apc  (Alomone Labs)


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    Alomone Labs apc
    Apc, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    apc  (Alomone Labs)


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    Structured Review

    Alomone Labs apc
    Apc, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apc/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
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    kv3 2  (Alomone Labs)


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    Alomone Labs kv3 2
    Primary antibodies used in this study.
    Kv3 2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Distinct Distribution Patterns of Potassium Channel Sub-Units in Somato-Dendritic Compartments of Neurons of the Medial Superior Olive"

    Article Title: Distinct Distribution Patterns of Potassium Channel Sub-Units in Somato-Dendritic Compartments of Neurons of the Medial Superior Olive

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2019.00038

    Primary antibodies used in this study.
    Figure Legend Snippet: Primary antibodies used in this study.

    Techniques Used:

    High voltage-activated potassium channels in MSO neurons. (A) Immunofluorescent staining of Kv3.1b, MAP2 (co-staining left, magnified Kv3.1b image middle) and Kv3.1b blocking peptide (+P, right). Intensity scaling of the gray scaled images is identical, to indicate the effect of the blocking peptide. Line scan intensity profiles are shown as arbitrary units (a.u.) on the right. The line scan was taken from the left image at the position of the gray dotted line. Colors match the color code in the left image. The black line indicates a Gaussian fit on the intensity distributions. Scale bars: left 50 μm, middle 10 μm, right 50 μm. (B) Same as in (A) but for Kv3.2 sub-unit staining. (C) Quantification of the intensity distributions shown in (A,B) and for Kv2.1 and Kv2.2 staining shown in . The half width of the Gaussian fit was used to calculate the potassium channel to MAP2 profile for Kv3.1b ( n = 7), Kv3.2 ( n = 8), Kv2.1 ( n = 6), and Kv2.2 ( n = 7). Black symbols represent single images, red symbols represent average values. The gray dotted line indicates a distribution profile equivalent to that of MAP2. Larger values indicate a broader, more dendritic distribution profile. (D) Single, digitally extracted MSO neuron stained for Kv3.1b. The position of the line scan is given by the red dotted line. (E) Intensity profile of the line scan shown in (D) . Gray area indicates the region of the cell’s nucleus. (F) Normalized intensity distribution of Kv2.1 ( n = 13) in single MSO neurons. The edge of the nucleus was defined as zero position. Gray area indicates the region of the soma. Dotted horizontal line indicates the half decay of the normalized intensity. (G) Same as in (F) but for Kv3.1b ( n = 6) sub-unit staining. Dotted vertical line indicates the position the intensity reached half of its initial value. (H) Same as in (F) but for Kv3.2 ( n = 9) sub-unit staining. Dotted vertical line indicates the position the intensity reached half of its initial value.
    Figure Legend Snippet: High voltage-activated potassium channels in MSO neurons. (A) Immunofluorescent staining of Kv3.1b, MAP2 (co-staining left, magnified Kv3.1b image middle) and Kv3.1b blocking peptide (+P, right). Intensity scaling of the gray scaled images is identical, to indicate the effect of the blocking peptide. Line scan intensity profiles are shown as arbitrary units (a.u.) on the right. The line scan was taken from the left image at the position of the gray dotted line. Colors match the color code in the left image. The black line indicates a Gaussian fit on the intensity distributions. Scale bars: left 50 μm, middle 10 μm, right 50 μm. (B) Same as in (A) but for Kv3.2 sub-unit staining. (C) Quantification of the intensity distributions shown in (A,B) and for Kv2.1 and Kv2.2 staining shown in . The half width of the Gaussian fit was used to calculate the potassium channel to MAP2 profile for Kv3.1b ( n = 7), Kv3.2 ( n = 8), Kv2.1 ( n = 6), and Kv2.2 ( n = 7). Black symbols represent single images, red symbols represent average values. The gray dotted line indicates a distribution profile equivalent to that of MAP2. Larger values indicate a broader, more dendritic distribution profile. (D) Single, digitally extracted MSO neuron stained for Kv3.1b. The position of the line scan is given by the red dotted line. (E) Intensity profile of the line scan shown in (D) . Gray area indicates the region of the cell’s nucleus. (F) Normalized intensity distribution of Kv2.1 ( n = 13) in single MSO neurons. The edge of the nucleus was defined as zero position. Gray area indicates the region of the soma. Dotted horizontal line indicates the half decay of the normalized intensity. (G) Same as in (F) but for Kv3.1b ( n = 6) sub-unit staining. Dotted vertical line indicates the position the intensity reached half of its initial value. (H) Same as in (F) but for Kv3.2 ( n = 9) sub-unit staining. Dotted vertical line indicates the position the intensity reached half of its initial value.

    Techniques Used: Staining, Blocking Assay

    anti kv3 2  (Alomone Labs)


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    Alomone Labs anti kv3 2
    Knockdown of <t>Kv3.4</t> inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
    Anti Kv3 2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv3 2/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    anti kv3 2 - by Bioz Stars, 2023-01
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    1) Product Images from "K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones"

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).

    Techniques Used: Construct, Cell Culture, Immunolabeling, Labeling, Expressing, Cotransfection, Fluorescence, Transfection

    Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.

    Techniques Used: In Vivo, Construct, Western Blot, Transfection, In Utero, Expressing, Fluorescence, shRNA

    Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .
    Figure Legend Snippet: Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .

    Techniques Used: Cell Culture, Incubation, Time-lapse Microscopy, Immunolabeling

    Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.

    Techniques Used: Double Staining, Cell Culture, Isolation, Fluorescence

    Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.

    Techniques Used: Cell Culture

    Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.
    Figure Legend Snippet: Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.

    Techniques Used: Imaging

    Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).
    Figure Legend Snippet: Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).

    Techniques Used: Isolation, Cell Culture

    A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.
    Figure Legend Snippet: A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.

    Techniques Used: Activity Assay, Binding Assay, Inhibition, Activation Assay, Concentration Assay

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    Knockdown of <t>Kv3.4</t> inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
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    1) Product Images from "K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones"

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).

    Techniques Used: Construct, Cell Culture, Immunolabeling, Labeling, Expressing, Cotransfection, Fluorescence, Transfection

    Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.

    Techniques Used: In Vivo, Construct, Western Blot, Transfection, In Utero, Expressing, Fluorescence, shRNA

    Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .
    Figure Legend Snippet: Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .

    Techniques Used: Cell Culture, Incubation, Time-lapse Microscopy, Immunolabeling

    Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.

    Techniques Used: Double Staining, Cell Culture, Isolation, Fluorescence

    Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.

    Techniques Used: Cell Culture

    Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.
    Figure Legend Snippet: Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.

    Techniques Used: Imaging

    Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).
    Figure Legend Snippet: Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).

    Techniques Used: Isolation, Cell Culture

    A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.
    Figure Legend Snippet: A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.

    Techniques Used: Activity Assay, Binding Assay, Inhibition, Activation Assay, Concentration Assay

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    Knockdown of <t>Kv3.4</t> inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
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    1) Product Images from "K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones"

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).

    Techniques Used: Construct, Cell Culture, Immunolabeling, Labeling, Expressing, Cotransfection, Fluorescence, Transfection

    Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.

    Techniques Used: In Vivo, Construct, Western Blot, Transfection, In Utero, Expressing, Fluorescence, shRNA

    Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .
    Figure Legend Snippet: Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .

    Techniques Used: Cell Culture, Incubation, Time-lapse Microscopy, Immunolabeling

    Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.

    Techniques Used: Double Staining, Cell Culture, Isolation, Fluorescence

    Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.

    Techniques Used: Cell Culture

    Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.
    Figure Legend Snippet: Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.

    Techniques Used: Imaging

    Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).
    Figure Legend Snippet: Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).

    Techniques Used: Isolation, Cell Culture

    A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.
    Figure Legend Snippet: A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.

    Techniques Used: Activity Assay, Binding Assay, Inhibition, Activation Assay, Concentration Assay

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    Alomone Labs anti kv3 2
    Knockdown of <t>Kv3.4</t> inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
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    1) Product Images from "K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones"

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).

    Techniques Used: Construct, Cell Culture, Immunolabeling, Labeling, Expressing, Cotransfection, Fluorescence, Transfection

    Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.
    Figure Legend Snippet: Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.

    Techniques Used: In Vivo, Construct, Western Blot, Transfection, In Utero, Expressing, Fluorescence, shRNA

    Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .
    Figure Legend Snippet: Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .

    Techniques Used: Cell Culture, Incubation, Time-lapse Microscopy, Immunolabeling

    Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.

    Techniques Used: Double Staining, Cell Culture, Isolation, Fluorescence

    Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.
    Figure Legend Snippet: Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.

    Techniques Used: Cell Culture

    Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.
    Figure Legend Snippet: Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.

    Techniques Used: Imaging

    Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).
    Figure Legend Snippet: Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).

    Techniques Used: Isolation, Cell Culture

    A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.
    Figure Legend Snippet: A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.

    Techniques Used: Activity Assay, Binding Assay, Inhibition, Activation Assay, Concentration Assay

    anti kv3 2  (Alomone Labs)


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    Alomone Labs anti kv3 2
    <t>Kv3.1</t> and <t>Kv3.2</t> channel proteins are not expressed in the SCN of mice deficient in both Kcnc1 and Kcnc2. IHC was used to determine expression of Kv3.1 and Kv3.2 channel proteins in the mouse SCN. Panels show photomicrograms (400×) of immuno-reactivity seen with each of the three genotypes: C57BL/6 (WT, top panels), Kcnc2-null (sKO, middle panels), Kcnc1- and Kcnc2-null (dKO, bottom panels). The sKO and dKO mice shown in this and subsequent figures are littermates on an ICR background. Tissue was collected at ZT 6. The scale bar represents 100 µm.
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    1) Product Images from "Fast delayed rectifier potassium current: critical for input and output of the circadian system"

    Article Title: Fast delayed rectifier potassium current: critical for input and output of the circadian system

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.5792-10.2011

    Kv3.1 and Kv3.2 channel proteins are not expressed in the SCN of mice deficient in both Kcnc1 and Kcnc2. IHC was used to determine expression of Kv3.1 and Kv3.2 channel proteins in the mouse SCN. Panels show photomicrograms (400×) of immuno-reactivity seen with each of the three genotypes: C57BL/6 (WT, top panels), Kcnc2-null (sKO, middle panels), Kcnc1- and Kcnc2-null (dKO, bottom panels). The sKO and dKO mice shown in this and subsequent figures are littermates on an ICR background. Tissue was collected at ZT 6. The scale bar represents 100 µm.
    Figure Legend Snippet: Kv3.1 and Kv3.2 channel proteins are not expressed in the SCN of mice deficient in both Kcnc1 and Kcnc2. IHC was used to determine expression of Kv3.1 and Kv3.2 channel proteins in the mouse SCN. Panels show photomicrograms (400×) of immuno-reactivity seen with each of the three genotypes: C57BL/6 (WT, top panels), Kcnc2-null (sKO, middle panels), Kcnc1- and Kcnc2-null (dKO, bottom panels). The sKO and dKO mice shown in this and subsequent figures are littermates on an ICR background. Tissue was collected at ZT 6. The scale bar represents 100 µm.

    Techniques Used: Expressing

    Detailed analysis of activity data.
    Figure Legend Snippet: Detailed analysis of activity data.

    Techniques Used: Activity Assay

    anti kv3 2  (Alomone Labs)


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    Alomone Labs anti kv3 2
    <t>Kv3.1</t> and <t>Kv3.2</t> channel proteins are not expressed in the SCN of mice deficient in both Kcnc1 and Kcnc2. IHC was used to determine expression of Kv3.1 and Kv3.2 channel proteins in the mouse SCN. Panels show photomicrograms (400×) of immuno-reactivity seen with each of the three genotypes: C57BL/6 (WT, top panels), Kcnc2-null (sKO, middle panels), Kcnc1- and Kcnc2-null (dKO, bottom panels). The sKO and dKO mice shown in this and subsequent figures are littermates on an ICR background. Tissue was collected at ZT 6. The scale bar represents 100 µm.
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    1) Product Images from "Fast delayed rectifier potassium current: critical for input and output of the circadian system"

    Article Title: Fast delayed rectifier potassium current: critical for input and output of the circadian system

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.5792-10.2011

    Kv3.1 and Kv3.2 channel proteins are not expressed in the SCN of mice deficient in both Kcnc1 and Kcnc2. IHC was used to determine expression of Kv3.1 and Kv3.2 channel proteins in the mouse SCN. Panels show photomicrograms (400×) of immuno-reactivity seen with each of the three genotypes: C57BL/6 (WT, top panels), Kcnc2-null (sKO, middle panels), Kcnc1- and Kcnc2-null (dKO, bottom panels). The sKO and dKO mice shown in this and subsequent figures are littermates on an ICR background. Tissue was collected at ZT 6. The scale bar represents 100 µm.
    Figure Legend Snippet: Kv3.1 and Kv3.2 channel proteins are not expressed in the SCN of mice deficient in both Kcnc1 and Kcnc2. IHC was used to determine expression of Kv3.1 and Kv3.2 channel proteins in the mouse SCN. Panels show photomicrograms (400×) of immuno-reactivity seen with each of the three genotypes: C57BL/6 (WT, top panels), Kcnc2-null (sKO, middle panels), Kcnc1- and Kcnc2-null (dKO, bottom panels). The sKO and dKO mice shown in this and subsequent figures are littermates on an ICR background. Tissue was collected at ZT 6. The scale bar represents 100 µm.

    Techniques Used: Expressing

    Detailed analysis of activity data.
    Figure Legend Snippet: Detailed analysis of activity data.

    Techniques Used: Activity Assay

    kv3 1 b amino acid residues 567 585  (Alomone Labs)


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    Alomone Labs kv3 1 b amino acid residues 567 585
    Kv3 1 B Amino Acid Residues 567 585, 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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    anti kv3 2  (Alomone Labs)


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    Primary antibodies used in this study.
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    Knockdown of <t>Kv3.4</t> inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
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    Alomone Labs kv3 1 b amino acid residues 567 585
    Knockdown of <t>Kv3.4</t> inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).
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    Primary antibodies used in this study.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Distinct Distribution Patterns of Potassium Channel Sub-Units in Somato-Dendritic Compartments of Neurons of the Medial Superior Olive

    doi: 10.3389/fncel.2019.00038

    Figure Lengend Snippet: Primary antibodies used in this study.

    Article Snippet: Kv3.2 , Rabbit , Polyclonal , 184–204 , Cy3 , 1:200 , Alomone labs , APC-011.

    Techniques:

    High voltage-activated potassium channels in MSO neurons. (A) Immunofluorescent staining of Kv3.1b, MAP2 (co-staining left, magnified Kv3.1b image middle) and Kv3.1b blocking peptide (+P, right). Intensity scaling of the gray scaled images is identical, to indicate the effect of the blocking peptide. Line scan intensity profiles are shown as arbitrary units (a.u.) on the right. The line scan was taken from the left image at the position of the gray dotted line. Colors match the color code in the left image. The black line indicates a Gaussian fit on the intensity distributions. Scale bars: left 50 μm, middle 10 μm, right 50 μm. (B) Same as in (A) but for Kv3.2 sub-unit staining. (C) Quantification of the intensity distributions shown in (A,B) and for Kv2.1 and Kv2.2 staining shown in . The half width of the Gaussian fit was used to calculate the potassium channel to MAP2 profile for Kv3.1b ( n = 7), Kv3.2 ( n = 8), Kv2.1 ( n = 6), and Kv2.2 ( n = 7). Black symbols represent single images, red symbols represent average values. The gray dotted line indicates a distribution profile equivalent to that of MAP2. Larger values indicate a broader, more dendritic distribution profile. (D) Single, digitally extracted MSO neuron stained for Kv3.1b. The position of the line scan is given by the red dotted line. (E) Intensity profile of the line scan shown in (D) . Gray area indicates the region of the cell’s nucleus. (F) Normalized intensity distribution of Kv2.1 ( n = 13) in single MSO neurons. The edge of the nucleus was defined as zero position. Gray area indicates the region of the soma. Dotted horizontal line indicates the half decay of the normalized intensity. (G) Same as in (F) but for Kv3.1b ( n = 6) sub-unit staining. Dotted vertical line indicates the position the intensity reached half of its initial value. (H) Same as in (F) but for Kv3.2 ( n = 9) sub-unit staining. Dotted vertical line indicates the position the intensity reached half of its initial value.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Distinct Distribution Patterns of Potassium Channel Sub-Units in Somato-Dendritic Compartments of Neurons of the Medial Superior Olive

    doi: 10.3389/fncel.2019.00038

    Figure Lengend Snippet: High voltage-activated potassium channels in MSO neurons. (A) Immunofluorescent staining of Kv3.1b, MAP2 (co-staining left, magnified Kv3.1b image middle) and Kv3.1b blocking peptide (+P, right). Intensity scaling of the gray scaled images is identical, to indicate the effect of the blocking peptide. Line scan intensity profiles are shown as arbitrary units (a.u.) on the right. The line scan was taken from the left image at the position of the gray dotted line. Colors match the color code in the left image. The black line indicates a Gaussian fit on the intensity distributions. Scale bars: left 50 μm, middle 10 μm, right 50 μm. (B) Same as in (A) but for Kv3.2 sub-unit staining. (C) Quantification of the intensity distributions shown in (A,B) and for Kv2.1 and Kv2.2 staining shown in . The half width of the Gaussian fit was used to calculate the potassium channel to MAP2 profile for Kv3.1b ( n = 7), Kv3.2 ( n = 8), Kv2.1 ( n = 6), and Kv2.2 ( n = 7). Black symbols represent single images, red symbols represent average values. The gray dotted line indicates a distribution profile equivalent to that of MAP2. Larger values indicate a broader, more dendritic distribution profile. (D) Single, digitally extracted MSO neuron stained for Kv3.1b. The position of the line scan is given by the red dotted line. (E) Intensity profile of the line scan shown in (D) . Gray area indicates the region of the cell’s nucleus. (F) Normalized intensity distribution of Kv2.1 ( n = 13) in single MSO neurons. The edge of the nucleus was defined as zero position. Gray area indicates the region of the soma. Dotted horizontal line indicates the half decay of the normalized intensity. (G) Same as in (F) but for Kv3.1b ( n = 6) sub-unit staining. Dotted vertical line indicates the position the intensity reached half of its initial value. (H) Same as in (F) but for Kv3.2 ( n = 9) sub-unit staining. Dotted vertical line indicates the position the intensity reached half of its initial value.

    Article Snippet: Kv3.2 , Rabbit , Polyclonal , 184–204 , Cy3 , 1:200 , Alomone labs , APC-011.

    Techniques: Staining, Blocking Assay

    Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Knockdown of Kv3.4 inhibits neurite protrusion and axon elongation. A–D, The spinal cord of chick embryos at HH15-HH17 was electroporated with constructs encoding EYFP alone (control, A), LacZshRNA/EYFP (LacZshRNA, B), Kv3.4shRNA/EYFP (Kv3.4shRNA, C), or Kv3.4shRNA/resKv3.4[Kv3.4shRNA-resistant Kv3.4]/EYFP (Kv3.4shRNA + resKv3.4, D). The dorsal spinal cord was dissociated at HH21-HH23 and cultured for 20 h before immunolabeling Kv3.4 (red). A–D, A′–D′, Top, Neurons without neurites. Bottom, Axon-bearing neurons. Nontransfected neurons in each culture were used for comparison, and their nuclei were labeled by DAPI (blue). Scale bars: Top, 17 μm; Bottom, 20 μm. A, B, In the control or LacZshRNA+ neurons, Kv3.4-IR was strong in the somatic surfaces of neurons without neurites, but it became more evident in the growth cones of axon-bearing neurons. LacZshRNA did not suppress Kv3.4 expression. C, Kv3.4shRNA strongly reduced Kv3.4-IR in neurons without neurites (arrowhead) but had a weaker effect in axon-bearing neurons. D, Cotransfection of resKv3.4 rescued the knockdown effect caused by Kv3.4shRNA. E, After measuring the fluorescence intensity of neurons with or without neurites (30 neuron counts for each; total 60 counts), the relative Kv3.4 protein level was obtained by dividing the Kv3.4 fluorescent intensity of EYFP+ neurons by that of EYFP− neurons. F, The percentage of protrusion-bearing neurons. G, The percentage of axon-bearing neurons. H, The average of axon length. I, The cumulative distribution of axon length (Kolmogorov–Smirnov test) shows that the axons of Kv3.4shRNA-transfected neurons are shorter. Numbers in parentheses indicate total EYFP+ neuron counts pooled from three independent experiments. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). **p < 0.01, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA).

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( Huang et al., 2012 ).

    Techniques: Construct, Cell Culture, Immunolabeling, Labeling, Expressing, Cotransfection, Fluorescence, Transfection

    Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Knockdown of Kv3.4 inhibits axon elongation, pathfinding, and fasciculation in vivo. A–E, The right side spinal cord of chick embryo at HH15-HH17 was electroporated with constructs encoding EYFP (control, B), EYFP/LacZshRNA (C), EYFP/Kv3.4shRNA (D), or EYFP/ Kv3.4shRNA/Kv3.4shRNA-resistant Kv3.4 (resKv3.4) (E). Embryos were fixed at HH22-HH23, and their spinal cords in open-book configurations show the trajectories of EYFP+ commissural axons. A, Anterior; D, dorsal; P, posterior; V, ventral. B–E, Arrows indicate the bundle of commissural axons (ventral funiculus, VF). D, Arrowheads indicate stalling axons at the floor plate (FP). Asterisks indicate misguided axons. F, Summary of projection errors of spinal commissural axons. G, The percentage of EYFP+ axons with projection errors. H, The width of the ventral funiculus. I, Western blotting was performed using lysate of HEK-293 cells transfected with constructs encoding Kv3.4/LacZshRNA/EYFP, Kv3.4/Kv3.4shRNA/EYFP, or Kv3.4shRNA/resKv3.4/EYFP. The major protein band of Kv3.4 at position of 100 kDa was shown, and GAPDH was as used as a loading control. J–M, In E15.5 rat brain, the ventricular zone (green) adjacent to the lateral ventricle (LV, blue) was electroporated with constructs encoding EYFP/LacZshRNA (K), EYFP/Kv3.4shRNA (L), or EYFP/Kv3.4shRNA/resKv3.4 (M). The positive electrode paddle was located on the left side of brain. Coronal sections of E20.5 rat brain were analyzed after embryos were grown in utero. EYFP+ callosal axons, which project from the cingulate cortex (CgC) and frontal cortex (FC), only reach the contralateral cingulate cortex in the Kv3.4shRNA-expressing brain. PC, Parietal cortex. N, Measurement of axon projection to the contralateral side. Relative intensity in each region (J, −2, −1, 0, 1, 2) is obtained by normalizing its fluorescence intensity with that in region 2. G, H, N, Numbers in parentheses indicate the total number of embryos analyzed. Data are mean ± SEM. *p < 0.05, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). #p < 0.05, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ##p < 0.01, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). ###p < 0.001, comparing “Kv3.4shRNA + resKv3.4” with Kv3.4 shRNA (Tukey's post hoc test after one-way ANOVA). Scale bars: B–E, 60 μm; K–M, 670 μm.

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( Huang et al., 2012 ).

    Techniques: In Vivo, Construct, Western Blot, Transfection, In Utero, Expressing, Fluorescence, shRNA

    Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Blockade of Kv3.4 causes axon retraction and defasciculation. A–D, Axons of cultured chick dorsal spinal neurons grew over the control period (from −0.5 to 0 h), followed by bath incubation of vehicle (A) or BDSII (3 μm, B) from 0 to 0.5 h. Representative images were captured at −0.5, 0, and 0.5 h using time-lapse microscopy. A, B, Arrowheads indicate growth cones. BDSII causes axon retraction. E–H, Chick dorsal spinal cord explants, with netrin-1 addition soon after plating and BDSII (1.5 μm) addition at 30 h after plating, were fixed at 45 h after plating. Axons in explants were visualized by immunolabeling for axonin-1 (red). E, F, Boxed areas are magnified in E′ and F′, respectively. Quantification of the axonal area per explant (G) and the percentage of axon bundles with diameter >5 μm per explant (H). BDSII decreases netrin-1-induced axon growth and causes axon defasciculation. Numbers in parentheses indicate total number of neurons (C) or explants (G, H) analyzed. Data are mean ± SEM. **p < 0.01, compared with vehicle (D) (independent-samples t test). ***p < 0.001, compared with netrin-1 (G, H) (independent-samples t test). Scale bars: A, B, 25 μm; E, F, 200 μm; E′, F′, 80 μm .

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( Huang et al., 2012 ).

    Techniques: Cell Culture, Incubation, Time-lapse Microscopy, Immunolabeling

    Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F, Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A, Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons (B–F, arrowheads) is evident during HH19-HH25 but disappears at HH27. D, Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L, Transverse sections of the spinal cord at HH23 were immunostained as indicated. G, Absence of Kv1.5-IR. H, Kv4.2-IR in the somata and dendrites of motoneurons (MN). I, Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons (J, arrow) but weak in precrossing commissural axons (J, arrowhead). K, Absence of Kv3.2-IR. L, Kv3.3 in motoneurons. M–M″, Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″, Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″, Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone (O–O″) and Kv3.1b in the soma/axon shaft (P–P″) of cultured dorsal spinal neurons. Q–Q″, Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions (Q″). R, Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM (n = 8 neurons, pooled from three independent experiments done on different days). ***p < 0.001, comparison of the indicated pairs (Tukey's post hoc test after one-way ANOVA). S–U, Kv3.4-IR in spinal commissural axons (arrowheads) of rat embryos is evident during E12.5-E13.5 (S, T), but it disappears at E14.5 (U). Scale bar: (in U) A, B, 35 μm; C, D, 40 μm; E, 50 μm; F, 60 μm; G–L, 50 μm; M–M″, 30 μm; N–N″, 13 μm; O–O″, 20 μm; P–P″, 20 μm; Q–Q″, 16 μm; S–U, 100 μm.

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( Huang et al., 2012 ).

    Techniques: Double Staining, Cell Culture, Isolation, Fluorescence

    Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Kv3.4 in the axonal growth cones of motoneurons, DRG neurons, RGCs, and callosal projection neurons. A, B, Transverse sections of the spinal cord (SC) of HH23 chick embryos were immunostained, showing Kv3.4-IR in the axonal bundle (arrowhead) of motoneurons (MN) (A) and the bifurcation zone (BZ) of DRG neuron afferents (B). C, D, Motoneurons and DRG neurons of HH21-HH23 chick embryos were dissociated, cultured for 20 h, and double immunostained for Kv3.4 and Islet1/2. E, F, RGCs and callosal projection neurons (CPNs) were dissociated from the retina and cingulate/frontal cortices of E18.5 rat embryo, respectively. After 16 h of culture, cells were double immunostained for Kv3.4 and Islet1/2 (E′) or TAG-1 (F′). Kv3.4-IR is evident in axonal growth cones (C″–F″, arrows). Scale bars: A, 38 μm; B, 25 μm; C, 16 μm; D, 13 μm; E, F, 16 μm.

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( Huang et al., 2012 ).

    Techniques: Cell Culture

    Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Blockade of Kv3.4 increases the influx of Ca2+ in axonal growth cones. A–C, During 10 min Ca2+ imaging analysis, the baseline was obtained during 0–1 min, and vehicle or BDSII (5 μm) was focally applied (arrows) to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images show relative levels of [Ca2+]i (purple to red, low to high) in growth cones, which are taken at 0, 3, 6, and 10 min, respectively. D, BDSII induces a sustained Ca2+ elevation in some GCaMP3+ neurons. E, BDSII induces Ca2+ transients in some other GCaMP3+ neurons. F, The incidence (% of neurons examined) of Ca2+ elevation and Ca2+ transients. G–I, The frequency (G, per hour) and amplitude (H, ΔF/F, as shown in E) of Ca2+ transients are higher in BDSII-treated neurons than vehicle-treated neurons, but the duration (I, seconds) has no difference. J, In the representative growth cone of fura-2-loaded neurons, the ratio of 340/380 (pseudocolor) is low during 0–1 min, but it is increased after focal application (arrow) of BDSII (5 μm) during 1–10 min. K, Compared with vehicle, BDSII increases the peak amplitude of [Ca2+]i transients in axonal growth cones. L, Representative patterns of BDSII-induced Ca2+ transients after depleting extracellular Ca2+ (Ca2+-free) or depleting intracellular Ca2+ using thapsigargin (2 μm) in bath medium. M, Representative patterns of BDSII-induced Ca2+ transients after bath application of SKF-96535 (25 μm), TTA-P2 (3 μm), nifedipine (5 μm), or ω-conotoxin (2 μm). N, BDSII-induced Ca2+ transients are abolished by extracellular Ca2+ depletion, TTA-P2, or nifedipine. O, P, In bath medium preincubated with vehicle or BDSII (3 μm), the baseline is obtained during 0–1 min, and netrin-1 (arrows) is focally applied to the growth cone of GCaMP3+ neurons during 1–10 min. Representative pseudocolor images are taken at 0, 2, 5, and 10 min, respectively. Q, Netrin-1-induced Ca2+ elevation is increased by BDSII preincubation. Number in parentheses indicates the total number of neurons analyzed. Data are mean ± SEM. *p < 0.05, compared with vehicle (independent-samples t test). ***p < 0.001, compared with vehicle (independent-samples t test). Scale bar, 10 μm.

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( Huang et al., 2012 ).

    Techniques: Imaging

    Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: Kv3.4-mediated K+ currents reduce membrane excitability in the axonal growth cones. A, An outside-out patch, isolated from growth cones of cultured dorsal spinal neurons, was held at −70 mV and hyperpolarized to −90 mV for 1.5 s before test pulses ranging from −70 to 70 mV (800 ms, 20 mV increments). Representative total outward K+ currents are shown. B, C, Plot of 20%–80% rise time (B) or decay time constant (C) versus test pulse voltage. Number in parentheses indicates the total number of neurons analyzed. D, Normalized peak conductance (G/Gmax)-voltage relationship (n = 3). The solid curve is given by Boltzmann function raised to the fourth power: [1/(1 + exp [−(V − Vh)/k])]4, where V is the membrane potential, Vh is the potential at which the Boltzmann function is 0.5, and k is the slope factor. Vh is −35.4 mV, and the slope factor is 23.9 mV. This function reaches a midpoint at a value of 1.8 ± 6.6 mV. E, BDSII-sensitive K+ current (red) is obtained by subtracting the sustained component (green) from the total current (control, black). F, Summary of the effect of 0.5 μm BDSII on peak current amplitude (n = 6). G, H, Summary of 20%–80% rise time and decay time constant between the control and BDSII-sensitive currents (n = 4). I, J, Whole-cell current-clamp recording was performed in growth cones of cultured dorsal spinal neurons. Membrane potential (Vm) was measured before and after focal application of BDSII (n = 4) (I). Representative action potentials were elicited with 200 ms current pulse of 50 pA in the growth cone before (black trace) and after BDSII (red trace) (J). Peak is defined from the initial rising phase (blue arrow) to the first valley (J, black or red arrow). K–O, Properties of action potential before and after BDSII (n = 6). Data are mean ± SEM. n.s., no significant difference. *p < 0.05, compared with control (paired-samples t test). ***p < 0.001, compared with control (paired-samples t test).

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( Huang et al., 2012 ).

    Techniques: Isolation, Cell Culture

    A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.

    Journal: The Journal of Neuroscience

    Article Title: K + Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca 2+ into Growth Cones

    doi: 10.1523/JNEUROSCI.1076-16.2017

    Figure Lengend Snippet: A schematic summary of Kv3.4 function in the growth cone. A, In normal growing axons, the growth cone membrane is depolarized by spontaneous electrical activity (1) or after the binding of an attractive guidance cue (such as netrin-1) to its receptor (such as DCC) (2). Membrane depolarization allows Ca2+ influx through T-type and L-type Cav channels sequentially. Then, Kv3.4 channels are activated and Kv3.4-mediated A-type K+ outward currents reduce membrane excitability. B, After Kv3.4 knockdown by Kv3.4shRNA or Kv3.4 blockade by BDSII, excessive extracellular Ca2+ ions enter the growth cone, which leads to axon growth inhibition. BDSII-induced Ca2+ influx does not require the release of intracellular Ca2+ from the ER (endoplasmic reticulum). C, The membrane potential of growth cones can be depolarized by spontaneous electrical activity or by the binding of attractive guidance cues. Slight membrane depolarization induces sustained Ca2+ elevation, and the opening of Kv3.4 channels quickly reduces membrane excitability, which can inhibit the generation of action potentials. Substantial membrane depolarization evokes Ca2+-dependent action potentials to generate Ca2+ transients, and the activation of Kv3.4 channels repolarizes the membrane to reduce the amplitudes of action potentials, resulting in Ca2+ transients with smaller amplitudes. Thus, by controlling growth cone membrane excitability, Kv3.4 acts to maintain [Ca2+]i at an optimal concentration for normal axon growth. AP, Action potential; Cav, voltage-gated calcium channel; DCC, deleted in colorectal cancer; IP3R, inositol 1,4,5-triphosphate receptor.

    Article Snippet: The specificity of anti-Kv3.2 (Alomone Labs catalog #APC-011, RRID:AB_2040168), anti-Kv3.3 (Alomone Labs catalog #APC-102, RRID:AB_2040170), and anti-Kv3.4 (Alomone Labs catalog #APC-019, RRID:AB_ 2040172) has been described previously ( Huang et al., 2012 ).

    Techniques: Activity Assay, Binding Assay, Inhibition, Activation Assay, Concentration Assay