed at AISs of neocortical neurons (single black arrowheads), nodes of Ranvier within white matter (double black arrowheads), AISs of hippocampal pyramidal neurons (white arrowheads), mossy fibers of dentate granule cells (black arrows), etc. Note that, while Nav1.2 at AISs and nodes of Ranvier peaked at P15.5 and became less at 8-weeks, diffused Nav1.2 signals in neocortex continued to become dense until 8-weeks-old. The brain slices were processed in parallel. Representative images of four or more slices per stage are shown. MZ marginal zone, UCP upper cortical plate, LCP lower cortical plate, DG dentate gyrus, WM white matter, IZ intermediate zone, o stratum oriens, p stratum pyramidale, l stratum lucidum, r stratum radiatum. Scale bars: a – e 500 µm; f – o 50 µm; p – t 100 µm

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Mouse Assay, Staining

    A pathogenic Scn2a nonsense mutation inactivated the mutated allele. a Schematic of the voltage-gated sodium channel Nav1.2, showing the location of R102* (RX) nonsense mutation. Full-length wild type Nav1.2 is composed of 2006-amino acid (aa) residues with the predicted molecular weight of ~228 kD. The RX mutation can cause a truncated peptides, Nav1.2-RX, consisting of the first 101-aa residues of Nav1.2 with the theoretical molecular weight of ~12 kD. Epitope locations for the anti-Nav1.2 (EM-1, ASC-002) and anti-pan Nav1 (SP19) antibodies are indicated. b , c The RX allele was effectively inactivated in Scn2a knock-in mice. Western blot analyses of P0.5 whole brain membrane ( b ) and cytosolic ( c ) fractions were performed using EM-1. b Full-length Nav1.2 was moderate and negligible in Scn2a RX/+ (RX/+, N = 4) and Scn2a RX/RX (RX/RX, N = 3) mice, respectively, compared with that in Scn2a +/+ mice (+/+, N = 3) [one-way analysis of variance; genotype: F (2, 7) = 1737, *** P = 0.00000000036, Tukey’s test; Scn2a +/+ vs . Scn2a RX/+ *** P

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: A pathogenic Scn2a nonsense mutation inactivated the mutated allele. a Schematic of the voltage-gated sodium channel Nav1.2, showing the location of R102* (RX) nonsense mutation. Full-length wild type Nav1.2 is composed of 2006-amino acid (aa) residues with the predicted molecular weight of ~228 kD. The RX mutation can cause a truncated peptides, Nav1.2-RX, consisting of the first 101-aa residues of Nav1.2 with the theoretical molecular weight of ~12 kD. Epitope locations for the anti-Nav1.2 (EM-1, ASC-002) and anti-pan Nav1 (SP19) antibodies are indicated. b , c The RX allele was effectively inactivated in Scn2a knock-in mice. Western blot analyses of P0.5 whole brain membrane ( b ) and cytosolic ( c ) fractions were performed using EM-1. b Full-length Nav1.2 was moderate and negligible in Scn2a RX/+ (RX/+, N = 4) and Scn2a RX/RX (RX/RX, N = 3) mice, respectively, compared with that in Scn2a +/+ mice (+/+, N = 3) [one-way analysis of variance; genotype: F (2, 7) = 1737, *** P = 0.00000000036, Tukey’s test; Scn2a +/+ vs . Scn2a RX/+ *** P

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Mutagenesis, Molecular Weight, Knock-In, Mouse Assay, Western Blot

    The pathogenic Scn2a nonsense mutation caused absence-like seizures with SWDs in mice. a Representative traces of somatosensory ECoG/EMG recordings in 6–11 weeks-old Scn2a RX/+ mice ( N = 7). Behavioral arrest during waking state associated with ECoG epileptiform SWDs (left). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. Positivity was plotted up. b Frequencies of SWDs during a 24 h ECoG recording period in Scn2a +/+ ( N = 5) and Scn2a RX/+ ( N = 7) mice. c An episode of non-convulsive epileptiform discharges detected in 1 out of 7 Scn2a RX/+ mice. An arrowhead indicates the onset of epileptiform discharge. Positivity was plotted up. d , e Seizure susceptibility to PTZ in Scn2a RX/+ and Scn2a +/+ mice (10 weeks of age). The latencies to the first appearance of absence seizure-like sudden immobility, myoclonus, clonic convulsion and tonic-clonic convulsion after intraperitoneal administration of PTZ at dose of 50 ( d , N = 20, each genotype) and 25 ( e , N = 14, each genotype) mg per kg body weight. Scn2a RX/+ mice had significantly shorter latencies to absence-like sudden immobility (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 106, * P = 0.0100, myoclonus, U = 116.5, * P = 0.0231, clonic convulsion, U = 162, P = 0.3098, tonic-clonic convulsion, U = 175, P = 0.5023; 25 mg per kg, sudden immobility, U = 53, * P = 0.0383). Data represent means ± SEM, * P

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: The pathogenic Scn2a nonsense mutation caused absence-like seizures with SWDs in mice. a Representative traces of somatosensory ECoG/EMG recordings in 6–11 weeks-old Scn2a RX/+ mice ( N = 7). Behavioral arrest during waking state associated with ECoG epileptiform SWDs (left). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. Positivity was plotted up. b Frequencies of SWDs during a 24 h ECoG recording period in Scn2a +/+ ( N = 5) and Scn2a RX/+ ( N = 7) mice. c An episode of non-convulsive epileptiform discharges detected in 1 out of 7 Scn2a RX/+ mice. An arrowhead indicates the onset of epileptiform discharge. Positivity was plotted up. d , e Seizure susceptibility to PTZ in Scn2a RX/+ and Scn2a +/+ mice (10 weeks of age). The latencies to the first appearance of absence seizure-like sudden immobility, myoclonus, clonic convulsion and tonic-clonic convulsion after intraperitoneal administration of PTZ at dose of 50 ( d , N = 20, each genotype) and 25 ( e , N = 14, each genotype) mg per kg body weight. Scn2a RX/+ mice had significantly shorter latencies to absence-like sudden immobility (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 106, * P = 0.0100, myoclonus, U = 116.5, * P = 0.0231, clonic convulsion, U = 162, P = 0.3098, tonic-clonic convulsion, U = 175, P = 0.5023; 25 mg per kg, sudden immobility, U = 53, * P = 0.0383). Data represent means ± SEM, * P

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Mutagenesis, Mouse Assay, MANN-WHITNEY

    Scn2a haplodeficiency did not alter expression levels of other sodium channel subunits. Quantitative RT-PCR analyses of brain mRNAs prepared from P14.5 Scn2a +/+ and Scn2a KO/+ mice ( N = 11, each genotype). Scn2a mRNA expression in Scn2a KO/+ whole brain was reduced to about 50% level of that in Scn2a +/+ mice while there were no significant changes in Scn1a , Scn3a , Scn5a , Scn8a , Scn1b , Scn2b , Scn3b , and Scn4b mRNA expression levels in Scn2a KO/+ , compared with those in Scn2a +/+ mice [unpaired t-test, Scn1a ; t (20) = 1.461, P = 0.1595, Scn2a ; t (20) = 5.250, *** P = 0.000039, Scn3a ; t (20) = 0.5066, P = 0.6180, Scn5a ; t (20) = 0.4223, P = 0.6773, Scn8a ; t (20) = 0.3952, P = 0.6969, Scn1b ; t (20) = 0.7407, P = 0.4675, Scn2b ; t (20) = 0.4259, P = 0.6747, Scn3b ; t (20) = 0.8664, P = 0.3965, Scn4b ; t (20) = 0.5273, P = 0.6038]. White and gray bars represent Scn2a +/+ and Scn2a KO/+ mice, respectively. Data represent means ± SEM, *** P

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: Scn2a haplodeficiency did not alter expression levels of other sodium channel subunits. Quantitative RT-PCR analyses of brain mRNAs prepared from P14.5 Scn2a +/+ and Scn2a KO/+ mice ( N = 11, each genotype). Scn2a mRNA expression in Scn2a KO/+ whole brain was reduced to about 50% level of that in Scn2a +/+ mice while there were no significant changes in Scn1a , Scn3a , Scn5a , Scn8a , Scn1b , Scn2b , Scn3b , and Scn4b mRNA expression levels in Scn2a KO/+ , compared with those in Scn2a +/+ mice [unpaired t-test, Scn1a ; t (20) = 1.461, P = 0.1595, Scn2a ; t (20) = 5.250, *** P = 0.000039, Scn3a ; t (20) = 0.5066, P = 0.6180, Scn5a ; t (20) = 0.4223, P = 0.6773, Scn8a ; t (20) = 0.3952, P = 0.6969, Scn1b ; t (20) = 0.7407, P = 0.4675, Scn2b ; t (20) = 0.4259, P = 0.6747, Scn3b ; t (20) = 0.8664, P = 0.3965, Scn4b ; t (20) = 0.5273, P = 0.6038]. White and gray bars represent Scn2a +/+ and Scn2a KO/+ mice, respectively. Data represent means ± SEM, *** P

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Expressing, Quantitative RT-PCR, Mouse Assay

    Heterozygous Scn2a knockout mice showed absence-like seizures with SWDs. a Representative traces of ECoG/EMG recordings from 10–27 weeks-old Scn2a KO/+ (KO/+) mice ( N = 6). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. b A representative trace of prolonged non-convulsive seizure. ECoG recordings detected 2 episodes of prolonged non-convulsive seizure with duration of 30–45 s in 2 out of 6 Scn2a KO/+ mice, which were neither accompanied by convulsions, nor followed by post-ictal depression. c Representative ECoG/EMG/LFP recordings during an SWD episode in Scn2a KO/+ mice. Epileptiform discharges with large amplitudes are seen in mPFC and CPu. Positivity was plotted up ( a , b , c ). d Quantification of ECoG SWDs and LFP epileptiform discharges [3-hour recording, light period, Scn2a +/+ , Scn2a KO/+ ( N = 4, each genotype)]. Mann–Whitney test, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 8, P > 0.9999; basolateral amygdala: U = 3, P = 0.2571; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0286; ventroposterior thalamus: U = 7.5, P > 0.9999. e Thresholds of body temperature for hyperthermia-induced seizures did not differ between Scn2a KO/+ and Scn2a +/+ mice (4-week-old, N = 10, each genotype) [unpaired t -test, t (18) = 1.149, P = 0.2658]. f , g Increased seizure susceptibility to PTZ in 10-week-old Scn2a KO/+ mice. Latencies to the first appearance of sudden immobility, myoclonus, clonic convulsion, and tonic-clonic convulsion after administrating of PTZ at doses of 50 ( f , N = 20, each genotype) or 25 ( g , N = 12, each genotype) mg per kg body weight. The latencies to the appearance of sudden immobility, myoclonus and clonic convulsion were shorter in Scn2a KO/+ than in Scn2a +/+ mice (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 69.5, *** P = 0.0002, myoclonus, U = 110.5, * P = 0.0145, clonic convulsion, U = 119, * P = 0.0278, tonic-clonic convulsion, U = 188.5, P = 0.7624; 25 mg per kg, sudden immobility, U = 26.5, ** P = 0.0071). Data represent means ± SEM, * P

    Journal: Communications Biology

    Article Title: Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice

    doi: 10.1038/s42003-018-0099-2

    Figure Lengend Snippet: Heterozygous Scn2a knockout mice showed absence-like seizures with SWDs. a Representative traces of ECoG/EMG recordings from 10–27 weeks-old Scn2a KO/+ (KO/+) mice ( N = 6). Black arrowheads indicate the onset of SWD. Gray arrowheads indicate the onset and end of EMG suppression. b A representative trace of prolonged non-convulsive seizure. ECoG recordings detected 2 episodes of prolonged non-convulsive seizure with duration of 30–45 s in 2 out of 6 Scn2a KO/+ mice, which were neither accompanied by convulsions, nor followed by post-ictal depression. c Representative ECoG/EMG/LFP recordings during an SWD episode in Scn2a KO/+ mice. Epileptiform discharges with large amplitudes are seen in mPFC and CPu. Positivity was plotted up ( a , b , c ). d Quantification of ECoG SWDs and LFP epileptiform discharges [3-hour recording, light period, Scn2a +/+ , Scn2a KO/+ ( N = 4, each genotype)]. Mann–Whitney test, medial prefrontal cortex: U = 0, * P = 0.0286; visual cortex: U = 8, P > 0.9999; basolateral amygdala: U = 3, P = 0.2571; hippocampus CA1: U = 4, P = 0.4286; caudate putamen: U = 0, * P = 0.0286; ventroposterior thalamus: U = 7.5, P > 0.9999. e Thresholds of body temperature for hyperthermia-induced seizures did not differ between Scn2a KO/+ and Scn2a +/+ mice (4-week-old, N = 10, each genotype) [unpaired t -test, t (18) = 1.149, P = 0.2658]. f , g Increased seizure susceptibility to PTZ in 10-week-old Scn2a KO/+ mice. Latencies to the first appearance of sudden immobility, myoclonus, clonic convulsion, and tonic-clonic convulsion after administrating of PTZ at doses of 50 ( f , N = 20, each genotype) or 25 ( g , N = 12, each genotype) mg per kg body weight. The latencies to the appearance of sudden immobility, myoclonus and clonic convulsion were shorter in Scn2a KO/+ than in Scn2a +/+ mice (Mann-Whitney test, 50 mg per kg, sudden immobility, U = 69.5, *** P = 0.0002, myoclonus, U = 110.5, * P = 0.0145, clonic convulsion, U = 119, * P = 0.0278, tonic-clonic convulsion, U = 188.5, P = 0.7624; 25 mg per kg, sudden immobility, U = 26.5, ** P = 0.0071). Data represent means ± SEM, * P

    Article Snippet: For immunofluorescence histochemistry, the sections were incubated with the rabbit anti-internal-region Nav1.2 (1:500; ASC-002, Alomone), the goat anti-internal-region Nav1.2 (1:500; SC-31371, G-20, Santa Cruz Biotechnology), the mouse anti-ankyrinG (1:250; SC-12719, Santa Cruz Biotechnology), the goat anti-ankyrinG (1:250; SC-31778, Santa Cruz Biotechnology), the rabbit anti-Tbr1 antibody (1:2,000; ab31940, Abcam), and the rabbit anti-Nav1.6 antibody (10 μg mL−1 ) .

    Techniques: Knock-Out, Mouse Assay, MANN-WHITNEY

    Elevated neuronal firing is reversible by FlpO-mediated restoration of Na V 1.2 expression in adult Na V 1.2-deficient mice (A) Cartoon illustration of mice systemically administered PHP.eB.AAV-control or PHP.eB.AAV-FlpO via tail vein injection. (B) Coronal views of LacZ staining of the striatum from WT and Scn2a gt/gt (HOM) mice injected with AAV-control or AAV-FlpO. Blue staining of HOM mice largely disappeared in the AAV-FlpO group. CPu, caudate nucleus and the putamen (dorsal striatum). Scale bar, 1 mm. (C) Western blot analysis showing Na V 1.2 protein levels in whole-brain homogenates from HOM mice in the AAV-control or AAV-FlpO group. One-way ANOVA with multiple comparisons. (D) Representative current-clamp recordings of MSNs from WT mice transduced with AAV-FlpO (red), HOM mice transduced with AAV-Control (blue), and HOM mice transduced with AAV-Control (magenta) obtained at the RMP. Inset: representative trace in response to +350 pA injection. (E) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired Mann-Whitney U test for each current pulse. (F) Typical spikes of MSNs were obtained at the normal RMP. (G) Associated phase-plane plots. (H) Individuals and average spike rheobase. Unpaired t test. (I) Typical spikes of MSNs at a fixed MP of −80 mV. (J) Associated phase-plane plots at −80 mV. Data were shown as mean ± SEM.

    Journal: Cell reports

    Article Title: Severe deficiency of the voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice

    doi: 10.1016/j.celrep.2021.109495

    Figure Lengend Snippet: Elevated neuronal firing is reversible by FlpO-mediated restoration of Na V 1.2 expression in adult Na V 1.2-deficient mice (A) Cartoon illustration of mice systemically administered PHP.eB.AAV-control or PHP.eB.AAV-FlpO via tail vein injection. (B) Coronal views of LacZ staining of the striatum from WT and Scn2a gt/gt (HOM) mice injected with AAV-control or AAV-FlpO. Blue staining of HOM mice largely disappeared in the AAV-FlpO group. CPu, caudate nucleus and the putamen (dorsal striatum). Scale bar, 1 mm. (C) Western blot analysis showing Na V 1.2 protein levels in whole-brain homogenates from HOM mice in the AAV-control or AAV-FlpO group. One-way ANOVA with multiple comparisons. (D) Representative current-clamp recordings of MSNs from WT mice transduced with AAV-FlpO (red), HOM mice transduced with AAV-Control (blue), and HOM mice transduced with AAV-Control (magenta) obtained at the RMP. Inset: representative trace in response to +350 pA injection. (E) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired Mann-Whitney U test for each current pulse. (F) Typical spikes of MSNs were obtained at the normal RMP. (G) Associated phase-plane plots. (H) Individuals and average spike rheobase. Unpaired t test. (I) Typical spikes of MSNs at a fixed MP of −80 mV. (J) Associated phase-plane plots at −80 mV. Data were shown as mean ± SEM.

    Article Snippet: Primary antibodies used were: Rabbit anti-SCN2A (NaV1.2) (1: 1000, Alomone Labs, ASC-002), β-Actin (8H10D10) Mouse mAb (1:2000, Cell Signaling Technology, 3700S), and GAPDH (D16H11) XP® Rabbit mAb (1:2000, Cell Signaling Technology, 5174S).

    Techniques: Expressing, Mouse Assay, Injection, Staining, Western Blot, Transduction, Generated, MANN-WHITNEY

    Elevated neuronal firing of striatal medium spiny neurons (MSNs) in adult Na V 1.2-deficient mice (A) A typical MSN labeled by neurobiotin. Scale bar, 40 μm. (B) Representative current-clamp recordings of MSNs from wild-type (WT, black) and homozygous (HOM) Scn2a gt/gt (blue) mice were obtained at the RMP. Inset: representative trace in response to +350 pA injection. (C) The average number of action potentials (APs) generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U test for each current pulse. (D) Individuals and mean RMP values. Unpaired t test. (Ei) Representative traces in response to −100 pA injection. V steady-state (V ss ) is the voltage recorded 0–10 ms before the end of the stimulus. (Eii) Individuals and mean input resistance values at the RMP. Unpaired t test. (F) Typical spikes of MSNs from WT (black) and HOM (blue) mice were obtained at the normal RMP. (G) Associated phase-plane plots. (H–L) Individual and mean spike rheobase, voltage threshold, amplitude, fast after-hyperpolarization (AHP), and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Journal: Cell reports

    Article Title: Severe deficiency of the voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice

    doi: 10.1016/j.celrep.2021.109495

    Figure Lengend Snippet: Elevated neuronal firing of striatal medium spiny neurons (MSNs) in adult Na V 1.2-deficient mice (A) A typical MSN labeled by neurobiotin. Scale bar, 40 μm. (B) Representative current-clamp recordings of MSNs from wild-type (WT, black) and homozygous (HOM) Scn2a gt/gt (blue) mice were obtained at the RMP. Inset: representative trace in response to +350 pA injection. (C) The average number of action potentials (APs) generated in response to depolarizing current pulses. Unpaired two-tailed non-parametric Mann-Whitney U test for each current pulse. (D) Individuals and mean RMP values. Unpaired t test. (Ei) Representative traces in response to −100 pA injection. V steady-state (V ss ) is the voltage recorded 0–10 ms before the end of the stimulus. (Eii) Individuals and mean input resistance values at the RMP. Unpaired t test. (F) Typical spikes of MSNs from WT (black) and HOM (blue) mice were obtained at the normal RMP. (G) Associated phase-plane plots. (H–L) Individual and mean spike rheobase, voltage threshold, amplitude, fast after-hyperpolarization (AHP), and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Article Snippet: Primary antibodies used were: Rabbit anti-SCN2A (NaV1.2) (1: 1000, Alomone Labs, ASC-002), β-Actin (8H10D10) Mouse mAb (1:2000, Cell Signaling Technology, 3700S), and GAPDH (D16H11) XP® Rabbit mAb (1:2000, Cell Signaling Technology, 5174S).

    Techniques: Mouse Assay, Labeling, Injection, Generated, Two Tailed Test, MANN-WHITNEY

    Elevated neuronal excitability is autonomous in adult Na V 1.2-deficient mice (A) Scn2a gt/gt (HOM) mice were injected with a dilute FlpO virus, sparsely transducing a subset of neurons in the striatum. Dashed circles highlight two neighboring AAV-negative (blue circle) and AAV-FlpO-positive (magenta circle) neurons. Scale bar, 10 μm. (B) Representative current-clamp recordings of AAV-negative (blue) and AAV-FlpO-positive (magenta) MSNs in the CPu of HOM mice were obtained at the RMP. Inset: representative trace in response to +350 pA injection. (C) The average number of APs generated in response to depolarizing current pulses. Unpaired Mann-Whitney U test for each current pulse. (D) Individuals and average RMP values. Unpaired t test. (E) Individuals and average input resistance values at the RMP. Unpaired t test. (F) Typical spikes were obtained at the RMP. (G) Associated phase-plane plots. (H–L) Individual and average spike rheobase, voltage threshold, amplitude, AHP, and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Journal: Cell reports

    Article Title: Severe deficiency of the voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice

    doi: 10.1016/j.celrep.2021.109495

    Figure Lengend Snippet: Elevated neuronal excitability is autonomous in adult Na V 1.2-deficient mice (A) Scn2a gt/gt (HOM) mice were injected with a dilute FlpO virus, sparsely transducing a subset of neurons in the striatum. Dashed circles highlight two neighboring AAV-negative (blue circle) and AAV-FlpO-positive (magenta circle) neurons. Scale bar, 10 μm. (B) Representative current-clamp recordings of AAV-negative (blue) and AAV-FlpO-positive (magenta) MSNs in the CPu of HOM mice were obtained at the RMP. Inset: representative trace in response to +350 pA injection. (C) The average number of APs generated in response to depolarizing current pulses. Unpaired Mann-Whitney U test for each current pulse. (D) Individuals and average RMP values. Unpaired t test. (E) Individuals and average input resistance values at the RMP. Unpaired t test. (F) Typical spikes were obtained at the RMP. (G) Associated phase-plane plots. (H–L) Individual and average spike rheobase, voltage threshold, amplitude, AHP, and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Article Snippet: Primary antibodies used were: Rabbit anti-SCN2A (NaV1.2) (1: 1000, Alomone Labs, ASC-002), β-Actin (8H10D10) Mouse mAb (1:2000, Cell Signaling Technology, 3700S), and GAPDH (D16H11) XP® Rabbit mAb (1:2000, Cell Signaling Technology, 5174S).

    Techniques: Mouse Assay, Injection, Generated, MANN-WHITNEY

    Activation of K V channels reverses elevated neuronal firing in adult Na V 1.2-deficient mice (A) Volcano plot displaying Scn2a and Scn8a as well as potassium channels that are statistically downregulated in Scn2a gt/gt (HOM) mice compared with WT mice identified by RNA-seq. Significantly upregulated genes are shown in yellow, and downregulated genes are shown in blue. (B) List of potassium channels that are significantly downregulated in HOM mice compared with the WT. Hits were identified from both DESeq2 and edgeR differential expression analysis with a false discovery rate of less than 0.05. “% expression” of WT level (100%). n = 4 mice for each group. (C) Representative whole-cell voltage-clamp recordings of MSNs in brain slices from WT and HOM mice, showing potassium currents at voltage steps from −120 mV to +50 mV. Voltage-gated Ca 2+ channels were not blocked to allow activation of Ca 2+ -dependent K + channels. (D) Summary of the total sustained current (measured at the end of steps). Two-way ANOVA with repeated measures. (E) Representative current-clamp recordings of MSNs from WT slices perfused with 0.1% DMSO in artificial cerebrospinal fluid (aCSF) (WT control, black), HOM slices perfused with 0.1% DMSO in aCSF (HOM control, blue), and HOM slices perfused with 0.1% DMSO in aCSF containing PiMA (HOM 10 μM PiMA, magenta) at the RMP. Inset: representative trace in response to +400 pA injection. (F) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired Mann-Whitney U test for each current pulse. (G) Individuals and average RMP values. Unpaired t test. (H) Individuals and average input resistance values at the RMP. Unpaired t test. (I) Typical spikes of MSNs were obtained at the RMP. (J) Associated phase-plane plots. (K–O) Individuals and average spike rheobase, voltage threshold, amplitude, AHP, and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Journal: Cell reports

    Article Title: Severe deficiency of the voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice

    doi: 10.1016/j.celrep.2021.109495

    Figure Lengend Snippet: Activation of K V channels reverses elevated neuronal firing in adult Na V 1.2-deficient mice (A) Volcano plot displaying Scn2a and Scn8a as well as potassium channels that are statistically downregulated in Scn2a gt/gt (HOM) mice compared with WT mice identified by RNA-seq. Significantly upregulated genes are shown in yellow, and downregulated genes are shown in blue. (B) List of potassium channels that are significantly downregulated in HOM mice compared with the WT. Hits were identified from both DESeq2 and edgeR differential expression analysis with a false discovery rate of less than 0.05. “% expression” of WT level (100%). n = 4 mice for each group. (C) Representative whole-cell voltage-clamp recordings of MSNs in brain slices from WT and HOM mice, showing potassium currents at voltage steps from −120 mV to +50 mV. Voltage-gated Ca 2+ channels were not blocked to allow activation of Ca 2+ -dependent K + channels. (D) Summary of the total sustained current (measured at the end of steps). Two-way ANOVA with repeated measures. (E) Representative current-clamp recordings of MSNs from WT slices perfused with 0.1% DMSO in artificial cerebrospinal fluid (aCSF) (WT control, black), HOM slices perfused with 0.1% DMSO in aCSF (HOM control, blue), and HOM slices perfused with 0.1% DMSO in aCSF containing PiMA (HOM 10 μM PiMA, magenta) at the RMP. Inset: representative trace in response to +400 pA injection. (F) The average number of APs generated in response to depolarizing current pulses at the RMP. Unpaired Mann-Whitney U test for each current pulse. (G) Individuals and average RMP values. Unpaired t test. (H) Individuals and average input resistance values at the RMP. Unpaired t test. (I) Typical spikes of MSNs were obtained at the RMP. (J) Associated phase-plane plots. (K–O) Individuals and average spike rheobase, voltage threshold, amplitude, AHP, and half-width values. Unpaired t test. Data are shown as mean ± SEM.

    Article Snippet: Primary antibodies used were: Rabbit anti-SCN2A (NaV1.2) (1: 1000, Alomone Labs, ASC-002), β-Actin (8H10D10) Mouse mAb (1:2000, Cell Signaling Technology, 3700S), and GAPDH (D16H11) XP® Rabbit mAb (1:2000, Cell Signaling Technology, 5174S).

    Techniques: Activation Assay, Mouse Assay, RNA Sequencing Assay, Expressing, Injection, Generated, MANN-WHITNEY

    Altered anxiety-related behavior and rotarod performance in Scn2a E/+ mice. A) Percent time spent in the open arms of a zero-maze apparatus in Scn2a E/+ mice compared to WT at 6 weeks of age. Scn2a E/+ males spent significantly more time in the open arms compared to WT (WT: 15.3 ± 2.2%, Scn2a E/+ : 30.5 ± 4.1%, **p=0.0023; Student’s t-test). Scn2a E/+ females spent significantly more time in the open arms compared to WT (WT: 22.0 ± 2.0%, Scn2a E/+ : 34.8 ± 4.9%, *p=0.0297; Welch’s t test). B) Percent time spent in the light zone of a light/dark box in Scn2a E/+ mice compared to WT at 7 weeks of age. Scn2a E/+ males spent significantly more time in the light zone compared to WT (WT: 20.9 ± 2.0%, Scn2a E/+ : 29.2 ± 3.2%, *p=0.0367; Student’s t-test). Scn2a E/+ females also spent significantly more time in the light zone compared to WT (WT: 21.1 ± 1.7%, Scn2a E/+ : 32.1 ± 3.5%, *p=0.0367; Mann-Whitney test). C) Percent time spent in the center zone of an open field apparatus in Scn2a E/+ mice compared to WT at 8 weeks of age. There was not a significant difference in the amount of time spent in the center zone between Scn2a E/+ and WT males (p=0.0556, Mann-Whitney test). However, Scn2a E/+ females spent significantly more time in the center zone compared to WT (WT: 6.27 ± 0.9%, Scn2a E/+ : 10.1 ± 1.2%, *p=0.0167; Mann-Whitney test). D) Number of marbles buried during a 30-minute trial by Scn2a E/+ mice compared to WT at 6 weeks of age is displayed. Scn2a E/+ males buried significantly fewer marbles compared to WT (WT: 12 ± 1, Scn2a E/+ : 5 ± 2, **p=0.0019; Mann-Whitney test). Scn2a E/+ females also buried significantly fewer marbles compared to WT (WT: 13 ± 2, Scn2a E/+ : 2 ± 1, ****p=0.0001; Mann-Whitney test). E) Average latency to fall during an accelerating rotarod task measured on three consecutive days in Scn2a E/+ mice compared to WT at 9 weeks of age. Daily performance for each animal was assessed by averaging across three trials. Two-way repeated measures ANOVA comparing average latency to fall between Scn2a E/+ and WT males showed significant main effects of test day [ F (1.511,34.76) = 8.450, **p=0.0022] and genotype [ F (1,23) = 10.18, *p=0.0041]. Scn2a E/+ males took significantly longer to fall compared to WT on day 3 (WT: 121 ± 6 sec, Scn2a E/+ : 152 ± 7 sec, p=0.0106; Sidak’s post-hoc test). Two-way repeated measures ANOVA comparing average latency to fall between Scn2a E/+ and WT females showed a significant main effect of test day only [ F (1.594,36.65) = 6.646, **p=0.0059]. For panels A-D symbols represent individual mice, horizontal lines represent mean, and error bars represent SEM. For panel E symbols and error bars represent mean ± SEM. Males and females were analyzed separately, with n=12-14 per genotype for males and n=11-15 per genotype for females.

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: Altered anxiety-related behavior and rotarod performance in Scn2a E/+ mice. A) Percent time spent in the open arms of a zero-maze apparatus in Scn2a E/+ mice compared to WT at 6 weeks of age. Scn2a E/+ males spent significantly more time in the open arms compared to WT (WT: 15.3 ± 2.2%, Scn2a E/+ : 30.5 ± 4.1%, **p=0.0023; Student’s t-test). Scn2a E/+ females spent significantly more time in the open arms compared to WT (WT: 22.0 ± 2.0%, Scn2a E/+ : 34.8 ± 4.9%, *p=0.0297; Welch’s t test). B) Percent time spent in the light zone of a light/dark box in Scn2a E/+ mice compared to WT at 7 weeks of age. Scn2a E/+ males spent significantly more time in the light zone compared to WT (WT: 20.9 ± 2.0%, Scn2a E/+ : 29.2 ± 3.2%, *p=0.0367; Student’s t-test). Scn2a E/+ females also spent significantly more time in the light zone compared to WT (WT: 21.1 ± 1.7%, Scn2a E/+ : 32.1 ± 3.5%, *p=0.0367; Mann-Whitney test). C) Percent time spent in the center zone of an open field apparatus in Scn2a E/+ mice compared to WT at 8 weeks of age. There was not a significant difference in the amount of time spent in the center zone between Scn2a E/+ and WT males (p=0.0556, Mann-Whitney test). However, Scn2a E/+ females spent significantly more time in the center zone compared to WT (WT: 6.27 ± 0.9%, Scn2a E/+ : 10.1 ± 1.2%, *p=0.0167; Mann-Whitney test). D) Number of marbles buried during a 30-minute trial by Scn2a E/+ mice compared to WT at 6 weeks of age is displayed. Scn2a E/+ males buried significantly fewer marbles compared to WT (WT: 12 ± 1, Scn2a E/+ : 5 ± 2, **p=0.0019; Mann-Whitney test). Scn2a E/+ females also buried significantly fewer marbles compared to WT (WT: 13 ± 2, Scn2a E/+ : 2 ± 1, ****p=0.0001; Mann-Whitney test). E) Average latency to fall during an accelerating rotarod task measured on three consecutive days in Scn2a E/+ mice compared to WT at 9 weeks of age. Daily performance for each animal was assessed by averaging across three trials. Two-way repeated measures ANOVA comparing average latency to fall between Scn2a E/+ and WT males showed significant main effects of test day [ F (1.511,34.76) = 8.450, **p=0.0022] and genotype [ F (1,23) = 10.18, *p=0.0041]. Scn2a E/+ males took significantly longer to fall compared to WT on day 3 (WT: 121 ± 6 sec, Scn2a E/+ : 152 ± 7 sec, p=0.0106; Sidak’s post-hoc test). Two-way repeated measures ANOVA comparing average latency to fall between Scn2a E/+ and WT females showed a significant main effect of test day only [ F (1.594,36.65) = 6.646, **p=0.0059]. For panels A-D symbols represent individual mice, horizontal lines represent mean, and error bars represent SEM. For panel E symbols and error bars represent mean ± SEM. Males and females were analyzed separately, with n=12-14 per genotype for males and n=11-15 per genotype for females.

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Mouse Assay, MANN-WHITNEY

    Altered social behavior in Scn2a E/+ mice. A) Sociability phase of three-chamber assay. Amount of time spent sniffing either an empty cup (EC) or unfamiliar mouse (S1) in Scn2a E/+ mice compared to WT at 10 weeks of age. Two-way ANOVA (using target as a within-subject variable) comparing average sniffing time between Scn2a E/+ and WT males showed a significant main effect of target [ F (1,24) = 57.28, p

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: Altered social behavior in Scn2a E/+ mice. A) Sociability phase of three-chamber assay. Amount of time spent sniffing either an empty cup (EC) or unfamiliar mouse (S1) in Scn2a E/+ mice compared to WT at 10 weeks of age. Two-way ANOVA (using target as a within-subject variable) comparing average sniffing time between Scn2a E/+ and WT males showed a significant main effect of target [ F (1,24) = 57.28, p

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Mouse Assay, Boyden Chamber Assay

    EEG abnormalities and altered susceptibility to induced seizures in Scn2a E/+ mice. A) Representative 5-minute epoch of EEG from Scn2a E/+ mice. A localized seizure occurred as an abrupt onset of rhythmic 2 Hz sharp waves with overriding fast activity that evolve in amplitude and frequency for ∼45 seconds before abruptly terminating with return to typical sleep background. B) 30-second epoch corresponding to the purple bar segment from A . The top line in both A and B corresponds to channel 1 (right posterior-left posterior) and second line is channel 2 (right anterior-left posterior). C) Example of an isolated high amplitude sharp wave with overriding fast activity corresponding to the blue bar segment in B . D) Power spectrum for the sharp wave in C showing elevated power in decibels across the 1-170 Hz frequency range at the time of discharge. E) Latency to flurothyl-induced GTCS in Scn2a E/+ mice compared to WT at 6-9 weeks of age. Scn2a E/+ males had an elevated threshold for flurothyl-induced seizures compared to WT (WT: 175 ± 8 sec, Scn2a E/+ : 247 ± 13 sec, ****p

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: EEG abnormalities and altered susceptibility to induced seizures in Scn2a E/+ mice. A) Representative 5-minute epoch of EEG from Scn2a E/+ mice. A localized seizure occurred as an abrupt onset of rhythmic 2 Hz sharp waves with overriding fast activity that evolve in amplitude and frequency for ∼45 seconds before abruptly terminating with return to typical sleep background. B) 30-second epoch corresponding to the purple bar segment from A . The top line in both A and B corresponds to channel 1 (right posterior-left posterior) and second line is channel 2 (right anterior-left posterior). C) Example of an isolated high amplitude sharp wave with overriding fast activity corresponding to the blue bar segment in B . D) Power spectrum for the sharp wave in C showing elevated power in decibels across the 1-170 Hz frequency range at the time of discharge. E) Latency to flurothyl-induced GTCS in Scn2a E/+ mice compared to WT at 6-9 weeks of age. Scn2a E/+ males had an elevated threshold for flurothyl-induced seizures compared to WT (WT: 175 ± 8 sec, Scn2a E/+ : 247 ± 13 sec, ****p

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Mouse Assay, Activity Assay, Isolation

    Scn2a K1422E prefrontal pyramidal cell AP waveform has loss-of-function characteristics. A) Example whole-cell voltage response to somatic current injection in WT (black) and Scn2a E/+ (K1422E, cyan) neurons. Numbers between examples correspond to current injection amplitude. B) AP number per 300 ms current injection, color coded as in A. Bars are mean ± SEM. n = 13 cells each. C) Rheobase AP as dV/dt vs. voltage (phase-plane plot). Note reduction of peak dV/dt for Scn2a E/+ cells compared to WT, indicative of loss-of-function in Na V 1.2-mediated somatic depolarization. D) Summaries of AP waveform and intrinsic excitability characteristics. Circles are single cells, bars are mean ± SEM. Peak dV/dt is reduced in Scn2a E/+ cells, unpaired t-test. n = 14 WT, 13 Scn2a E/+ cells.

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: Scn2a K1422E prefrontal pyramidal cell AP waveform has loss-of-function characteristics. A) Example whole-cell voltage response to somatic current injection in WT (black) and Scn2a E/+ (K1422E, cyan) neurons. Numbers between examples correspond to current injection amplitude. B) AP number per 300 ms current injection, color coded as in A. Bars are mean ± SEM. n = 13 cells each. C) Rheobase AP as dV/dt vs. voltage (phase-plane plot). Note reduction of peak dV/dt for Scn2a E/+ cells compared to WT, indicative of loss-of-function in Na V 1.2-mediated somatic depolarization. D) Summaries of AP waveform and intrinsic excitability characteristics. Circles are single cells, bars are mean ± SEM. Peak dV/dt is reduced in Scn2a E/+ cells, unpaired t-test. n = 14 WT, 13 Scn2a E/+ cells.

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Injection

    Generation and molecular characterization of Scn2a K1422E mice. A) Sequencing chromatograms of Scn2a genomic PCR products with the first nucleotide of the K1422E codon highlighted in black. Top chromatogram from a WT littermate control mouse shows homozygosity for the WT nucleotide at the K1422E codon. Bottom chromatogram from a heterozygous Scn2a E/+ mouse shows heterozygosity for the single nucleotide change introduced by CRISPR/Cas9 genome editing and homology directed repair. B) Relative expression of whole brain Scn2a transcript in WT and Scn2a E/+ mice assayed by RT-ddPCR. Relative transcript levels are expressed as a ratio of Scn2a concentration to Tbp concentration (normalized to WT average). There was no difference in transcript expression between genotypes (p=0.6295; n=8 mice per genotype). C) Relative expression of whole brain Na V 1.2 protein in WT and Scn2a E/+ mice assayed by immunoblotting. Quantification is expressed as a ratio of Na V 1.2 immunofluorescence relative to GRP75/Mortalin (normalized to WT average). There was no difference in protein expression between genotypes (p=0.2937; n=8 mice per genotype). For both B and C , circles represent samples from individual mice, horizontal lines rep resent mean, and error bars represent SEM. D) Representative immunoblot. Bands corresponding to Na V 1.2 (MW: 260 kDa) are visualized in green (Alexa Fluor 790) while bands corresponding to GRP75 (MW: 73 kDa) are visualized in red (Alexa Fluor 680).

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: Generation and molecular characterization of Scn2a K1422E mice. A) Sequencing chromatograms of Scn2a genomic PCR products with the first nucleotide of the K1422E codon highlighted in black. Top chromatogram from a WT littermate control mouse shows homozygosity for the WT nucleotide at the K1422E codon. Bottom chromatogram from a heterozygous Scn2a E/+ mouse shows heterozygosity for the single nucleotide change introduced by CRISPR/Cas9 genome editing and homology directed repair. B) Relative expression of whole brain Scn2a transcript in WT and Scn2a E/+ mice assayed by RT-ddPCR. Relative transcript levels are expressed as a ratio of Scn2a concentration to Tbp concentration (normalized to WT average). There was no difference in transcript expression between genotypes (p=0.6295; n=8 mice per genotype). C) Relative expression of whole brain Na V 1.2 protein in WT and Scn2a E/+ mice assayed by immunoblotting. Quantification is expressed as a ratio of Na V 1.2 immunofluorescence relative to GRP75/Mortalin (normalized to WT average). There was no difference in protein expression between genotypes (p=0.2937; n=8 mice per genotype). For both B and C , circles represent samples from individual mice, horizontal lines rep resent mean, and error bars represent SEM. D) Representative immunoblot. Bands corresponding to Na V 1.2 (MW: 260 kDa) are visualized in green (Alexa Fluor 790) while bands corresponding to GRP75 (MW: 73 kDa) are visualized in red (Alexa Fluor 680).

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Mouse Assay, Sequencing, Polymerase Chain Reaction, CRISPR, Expressing, Concentration Assay, Immunofluorescence

    Whole-cell sodium currents of acutely isolated neuron. Representative whole-cell sodium currents and current voltage relationships of A) total sodium current, B) TTX-resistant currents, and C) TTX-sensitive currents from acutely dissociated hippocampal pyramidal neurons from WT and Scn2a E/+ mice, D) Sodium reversal potential of total sodium current (left, p=0.0364), TTX-resistant current (middle, p=0.0006), and TTX-sensitive currents (right, p=0.6964). All data are plotted as mean ± SEM of n=8-11 cells.

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: Whole-cell sodium currents of acutely isolated neuron. Representative whole-cell sodium currents and current voltage relationships of A) total sodium current, B) TTX-resistant currents, and C) TTX-sensitive currents from acutely dissociated hippocampal pyramidal neurons from WT and Scn2a E/+ mice, D) Sodium reversal potential of total sodium current (left, p=0.0364), TTX-resistant current (middle, p=0.0006), and TTX-sensitive currents (right, p=0.6964). All data are plotted as mean ± SEM of n=8-11 cells.

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Isolation, Mouse Assay

    Heterologous expression of K1422E in HEK293T cells reveals altered ion selectivity. A) Homology model of voltage gated sodium channel alpha subunit (PDB 6MWA Na V Ab, residues1-239). Four internally homologous domains coalesce with four-fold symmetry around an ion conducting pore denoted by an asterisk. The Cα carbons from each residue (DEKA) of the highly conserved ion selectivity filter are represented by colored ellipses. The red ellipse denotes the Cα carbon from the K1422 residue that is substituted for glutamate (E) in the Scn2a K1422E model. B) Representative whole-cell sodium currents (top) and calcium currents (bottom) from WT (left) and K1422E (right) expressing cells. C) Summary current-voltage relationship of whole cells sodium current showing reduced sodium current density of K1422E at -10 mV (p=0.0033). D) Sodium reversal potential of WT and K1422E expressing cells (p

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: Heterologous expression of K1422E in HEK293T cells reveals altered ion selectivity. A) Homology model of voltage gated sodium channel alpha subunit (PDB 6MWA Na V Ab, residues1-239). Four internally homologous domains coalesce with four-fold symmetry around an ion conducting pore denoted by an asterisk. The Cα carbons from each residue (DEKA) of the highly conserved ion selectivity filter are represented by colored ellipses. The red ellipse denotes the Cα carbon from the K1422 residue that is substituted for glutamate (E) in the Scn2a K1422E model. B) Representative whole-cell sodium currents (top) and calcium currents (bottom) from WT (left) and K1422E (right) expressing cells. C) Summary current-voltage relationship of whole cells sodium current showing reduced sodium current density of K1422E at -10 mV (p=0.0033). D) Sodium reversal potential of WT and K1422E expressing cells (p

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Expressing

    Lower olfactory dishabituation to social odors and intact olfactory-guided behavior in Scn2a E/+ mice. A) Average sniffing times during an odor habituation/dishabituation assay in Scn2a E/+ mice compared to WT at 11 weeks of age. Overall, olfactory discrimination in Scn2a E/+ males was not significantly different from WT males. However, Scn2a E/+ males spent significantly less time sniffing same sex urine during the first two presentations compared to WT males. During the first presentation, average sniffing time was 42.3 ± 3.3 sec for WT males and 18.8 ± 3.4 sec for Scn2a E/+ males (***p=0.0005, multiple t-tests). During the second presentation, average sniffing time was 26.3 ± 8.9 sec for WT males and 8.9 ± 2.1 sec for Scn2a E/+ males (*p=0.0307, multiple t-tests). Olfactory discrimination in Scn2a E/+ females was also not significantly different from WT females. However, Scn2a E/+ females spent significantly less time sniffing same sex urine during the first presentation compared to WT females (WT: 46.3 ± 4.6 sec, Scn2a E/+ : 19.9 ± 4.2 sec, **p=0.0045, multiple t-tests). Symbols and error bars represent mean ± SEM. Males and females were analyzed separately, with n=14 per genotype for males and n=12-13 per genotype for females. B) Latency to find a buried food item in Scn2a E/+ mice compared to WT at 8 weeks of age. Latency to find a buried food item was not significantly different between Scn2a E/+ and WT males (WT: 44.86 ± 11.58 sec, Scn2a E/+ : 32.46 ± 9.95 sec, p = 0.7471; Mann-Whitney test). Latency to find a buried food item was not significantly different between Scn2a E/+ and WT females (WT: 89.00 ± 44.31 sec, Scn2a E/+ : 161.7 ± 70.02, p = 0.2927; Mann-Whitney test). Symbols represent measured values from individual mice, horizontal lines represent mean, and error bars represent SEM. Males and females were analyzed separately, with n=13-14 per genotype for males and n=13 per genotype for females.

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: Lower olfactory dishabituation to social odors and intact olfactory-guided behavior in Scn2a E/+ mice. A) Average sniffing times during an odor habituation/dishabituation assay in Scn2a E/+ mice compared to WT at 11 weeks of age. Overall, olfactory discrimination in Scn2a E/+ males was not significantly different from WT males. However, Scn2a E/+ males spent significantly less time sniffing same sex urine during the first two presentations compared to WT males. During the first presentation, average sniffing time was 42.3 ± 3.3 sec for WT males and 18.8 ± 3.4 sec for Scn2a E/+ males (***p=0.0005, multiple t-tests). During the second presentation, average sniffing time was 26.3 ± 8.9 sec for WT males and 8.9 ± 2.1 sec for Scn2a E/+ males (*p=0.0307, multiple t-tests). Olfactory discrimination in Scn2a E/+ females was also not significantly different from WT females. However, Scn2a E/+ females spent significantly less time sniffing same sex urine during the first presentation compared to WT females (WT: 46.3 ± 4.6 sec, Scn2a E/+ : 19.9 ± 4.2 sec, **p=0.0045, multiple t-tests). Symbols and error bars represent mean ± SEM. Males and females were analyzed separately, with n=14 per genotype for males and n=12-13 per genotype for females. B) Latency to find a buried food item in Scn2a E/+ mice compared to WT at 8 weeks of age. Latency to find a buried food item was not significantly different between Scn2a E/+ and WT males (WT: 44.86 ± 11.58 sec, Scn2a E/+ : 32.46 ± 9.95 sec, p = 0.7471; Mann-Whitney test). Latency to find a buried food item was not significantly different between Scn2a E/+ and WT females (WT: 89.00 ± 44.31 sec, Scn2a E/+ : 161.7 ± 70.02, p = 0.2927; Mann-Whitney test). Symbols represent measured values from individual mice, horizontal lines represent mean, and error bars represent SEM. Males and females were analyzed separately, with n=13-14 per genotype for males and n=13 per genotype for females.

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Mouse Assay, MANN-WHITNEY

    AP-evoked Ca 2+ influx during the rising phase of the AP in the proximal AIS of Scn2a E/+ cells. A) Pyramidal cell initial segments are enriched with Na V 1.2 proximal to the soma and Na V 1.6 more distal to the soma. Pointscan imaging was performed 5 and 30 µm from the axon hillock, corresponding to Na V 1.2 and Na V 1.6-enriched regions, respectively. B) Examples of AP-evoked (2 nA, 2 ms stimulus; black, top) calcium transients imaged in pointscan mode in the proximal (green, middle) and distal (violet, bottom) AIS in WT (left) and Scn2a E/+ cells (right). Vertical line is aligned to peak AP voltage. Grey shaded area encompasses imaging signal root-mean-squared error (RMSE) during baseline, before AP. Consistent deviation above this error value defines onset of Ca 2+ transient. Note Ca 2+ influx before AP peak in proximal AIS of K1422E condition, only. C) Amplitude of Ca 2+ transient is higher in proximal AIS of Scn2a E/+ cells, consistent with influx from both local voltage-gated calcium channels and additional influx through K1422E Na V 1.2 channels. Circles are single cells and bars are mean ± SEM. p values from unpaired t-tests. D) Ca 2+ transient onset occurs earlier in the proximal AIS of Scn2a E/+ cells, consistent with Ca 2+ influx through K1422E Na V 1.2 channels. Display as in C .

    Journal: bioRxiv

    Article Title: Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice

    doi: 10.1101/2021.07.19.452930

    Figure Lengend Snippet: AP-evoked Ca 2+ influx during the rising phase of the AP in the proximal AIS of Scn2a E/+ cells. A) Pyramidal cell initial segments are enriched with Na V 1.2 proximal to the soma and Na V 1.6 more distal to the soma. Pointscan imaging was performed 5 and 30 µm from the axon hillock, corresponding to Na V 1.2 and Na V 1.6-enriched regions, respectively. B) Examples of AP-evoked (2 nA, 2 ms stimulus; black, top) calcium transients imaged in pointscan mode in the proximal (green, middle) and distal (violet, bottom) AIS in WT (left) and Scn2a E/+ cells (right). Vertical line is aligned to peak AP voltage. Grey shaded area encompasses imaging signal root-mean-squared error (RMSE) during baseline, before AP. Consistent deviation above this error value defines onset of Ca 2+ transient. Note Ca 2+ influx before AP peak in proximal AIS of K1422E condition, only. C) Amplitude of Ca 2+ transient is higher in proximal AIS of Scn2a E/+ cells, consistent with influx from both local voltage-gated calcium channels and additional influx through K1422E Na V 1.2 channels. Circles are single cells and bars are mean ± SEM. p values from unpaired t-tests. D) Ca 2+ transient onset occurs earlier in the proximal AIS of Scn2a E/+ cells, consistent with Ca 2+ influx through K1422E Na V 1.2 channels. Display as in C .

    Article Snippet: Blots were probed with anti-NaV 2.1 pAb (Alomone Labs, Jerusalem, ISR; #ASC-002, RRID: AB_2040005; 1:200 dilution) and anti-mortalin/GRP75 mAb (Antibodies Inc, Davis, CA, USA; Neuromab N52A/42, RRID: 2120479; 1:1000 dilution), which served as a normalization standard.

    Techniques: Imaging