ω conotoxin gvia  (Alomone Labs)


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    Structured Review

    Alomone Labs ω conotoxin gvia
    GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM <t>ω-conotoxin-GVIA</t> (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P
    ω Conotoxin Gvia, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons"

    Article Title: Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201511383

    GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P
    Figure Legend Snippet: GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P

    Techniques Used: Activity Assay, Mouse Assay, Activation Assay

    GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P
    Figure Legend Snippet: GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P

    Techniques Used: Activity Assay, Transfection, Inhibition

    2) Product Images from "Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons"

    Article Title: Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2011.181

    Rb1 inhibited the I Ba in hippocampal neurons, and this inhibitory effect was eliminated after the application of nifedipine (A). Neither ω-conotoxin-GVIA nor ω-agatoxin IVA diminished the Rb1-sensitive I Ba (B and C, respectively). Upper panel, pairs of the inward currents evoked by pulses from −60 to +0 mV (0–+20 mV) at the times indicated in the lower panel. Lower panel, time course of the effects of 10 μmol/L Rb1 on the I Ba amplitude before and after application of the Ca 2+ channel antagonists (10 μmol/L nifedipine, 1 μmol/L ω-conotoxin GVIA and 30 nmol/L ω-agatoxin IVA). The bar graphs for Rb1 inhibition (mean±SEM, n =5 for Rb1) on the I Ba in cells untreated or treated with Ca 2+ channel antagonists. c P
    Figure Legend Snippet: Rb1 inhibited the I Ba in hippocampal neurons, and this inhibitory effect was eliminated after the application of nifedipine (A). Neither ω-conotoxin-GVIA nor ω-agatoxin IVA diminished the Rb1-sensitive I Ba (B and C, respectively). Upper panel, pairs of the inward currents evoked by pulses from −60 to +0 mV (0–+20 mV) at the times indicated in the lower panel. Lower panel, time course of the effects of 10 μmol/L Rb1 on the I Ba amplitude before and after application of the Ca 2+ channel antagonists (10 μmol/L nifedipine, 1 μmol/L ω-conotoxin GVIA and 30 nmol/L ω-agatoxin IVA). The bar graphs for Rb1 inhibition (mean±SEM, n =5 for Rb1) on the I Ba in cells untreated or treated with Ca 2+ channel antagonists. c P

    Techniques Used: Inhibition

    (A) Phase-contrast image showing a single patch recording from 7-d cultured hippocampal neurons for the recording of the VGCCs. Scale bar, 10 μm. (B) Pharmacological separation of the VGCC subtypes in hippocampal neurons. Upper panel, inward Ca 2+ channel Ba 2+ currents evoked by pulses from −60 mV to 0 mV at the times indicated in the lower panel. Lower panel, time course of effects of ω-conotoxin GVIA (1 μmol/L), ω-agatoxin IVA (30 nmol/L) and nifedipine (10 μmol/L) on the Ba 2+ current amplitude.
    Figure Legend Snippet: (A) Phase-contrast image showing a single patch recording from 7-d cultured hippocampal neurons for the recording of the VGCCs. Scale bar, 10 μm. (B) Pharmacological separation of the VGCC subtypes in hippocampal neurons. Upper panel, inward Ca 2+ channel Ba 2+ currents evoked by pulses from −60 mV to 0 mV at the times indicated in the lower panel. Lower panel, time course of effects of ω-conotoxin GVIA (1 μmol/L), ω-agatoxin IVA (30 nmol/L) and nifedipine (10 μmol/L) on the Ba 2+ current amplitude.

    Techniques Used: Cell Culture

    3) Product Images from "Voltage-dependent Ca2+ channels promote branching morphogenesis of salivary glands by patterning differential growth"

    Article Title: Voltage-dependent Ca2+ channels promote branching morphogenesis of salivary glands by patterning differential growth

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25957-w

    The effect of L-type voltage-dependent Ca 2+ channels (VDCCs) on branching morphogenesis. ( A ) Morphological changes of SMG cultures (E13.5) upon 500 μM LaCl 3 treatment. ( B ) Bud numbers of SMG cultures upon 500 μM LaCl 3 (La) and 1 M EGTA treatment. n = 7, Data are represented as mean ± SEM. ( C ) Representative images of SMG cultures treated with various Ca 2+ channel inhibitors. ( D ) Bud numbers of SMG cultures (E12) upon treatment with various Ca 2+ channel inhibitors. Nif: 100 μM nifedipine; Gd: 500 μM GdCl3; SKF: 10 μM SKF 96365, n = 7, Data are represented as mean ±SEM. ( E ) Bud numbers of SMG cultures (E13) upon different concentrations of nifedipine treatment for 48 h. n = 5. Data are represented as mean ± SEM. ( F ) Relative acinar size of SMGs (E13) upon different concentrations of nifedipine treatment. n = 5. Data are represented as mean ±SEM. ( G ) Epithelial bud numbers of SMGs (E13.5) upon treatment with antagonists for different types of VDCCs: 2 μM w-Agatoxin IVA (Aga, P-type); 2 μM SNX 482 (SNX, R-type); 10 μM w-Conotoxin GVIA (Cono, N-type). n = 6. Data are represented as mean ±SEM. ( H ) Time-course changes of bud outline of developing SMG cultures. Arrowheads indicate the cleft initiation points. ( I ) Time-lapse images of epithelial rudiment cultures (E13) upon 100 μM nifedipine treatment. Arrowheads indicate cleft sites. Panels below indicate the bud numbers. Scale bars: 200 ( A , C ), 100 μm ( H , I ).
    Figure Legend Snippet: The effect of L-type voltage-dependent Ca 2+ channels (VDCCs) on branching morphogenesis. ( A ) Morphological changes of SMG cultures (E13.5) upon 500 μM LaCl 3 treatment. ( B ) Bud numbers of SMG cultures upon 500 μM LaCl 3 (La) and 1 M EGTA treatment. n = 7, Data are represented as mean ± SEM. ( C ) Representative images of SMG cultures treated with various Ca 2+ channel inhibitors. ( D ) Bud numbers of SMG cultures (E12) upon treatment with various Ca 2+ channel inhibitors. Nif: 100 μM nifedipine; Gd: 500 μM GdCl3; SKF: 10 μM SKF 96365, n = 7, Data are represented as mean ±SEM. ( E ) Bud numbers of SMG cultures (E13) upon different concentrations of nifedipine treatment for 48 h. n = 5. Data are represented as mean ± SEM. ( F ) Relative acinar size of SMGs (E13) upon different concentrations of nifedipine treatment. n = 5. Data are represented as mean ±SEM. ( G ) Epithelial bud numbers of SMGs (E13.5) upon treatment with antagonists for different types of VDCCs: 2 μM w-Agatoxin IVA (Aga, P-type); 2 μM SNX 482 (SNX, R-type); 10 μM w-Conotoxin GVIA (Cono, N-type). n = 6. Data are represented as mean ±SEM. ( H ) Time-course changes of bud outline of developing SMG cultures. Arrowheads indicate the cleft initiation points. ( I ) Time-lapse images of epithelial rudiment cultures (E13) upon 100 μM nifedipine treatment. Arrowheads indicate cleft sites. Panels below indicate the bud numbers. Scale bars: 200 ( A , C ), 100 μm ( H , I ).

    Techniques Used:

    4) Product Images from "Binomial parameters differ across neocortical layers and with different classes of connections in adult rat and cat neocortex"

    Article Title: Binomial parameters differ across neocortical layers and with different classes of connections in adult rat and cat neocortex

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

    doi: 10.1073/pnas.0705661104

    A layer 3 depressing pyramid–pyramid connection ( A ) and a layer 3 depressing pyramid to fast spiking, multipolar interneuron connection ( B ). These pairs were first recorded under control conditions (filled circles) and then after addition of ω-conotoxin GVIA (open). After addition of this N-type Ca 2+ channel blocker, EPSPs decreased in M , and F increased. Shown are CV plotted against M ( Aa and Ba ) and F plotted against M ( Ab and Bb ). Control and ω-conotoxin GVIA data were fit separately. Estimates for n and q are given as Insets . For these plots, r 2 (coefficient of determination) was control 0.83, conotoxin 0.92 ( Aa ); control 0.90, conotoxin 0.95 ( Ab ); control 0.75, conotoxin 0.93 ( Ba ); and control 0.85, conotoxin 0.96 ( Bb ).
    Figure Legend Snippet: A layer 3 depressing pyramid–pyramid connection ( A ) and a layer 3 depressing pyramid to fast spiking, multipolar interneuron connection ( B ). These pairs were first recorded under control conditions (filled circles) and then after addition of ω-conotoxin GVIA (open). After addition of this N-type Ca 2+ channel blocker, EPSPs decreased in M , and F increased. Shown are CV plotted against M ( Aa and Ba ) and F plotted against M ( Ab and Bb ). Control and ω-conotoxin GVIA data were fit separately. Estimates for n and q are given as Insets . For these plots, r 2 (coefficient of determination) was control 0.83, conotoxin 0.92 ( Aa ); control 0.90, conotoxin 0.95 ( Ab ); control 0.75, conotoxin 0.93 ( Ba ); and control 0.85, conotoxin 0.96 ( Bb ).

    Techniques Used:

    5) Product Images from "Effects of IgG anti-GM1 monoclonal antibodies on neuromuscular transmission and calcium channel binding in rat neuromuscular junctions"

    Article Title: Effects of IgG anti-GM1 monoclonal antibodies on neuromuscular transmission and calcium channel binding in rat neuromuscular junctions

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2015.2575

    Effects of pretreatment with the N-type calcium channel blocker, ω-conotoxin GVIA, on the inhibitory effect of IgG anti-GM1 mAb (1:100) on spontaneous muscle action potentials (SMAPs) in the spinal cord-muscle co-culture system. (A) Inhibition
    Figure Legend Snippet: Effects of pretreatment with the N-type calcium channel blocker, ω-conotoxin GVIA, on the inhibitory effect of IgG anti-GM1 mAb (1:100) on spontaneous muscle action potentials (SMAPs) in the spinal cord-muscle co-culture system. (A) Inhibition

    Techniques Used: Co-Culture Assay, Inhibition

    6) Product Images from "CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy"

    Article Title: CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

    Journal: Molecular Pain

    doi: 10.1186/1744-8069-8-54

    Pharmacological and biophysical dissection of TAT-CBD3A6K-mediated block of T-and R-type calcium currents in DRG neurons. ( A ) Representative T- (top) and R-type (bottom) current traces obtained from two separate DRG neurons evoked by 200 ms steps in 5 mV increments from −60 mV to +50 mV, from a holding potential of −90 mV. The extracellular bath solution contained 5 mM Nifedipine (Nif), 200 nM ω-Agatoxin IVA (Aga) and 500 nM ω-Conotoxin GVIA (CTX) to block L-, P/Q-, and N-type calcium currents, respectively. ( B ) Summary of the normalized conductance (G) versus voltage relations for DRG neurons with T- (filled squares) or R- (open squares) type calcium currents. The dotted line at −10 mV highlights the clear discrimination in conductances between T- and R-type currents. ( C ) Representative currents, evoked by a ramp depolarizations from −60 mV to +20 mV for 2 s, illustrating the presence of both T- and R-type currents in the same DRG neuron before (left trace) and 2 min after application 10 μM TAT-CBD3A6K (right trace)
    Figure Legend Snippet: Pharmacological and biophysical dissection of TAT-CBD3A6K-mediated block of T-and R-type calcium currents in DRG neurons. ( A ) Representative T- (top) and R-type (bottom) current traces obtained from two separate DRG neurons evoked by 200 ms steps in 5 mV increments from −60 mV to +50 mV, from a holding potential of −90 mV. The extracellular bath solution contained 5 mM Nifedipine (Nif), 200 nM ω-Agatoxin IVA (Aga) and 500 nM ω-Conotoxin GVIA (CTX) to block L-, P/Q-, and N-type calcium currents, respectively. ( B ) Summary of the normalized conductance (G) versus voltage relations for DRG neurons with T- (filled squares) or R- (open squares) type calcium currents. The dotted line at −10 mV highlights the clear discrimination in conductances between T- and R-type currents. ( C ) Representative currents, evoked by a ramp depolarizations from −60 mV to +20 mV for 2 s, illustrating the presence of both T- and R-type currents in the same DRG neuron before (left trace) and 2 min after application 10 μM TAT-CBD3A6K (right trace)

    Techniques Used: Dissection, Blocking Assay, Mass Spectrometry

    Characterization of TAT-CBD3A6K-mediated inhibition of T- and R-type calcium currents. ( A ) Representative family of traces from a DRG neuron with both T- and R-type calcium currents before ( left ), 2 min ( middle ) and 5 min ( right ) after addition of 10 μM TAT-CBD3A6K. Currents were elicited in response to the voltage protocol described in the legend to Figure 5 A. To isolate T- and R-type calcium currents, the extracellular bath solution contained 5 mM Nifedipine (Nif), 200 nM ω-Agatoxin IVA (Aga) and 500 nM ω-Conotoxin GVIA (CTX) to block L-, P/Q-, and N-type calcium currents, respectively. ( B , C ) Time course of TAT-CBD3A6K mediated inhibition (“run-down”) of T-type ( B ) and R-type ( C ) calcium currents. Time course of inhibition is shown as averaged normalized current density (pA pF -1 ) before peptide addition and at intervals of 30 s for 5 min. Averaged values are shown with standard error for 4–6 control cells and 4 cells following addition of 10 μM TAT-CBD3A6K. The asterisk denotes statistical significance (p
    Figure Legend Snippet: Characterization of TAT-CBD3A6K-mediated inhibition of T- and R-type calcium currents. ( A ) Representative family of traces from a DRG neuron with both T- and R-type calcium currents before ( left ), 2 min ( middle ) and 5 min ( right ) after addition of 10 μM TAT-CBD3A6K. Currents were elicited in response to the voltage protocol described in the legend to Figure 5 A. To isolate T- and R-type calcium currents, the extracellular bath solution contained 5 mM Nifedipine (Nif), 200 nM ω-Agatoxin IVA (Aga) and 500 nM ω-Conotoxin GVIA (CTX) to block L-, P/Q-, and N-type calcium currents, respectively. ( B , C ) Time course of TAT-CBD3A6K mediated inhibition (“run-down”) of T-type ( B ) and R-type ( C ) calcium currents. Time course of inhibition is shown as averaged normalized current density (pA pF -1 ) before peptide addition and at intervals of 30 s for 5 min. Averaged values are shown with standard error for 4–6 control cells and 4 cells following addition of 10 μM TAT-CBD3A6K. The asterisk denotes statistical significance (p

    Techniques Used: Inhibition, Blocking Assay

    7) Product Images from "Alteration of the mu opioid receptor: Ca2+ channel signaling pathway in a subset of rat sensory neurons following chronic femoral artery occlusion"

    Article Title: Alteration of the mu opioid receptor: Ca2+ channel signaling pathway in a subset of rat sensory neurons following chronic femoral artery occlusion

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.00630.2014

    A and B : summary scatter plots of Ca 2+ current density (pA/pF) and membrane capacitance (pF) in acutely isolated DiI-labeled and EGFP-expressing DRG neurons from rats with freely perfused and 72 h-ligated femoral arteries. Current density was calculated from the peak Ca 2+ current amplitude normalized to membrane capacitance. The lines on the plots indicate the means (±SE), and the numbers in parentheses indicate the number of neurons tested. C : summary plot of the mean (±SE) Ca 2+ current inhibition produced by the N (ω-conotoxin GVIA; 10 μM)- and P/Q [ω-agatoxin IVA (AgaIVa]; 0.2 μM)-type Ca 2+ channel blockers in DRG neurons from freely perfused and ligated rats. The number of cells tested is indicated in parentheses.
    Figure Legend Snippet: A and B : summary scatter plots of Ca 2+ current density (pA/pF) and membrane capacitance (pF) in acutely isolated DiI-labeled and EGFP-expressing DRG neurons from rats with freely perfused and 72 h-ligated femoral arteries. Current density was calculated from the peak Ca 2+ current amplitude normalized to membrane capacitance. The lines on the plots indicate the means (±SE), and the numbers in parentheses indicate the number of neurons tested. C : summary plot of the mean (±SE) Ca 2+ current inhibition produced by the N (ω-conotoxin GVIA; 10 μM)- and P/Q [ω-agatoxin IVA (AgaIVa]; 0.2 μM)-type Ca 2+ channel blockers in DRG neurons from freely perfused and ligated rats. The number of cells tested is indicated in parentheses.

    Techniques Used: Isolation, Labeling, Expressing, Inhibition, Produced

    8) Product Images from "Midbrain dopaminergic neurons generate calcium and sodium currents and release dopamine in the striatum of pups"

    Article Title: Midbrain dopaminergic neurons generate calcium and sodium currents and release dopamine in the striatum of pups

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2012.00007

    Pharmacology of the spontaneous Ca 2+ activities of embryonic and early postnatal mDA neurons. Representative calcium fluorescence traces from mDA neurons and corresponding quantitative data at the indicated ages showing the effect of TTX (1 μ M)—nifedipin (10 μ M) (A) nifedipin (3 μ M) (B) ω-conotoxin GVIA (1 μ M) (C) and blockers of ionotropic glutamate and GABA receptors [APV (40 μ M)—CNQX (10 μ M)—Gabazine (5 μ M), D and E ].
    Figure Legend Snippet: Pharmacology of the spontaneous Ca 2+ activities of embryonic and early postnatal mDA neurons. Representative calcium fluorescence traces from mDA neurons and corresponding quantitative data at the indicated ages showing the effect of TTX (1 μ M)—nifedipin (10 μ M) (A) nifedipin (3 μ M) (B) ω-conotoxin GVIA (1 μ M) (C) and blockers of ionotropic glutamate and GABA receptors [APV (40 μ M)—CNQX (10 μ M)—Gabazine (5 μ M), D and E ].

    Techniques Used: Multiple Displacement Amplification, Fluorescence

    9) Product Images from "Stimulation of Synaptic Vesicle Exocytosis by the Mental Disease Gene DISC1 is Mediated by N-Type Voltage-Gated Calcium Channels"

    Article Title: Stimulation of Synaptic Vesicle Exocytosis by the Mental Disease Gene DISC1 is Mediated by N-Type Voltage-Gated Calcium Channels

    Journal: Frontiers in Synaptic Neuroscience

    doi: 10.3389/fnsyn.2016.00015

    DISC1 regulates Cav2.2-dependent SV exocytosis. (A) Average vGpH traces in scr (101 boutons, 6 fields, 2 exps) and DISC1-E (87 boutons, 5 fields, 2 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.2 blocker ω-Conotoxin GVIA (125 nM). (B) Boxplot of SV exocytic rates before and after ω-Conotoxin GVIA application. Exocytic rates were measured by linear fitting of the first six time points of the vGpH response. (C) Average vGpH traces in scr (1294 boutons, 9 fields, 3 exps) and DISC1-E (1282 boutons, 9 fields, 3 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.1 blocker ω-Agatoxin TK (125 nM). (D) Boxplot of SV exocytic rates before and after ω-Agatoxin TK application. (E) Table showing the percentage of inhibition of exocytosis rate by DISC1 knockdown before and after Cav2.2- or Cav2.1 blockade.
    Figure Legend Snippet: DISC1 regulates Cav2.2-dependent SV exocytosis. (A) Average vGpH traces in scr (101 boutons, 6 fields, 2 exps) and DISC1-E (87 boutons, 5 fields, 2 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.2 blocker ω-Conotoxin GVIA (125 nM). (B) Boxplot of SV exocytic rates before and after ω-Conotoxin GVIA application. Exocytic rates were measured by linear fitting of the first six time points of the vGpH response. (C) Average vGpH traces in scr (1294 boutons, 9 fields, 3 exps) and DISC1-E (1282 boutons, 9 fields, 3 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.1 blocker ω-Agatoxin TK (125 nM). (D) Boxplot of SV exocytic rates before and after ω-Agatoxin TK application. (E) Table showing the percentage of inhibition of exocytosis rate by DISC1 knockdown before and after Cav2.2- or Cav2.1 blockade.

    Techniques Used: shRNA, Expressing, Inhibition

    10) Product Images from "Heart failure-induced changes of voltage-gated Ca2+ channels and cell excitability in rat cardiac postganglionic neurons"

    Article Title: Heart failure-induced changes of voltage-gated Ca2+ channels and cell excitability in rat cardiac postganglionic neurons

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00223.2013

    Current threshold-inducing action potential ( A and B ) and frequency of action potentials ( C and D ) are shown before and after treatment of ω-conotoxin GVIA (1 μM) in the cardiac postganglionic vagal neurons from sham and CHF rats. Data
    Figure Legend Snippet: Current threshold-inducing action potential ( A and B ) and frequency of action potentials ( C and D ) are shown before and after treatment of ω-conotoxin GVIA (1 μM) in the cardiac postganglionic vagal neurons from sham and CHF rats. Data

    Techniques Used:

    11) Product Images from "Regulation of glutamatergic and GABAergic neurotransmission in the chick nucleus laminaris: role of N-type calcium channels"

    Article Title: Regulation of glutamatergic and GABAergic neurotransmission in the chick nucleus laminaris: role of N-type calcium channels

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2009.09.013

    Glutamatergic transmission in the NL is predominantly triggered by Ca 2+ entry through N-type VGCCs. A : Both the ipsilateral (open circles) and the contralateral (filled circles) EPSCs recorded from the same NL neuron were largely and irreversibly blocked by the N-type blocker ω-Conotoxin-GVIA (ω-CTx-GVIA, 2.5 μM), with nearly identical time courses. Averaged traces under control (thin traces) and drug (thick traces) conditions are shown on the right. Stimulus artifacts are truncated for clarity. B-C : No inhibition of EPSCs was produced by the P/Q-type blocker ω-Agatoxin-IVA (ω-Aga-IVA, 0.1 μM) or the L-type blocker nimodipine (10 μM). D: A small and reversible inhibition of the EPSCs by the R-type blocker SNX-482 (50 nM) was observed. The different time courses of the EPSCs observed in the sampled neurons may be due to different locations of the neurons along the tonotopic frequency axis. E : Summary of the effects of different VGCC blockers on EPSCs of NL neurons. ω-CTx-GVIA inhibited the ipsilateral and the contralateral EPSCs by 88% and 86%, respectively. Blockers for P/Q- and L-type VGCCs had no effects on EPSCs. SNX-482 produced a small inhibition with a large variation among cells. No differences in the percent inhibition by either drug were detected between the ipsilateral and the contralateral EPSCs. Error bars represent standard deviations. n.s.: non-significant (paired t-test, p > 0.05; the number of cells is indicated in parentheses).
    Figure Legend Snippet: Glutamatergic transmission in the NL is predominantly triggered by Ca 2+ entry through N-type VGCCs. A : Both the ipsilateral (open circles) and the contralateral (filled circles) EPSCs recorded from the same NL neuron were largely and irreversibly blocked by the N-type blocker ω-Conotoxin-GVIA (ω-CTx-GVIA, 2.5 μM), with nearly identical time courses. Averaged traces under control (thin traces) and drug (thick traces) conditions are shown on the right. Stimulus artifacts are truncated for clarity. B-C : No inhibition of EPSCs was produced by the P/Q-type blocker ω-Agatoxin-IVA (ω-Aga-IVA, 0.1 μM) or the L-type blocker nimodipine (10 μM). D: A small and reversible inhibition of the EPSCs by the R-type blocker SNX-482 (50 nM) was observed. The different time courses of the EPSCs observed in the sampled neurons may be due to different locations of the neurons along the tonotopic frequency axis. E : Summary of the effects of different VGCC blockers on EPSCs of NL neurons. ω-CTx-GVIA inhibited the ipsilateral and the contralateral EPSCs by 88% and 86%, respectively. Blockers for P/Q- and L-type VGCCs had no effects on EPSCs. SNX-482 produced a small inhibition with a large variation among cells. No differences in the percent inhibition by either drug were detected between the ipsilateral and the contralateral EPSCs. Error bars represent standard deviations. n.s.: non-significant (paired t-test, p > 0.05; the number of cells is indicated in parentheses).

    Techniques Used: Transmission Assay, Inhibition, Produced

    12) Product Images from "Presynaptic α4β2 Nicotinic Acetylcholine Receptors Increase Glutamate Release and Serotonin Neuron Excitability in the Dorsal Raphe Nucleus"

    Article Title: Presynaptic α4β2 Nicotinic Acetylcholine Receptors Increase Glutamate Release and Serotonin Neuron Excitability in the Dorsal Raphe Nucleus

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0941-12.2012

    Nicotinic effects depend on VGCCs and intracellular CICR. A , Time–frequency histogram shows the effect of nicotine on the sEPSC frequency in the presence of CdCl 2 (gray bar). B , Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of a mixture containing the Ca 2+ channel blockers ω-agatoxin-TK, ω-conotoxin-GVIA, and nitrendipine (gray bar). C , Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of the SERCA blocker thapsigargin (gray bar). D , Bar graph shows the effect of nicotine on the sEPSC frequency in slices pretreated with CdCl 2 , Ca 2+ channel blockers, thapsigargin, CPA, or ryanodine (** p
    Figure Legend Snippet: Nicotinic effects depend on VGCCs and intracellular CICR. A , Time–frequency histogram shows the effect of nicotine on the sEPSC frequency in the presence of CdCl 2 (gray bar). B , Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of a mixture containing the Ca 2+ channel blockers ω-agatoxin-TK, ω-conotoxin-GVIA, and nitrendipine (gray bar). C , Time–frequency histogram shows the lack of effect of nicotine on the sEPSC frequency in the presence of the SERCA blocker thapsigargin (gray bar). D , Bar graph shows the effect of nicotine on the sEPSC frequency in slices pretreated with CdCl 2 , Ca 2+ channel blockers, thapsigargin, CPA, or ryanodine (** p

    Techniques Used:

    13) Product Images from "Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density"

    Article Title: Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    Journal: Nature Communications

    doi: 10.1038/ncomms4628

    FMRP knockdown enhances synaptic vesicle exocytosis in presynaptic terminals of DRG neurons via Ca V 2.2 channels. ( a ) Presynaptic terminals of DRG neurons expressing vGpH (vGpH). Images show vGpH fluorescence (green) colocalized with endogenous synapsin 1 and 2 (left panel, red) and apposed to endogenous PSD-95 (right panel, red). Synapses are indicated by the white arrows. Scale bars, 5 μm. ( b ) Fluorescence changes (ΔF) of vGpH in presynaptic terminals of DRG neurons transfected with Ctrl shRNA (top panels) or FMRP shRNA (bottom panels) in response to electrical stimulation. Left panels: at rest; middle panels: after 40 action potentials (AP) at 10 Hz; right panels: after a brief application of NH 4 Cl. Responsive terminals are indicated by the black arrows. Pseudocolor scale is shown to the right (min, max:minimum and maximum fluorescence intensity). Scale bar, 10 μm. ( c , d ) vGpH response to 40 AP at 10 Hz from presynaptic terminals of DRG neurons transfected with Ctrl shRNA ( c ) or FMRP shRNA ( d ) before and after treatment with toxins (10 min with ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (300 nM)). Fluorescence intensities were normalized to the peak of a brief application of NH 4 Cl. ( e ) Normalized vGpH responses to 40 AP at 10 Hz from presynaptic terminals of DRG neurons transfected with Ctrl shRNA (black-filled bar, 100±10.6%, n =38) or FMRP shRNA (red open bar, 137.0±12.6%, n =25, P =0.027). ω-conotoxin GVIA (ConoTx, 1 μM) reduces Ctrl shRNA and FMRP shRNA responses to a similar level (44.7±4.9%, n =15 and 41.6±3.3%, n =24, respectively). ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (AgaTx, 300 nM) application reduces further the responses: Ctrl shRNA=17.3±3.2%, n =38, and FMRP shRNA=18.1±5.0%, n =27. A dot plot graph for the data is presented in Supplementary Fig. 9 . Means±s.e.m., * P
    Figure Legend Snippet: FMRP knockdown enhances synaptic vesicle exocytosis in presynaptic terminals of DRG neurons via Ca V 2.2 channels. ( a ) Presynaptic terminals of DRG neurons expressing vGpH (vGpH). Images show vGpH fluorescence (green) colocalized with endogenous synapsin 1 and 2 (left panel, red) and apposed to endogenous PSD-95 (right panel, red). Synapses are indicated by the white arrows. Scale bars, 5 μm. ( b ) Fluorescence changes (ΔF) of vGpH in presynaptic terminals of DRG neurons transfected with Ctrl shRNA (top panels) or FMRP shRNA (bottom panels) in response to electrical stimulation. Left panels: at rest; middle panels: after 40 action potentials (AP) at 10 Hz; right panels: after a brief application of NH 4 Cl. Responsive terminals are indicated by the black arrows. Pseudocolor scale is shown to the right (min, max:minimum and maximum fluorescence intensity). Scale bar, 10 μm. ( c , d ) vGpH response to 40 AP at 10 Hz from presynaptic terminals of DRG neurons transfected with Ctrl shRNA ( c ) or FMRP shRNA ( d ) before and after treatment with toxins (10 min with ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (300 nM)). Fluorescence intensities were normalized to the peak of a brief application of NH 4 Cl. ( e ) Normalized vGpH responses to 40 AP at 10 Hz from presynaptic terminals of DRG neurons transfected with Ctrl shRNA (black-filled bar, 100±10.6%, n =38) or FMRP shRNA (red open bar, 137.0±12.6%, n =25, P =0.027). ω-conotoxin GVIA (ConoTx, 1 μM) reduces Ctrl shRNA and FMRP shRNA responses to a similar level (44.7±4.9%, n =15 and 41.6±3.3%, n =24, respectively). ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (AgaTx, 300 nM) application reduces further the responses: Ctrl shRNA=17.3±3.2%, n =38, and FMRP shRNA=18.1±5.0%, n =27. A dot plot graph for the data is presented in Supplementary Fig. 9 . Means±s.e.m., * P

    Techniques Used: Expressing, Fluorescence, Transfection, shRNA

    14) Product Images from "Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex"

    Article Title: Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex

    Journal: Biology Open

    doi: 10.1242/bio.058840

    T-type Ca2+ channel blockers occlude the increment in mIPSC frequency induced by blocking HCN channels. (A) Representative traces of mIPSCs recorded in pyramidal cell. Holding potential: −70 mV. (B) The cumulative fraction distribution of inter-event intervals (left) and amplitude (right) of mIPSCs before (Control), during (ZD7288, 30 µM), and after co-application of ZD7288 with T-type Ca 2+ channel selective blocker mibefradil (Mib; 10 µM) ( ZD+Mib ). (C,D) Bar graph demonstrating the effects of co-application of ZD7288 and Ca 2+ channel blockers for T-type (pimozide, 1 µM; mibefradil, 10 µM), P/Q-type (ω-agatoxin IVA, 500 nM), N-type (ω-Conotoxin GVIA, 500 nM), and L-type (nifedipine, 2 mM) Ca 2+ channels on the frequency (C) and amplitude (D) of mIPSCs. Open circles for individual cells and bar for grouped data. ** P
    Figure Legend Snippet: T-type Ca2+ channel blockers occlude the increment in mIPSC frequency induced by blocking HCN channels. (A) Representative traces of mIPSCs recorded in pyramidal cell. Holding potential: −70 mV. (B) The cumulative fraction distribution of inter-event intervals (left) and amplitude (right) of mIPSCs before (Control), during (ZD7288, 30 µM), and after co-application of ZD7288 with T-type Ca 2+ channel selective blocker mibefradil (Mib; 10 µM) ( ZD+Mib ). (C,D) Bar graph demonstrating the effects of co-application of ZD7288 and Ca 2+ channel blockers for T-type (pimozide, 1 µM; mibefradil, 10 µM), P/Q-type (ω-agatoxin IVA, 500 nM), N-type (ω-Conotoxin GVIA, 500 nM), and L-type (nifedipine, 2 mM) Ca 2+ channels on the frequency (C) and amplitude (D) of mIPSCs. Open circles for individual cells and bar for grouped data. ** P

    Techniques Used: Blocking Assay

    15) Product Images from "Synchronous and asynchronous modes of synaptic transmission utilize different calcium sources"

    Article Title: Synchronous and asynchronous modes of synaptic transmission utilize different calcium sources

    Journal: eLife

    doi: 10.7554/eLife.01206

    The delayed asynchronous release and Ca 2+ rise in ω-conotoxin GVIA-treated fish is not due to slow local calcium accumulation at the distal boutons. ( A and B ) Two paired recordings for killswitch experiments are shown with expanded insets (boxed region). ( A ) In this example, the motor neuron stimulation (top trace) was terminated prior to the sudden onset of asynchronous release (bottom trace). ( B ) In this recording, the stimulus was terminated at the onset of asynchronous transmission, showing the persistence of the release. Both the experiments in ( A ) and ( B ) were performed with 5 mM EGTA in the intracellular solution. ( C ) An example calcium imaging experiment showing that the fluorescence signal peaked after the termination of the 100 Hz stimulation at 4 s. Left: an overlay of single imaging plane with the dye fill (red) and the Fluo-4 signal (green). The individual color coded ROIs were used to generate the associated ΔG/R vs time plot (right panel). The black bar shows the timing of stimulation, with a dashed line indicating the end the stimulation. This experiment was performed with 0.5 mM EGTA in the intracellular solution. DOI: http://dx.doi.org/10.7554/eLife.01206.012
    Figure Legend Snippet: The delayed asynchronous release and Ca 2+ rise in ω-conotoxin GVIA-treated fish is not due to slow local calcium accumulation at the distal boutons. ( A and B ) Two paired recordings for killswitch experiments are shown with expanded insets (boxed region). ( A ) In this example, the motor neuron stimulation (top trace) was terminated prior to the sudden onset of asynchronous release (bottom trace). ( B ) In this recording, the stimulus was terminated at the onset of asynchronous transmission, showing the persistence of the release. Both the experiments in ( A ) and ( B ) were performed with 5 mM EGTA in the intracellular solution. ( C ) An example calcium imaging experiment showing that the fluorescence signal peaked after the termination of the 100 Hz stimulation at 4 s. Left: an overlay of single imaging plane with the dye fill (red) and the Fluo-4 signal (green). The individual color coded ROIs were used to generate the associated ΔG/R vs time plot (right panel). The black bar shows the timing of stimulation, with a dashed line indicating the end the stimulation. This experiment was performed with 0.5 mM EGTA in the intracellular solution. DOI: http://dx.doi.org/10.7554/eLife.01206.012

    Techniques Used: Fluorescence In Situ Hybridization, Transmission Assay, Imaging, Fluorescence

    Simultaneous paired recordings and calcium imaging in ω-conotoxin GVIA-treated fish. The examples shown compare the effects of 0.5 mM ( A – D ) vs 5 mM ( E – H ) intracellular EGTA. ( A ) and ( E ) Image of the CaP dye filled with Alexa Fluor 647 (green) and target muscle filled with Alexa Fluor 555 (red). Multiple synaptic boutons contacting the target muscle cell are visible as yellow varicosities (scale bar = 10 μm). An enlarged view of the fill ( A1 and E1 ) and peak Fluo-5F calcium response ( A2 and E2 ) are shown for the color coded boutons. ( B ) and ( F ) The associated ΔG/R plots for each of the boutons in A and E as a function of time during 20 s, 100 Hz stimulus (indicated by black bar), which began at time 0. ( C ) and ( G ) The associated patch clamp recording showing the motor neuron action potential (top) and postsynaptic EPCs (bottom) during the 20 s, 100 Hz stimulation. The scale bars correspond to 40 mV, 1 nA, and 2 s. ( D ) and ( H ) An overlay plot showing the coincidence between the onset of the mean ΔG/R fluorescence for all ROIs (blue with gray SD) and the onset of asynchronous release (red). Release was shown as integrated synaptic charge entry for each consecutive half-second of stimulation. DOI: http://dx.doi.org/10.7554/eLife.01206.013
    Figure Legend Snippet: Simultaneous paired recordings and calcium imaging in ω-conotoxin GVIA-treated fish. The examples shown compare the effects of 0.5 mM ( A – D ) vs 5 mM ( E – H ) intracellular EGTA. ( A ) and ( E ) Image of the CaP dye filled with Alexa Fluor 647 (green) and target muscle filled with Alexa Fluor 555 (red). Multiple synaptic boutons contacting the target muscle cell are visible as yellow varicosities (scale bar = 10 μm). An enlarged view of the fill ( A1 and E1 ) and peak Fluo-5F calcium response ( A2 and E2 ) are shown for the color coded boutons. ( B ) and ( F ) The associated ΔG/R plots for each of the boutons in A and E as a function of time during 20 s, 100 Hz stimulus (indicated by black bar), which began at time 0. ( C ) and ( G ) The associated patch clamp recording showing the motor neuron action potential (top) and postsynaptic EPCs (bottom) during the 20 s, 100 Hz stimulation. The scale bars correspond to 40 mV, 1 nA, and 2 s. ( D ) and ( H ) An overlay plot showing the coincidence between the onset of the mean ΔG/R fluorescence for all ROIs (blue with gray SD) and the onset of asynchronous release (red). Release was shown as integrated synaptic charge entry for each consecutive half-second of stimulation. DOI: http://dx.doi.org/10.7554/eLife.01206.013

    Techniques Used: Imaging, Fluorescence In Situ Hybridization, Patch Clamp, Fluorescence

    Overall comparisons of release time course between control, ω-conotoxin GVIA-treated and tb204a mutant fish. ( A ) The time courses for paired recordings from wild-type (black), tb204a mutant (green) and ω-conotoxin GVIA-treated (red) fish. The total release was expressed as the integrated charge for each consecutive second of the recording and normalized for comparison. ( B ) Scatter plot for recordings from wild type (black; 3.4 ± 0.5 s, n = 15), tb204a mutant (green; 5.2 ± 1.0 s, n = 9) and ω-conotoxin GVIA-treated (red; 10.5 ± 2.4 s, n = 13) fish comparing the time to peak release. Bars indicate the mean value and SD for each group. Asterisks indicate p
    Figure Legend Snippet: Overall comparisons of release time course between control, ω-conotoxin GVIA-treated and tb204a mutant fish. ( A ) The time courses for paired recordings from wild-type (black), tb204a mutant (green) and ω-conotoxin GVIA-treated (red) fish. The total release was expressed as the integrated charge for each consecutive second of the recording and normalized for comparison. ( B ) Scatter plot for recordings from wild type (black; 3.4 ± 0.5 s, n = 15), tb204a mutant (green; 5.2 ± 1.0 s, n = 9) and ω-conotoxin GVIA-treated (red; 10.5 ± 2.4 s, n = 13) fish comparing the time to peak release. Bars indicate the mean value and SD for each group. Asterisks indicate p

    Techniques Used: Mutagenesis, Fluorescence In Situ Hybridization

    Distance-dependent delay in Ca 2+ rise in ω-conotoxin GVIA-treated CaP boutons. ( A ) Sample images of Fluo-5F fluorescence taken at 0.5 s intervals during 100 Hz stimulation. 5 ROIs are shown at different distances from the reference point at the ventral edge of the notochord. ( B ) The time course of fluorescence change, expressed as ΔG/R, for each of the ROIs shown in A . The black bar indicates the duration of stimulation. ( C ) Imaris Filament Tracer 3D reconstruction of the same motor neuron based on z-stacks of the Alexa Fluor 647 fill, with the ROIs in A and B overlaid. An arrowhead indicates the reference point for distance measurements. Scale bars in A and C correspond to 10 μm. ( D ) The time required for each ROI to reach 20% of peak as a function of the distance from the reference point. The distance measurements for each ROI were determined on the basis of Imaris 3D reconstruction. Colored symbols correspond to the individually colored ROIs shown in the A – C . The data points from the boutons, excluding the first distance measurement, were fit by a line with a slope corresponding to 57 µm/s. ( E ) Scatter plot of distance-dependent Ca 2+ rise for 61 ROIs in ω-conotoxin GVIA-treated neurons (n = 4 fish, colored markers) and 47 ROIs in control (n = 3 fish, gray markers). Each neuron was reconstructed using Imaris filament software to obtain the physical distances. Example cell in A – D is shown with green markers. Measurement was obtained with Fluo-5F and 0.5 mM EGTA in the intracellular solution. DOI: http://dx.doi.org/10.7554/eLife.01206.011
    Figure Legend Snippet: Distance-dependent delay in Ca 2+ rise in ω-conotoxin GVIA-treated CaP boutons. ( A ) Sample images of Fluo-5F fluorescence taken at 0.5 s intervals during 100 Hz stimulation. 5 ROIs are shown at different distances from the reference point at the ventral edge of the notochord. ( B ) The time course of fluorescence change, expressed as ΔG/R, for each of the ROIs shown in A . The black bar indicates the duration of stimulation. ( C ) Imaris Filament Tracer 3D reconstruction of the same motor neuron based on z-stacks of the Alexa Fluor 647 fill, with the ROIs in A and B overlaid. An arrowhead indicates the reference point for distance measurements. Scale bars in A and C correspond to 10 μm. ( D ) The time required for each ROI to reach 20% of peak as a function of the distance from the reference point. The distance measurements for each ROI were determined on the basis of Imaris 3D reconstruction. Colored symbols correspond to the individually colored ROIs shown in the A – C . The data points from the boutons, excluding the first distance measurement, were fit by a line with a slope corresponding to 57 µm/s. ( E ) Scatter plot of distance-dependent Ca 2+ rise for 61 ROIs in ω-conotoxin GVIA-treated neurons (n = 4 fish, colored markers) and 47 ROIs in control (n = 3 fish, gray markers). Each neuron was reconstructed using Imaris filament software to obtain the physical distances. Example cell in A – D is shown with green markers. Measurement was obtained with Fluo-5F and 0.5 mM EGTA in the intracellular solution. DOI: http://dx.doi.org/10.7554/eLife.01206.011

    Techniques Used: Fluorescence, Fluorescence In Situ Hybridization, Software

    Calcium signal onset is delayed at boutons in ω-conotoxin GVIA-treated fish. ( A and C ) Sample images of 100 Hz stimulus evoked Fluo-5F fluorescence increases taken at 1 s intervals for 4 ROIs for control ( A ) and ω-conotoxin GVIA-treated ( C ) fish. The scale bar corresponds to 20 μm. ( B and D ) The stimulus-driven fluorescence increases associated with each color-coded ROI in control ( B ) and ω-conotoxin GVIA-treated ( D ) fish. The fluorescence was baseline subtracted and the increase was expressed as ΔG/R. Black bars in ( B ) and ( D ) indicate the timing of 100 Hz stimulation. Experiments were performed with 0.5 mM EGTA in the intracellular solution. The entire videos for A and C are available as Video 1 and Video 2 respectively. For each video the timing of stimulation is indicated by the dot. DOI: http://dx.doi.org/10.7554/eLife.01206.007
    Figure Legend Snippet: Calcium signal onset is delayed at boutons in ω-conotoxin GVIA-treated fish. ( A and C ) Sample images of 100 Hz stimulus evoked Fluo-5F fluorescence increases taken at 1 s intervals for 4 ROIs for control ( A ) and ω-conotoxin GVIA-treated ( C ) fish. The scale bar corresponds to 20 μm. ( B and D ) The stimulus-driven fluorescence increases associated with each color-coded ROI in control ( B ) and ω-conotoxin GVIA-treated ( D ) fish. The fluorescence was baseline subtracted and the increase was expressed as ΔG/R. Black bars in ( B ) and ( D ) indicate the timing of 100 Hz stimulation. Experiments were performed with 0.5 mM EGTA in the intracellular solution. The entire videos for A and C are available as Video 1 and Video 2 respectively. For each video the timing of stimulation is indicated by the dot. DOI: http://dx.doi.org/10.7554/eLife.01206.007

    Techniques Used: Fluorescence In Situ Hybridization, Fluorescence

    Delayed release in ω-conotoxin GVIA-treated fish is also observed by means of the exocytotic indicator synaptopHluorin. ( A ) Images taken from a single focal plane of the CaP motor neuron terminals showing the motor neuron fill with Alexa Fluor 647 (gray), postsynaptic labeling with α-btx (red), peak stimulus-induced synaptopHluorin fluorescence (green) and α-btx/synaptopHluorin overlay. These images are shown for a single control (left) and ω-conotoxin GVIA-treated (right) fish. The scale bar corresponds to 10 μm. ( B ) Stimulus-driven fluorescence change, expressed as ΔF/F 0 for the three representative boutons shown for control (gray) and ω-conotoxin GVIA treated (colored) synaptopHluorin motor neurons. The synaptopHluorin signal was measured at each ROI during the time course of 100 Hz stimulation (indicated by the black bar in B ). ( C ) The histogram showing time required to reach 50% maximal fluorescence increase for boutons of control (gray, 127 boutons from 8 fish) and ω-conotoxin GVIA-treated fish (red, 55 boutons from 4 fish). Experiments were performed with 5 mM EGTA in the intracellular solution. DOI: http://dx.doi.org/10.7554/eLife.01206.005
    Figure Legend Snippet: Delayed release in ω-conotoxin GVIA-treated fish is also observed by means of the exocytotic indicator synaptopHluorin. ( A ) Images taken from a single focal plane of the CaP motor neuron terminals showing the motor neuron fill with Alexa Fluor 647 (gray), postsynaptic labeling with α-btx (red), peak stimulus-induced synaptopHluorin fluorescence (green) and α-btx/synaptopHluorin overlay. These images are shown for a single control (left) and ω-conotoxin GVIA-treated (right) fish. The scale bar corresponds to 10 μm. ( B ) Stimulus-driven fluorescence change, expressed as ΔF/F 0 for the three representative boutons shown for control (gray) and ω-conotoxin GVIA treated (colored) synaptopHluorin motor neurons. The synaptopHluorin signal was measured at each ROI during the time course of 100 Hz stimulation (indicated by the black bar in B ). ( C ) The histogram showing time required to reach 50% maximal fluorescence increase for boutons of control (gray, 127 boutons from 8 fish) and ω-conotoxin GVIA-treated fish (red, 55 boutons from 4 fish). Experiments were performed with 5 mM EGTA in the intracellular solution. DOI: http://dx.doi.org/10.7554/eLife.01206.005

    Techniques Used: Fluorescence In Situ Hybridization, Labeling, Fluorescence

    Asynchronous synaptic transmission remains intact in the P/Q calcium channel mutant tb204a and following treatment of wild-type fish with ω-conotoxin GVIA. ( A – C ) A representative paired recording from untreated wild-type fish. ( A ) Voltage clamp traces of EPCs in response to 20 s, 100 Hz stimulation of the motor neuron. Expanded views with both action potentials and associated postsynaptic EPCs showing early synchronous ( A1 ) and mixed synchronous and asynchronous release at the peak of release ( A2 ). ( B ) Quantitation of the time-dependence of synchronous (blue), asynchronous (red) and total (black) synaptic charge integrals determined using the methods described in Wen et al. (2010) . ( C ) Comparison of the stimulus evoked asynchronous event amplitudes recorded during the last 10 s of stimulation (black fill) and spontaneous synaptic current amplitudes (gray fill, 402 events from 17 cells). The distributions are fit by a Gaussian function with means corresponding to 637 pA and 556 pA. ( D – F ) A representative paired recording from fish treated with 1 µM ω-conotoxin GVIA. ( D ) Traces of EPCs with expanded views showing near elimination of synchronous release ( D1 ) and intact asynchronous release ( D2 ) in ω-conotoxin GVIA-treated fish. ( E ) Time course of release for the recording shown in D . ( F ) Comparison of its asynchronous event amplitude (black fill) and the same spontaneous synaptic current amplitudes used for 1C and 1I (gray fill). Events during the last 5 s of stimulation were included in the analysis. The mean value from a Gaussian fit for ω-conotoxin GVIA-treated fish was 620 pA. ( G – I ) A representative paired recording from the mutant line tb204a . ( G ) Traces of action potentials and EPCs from a homozygous tb204a mutant showing greatly reduced synchronous release ( G1 ) and intact late asynchronous release ( G2 ). ( H ) The time course of release for the recording shown in G . ( I ) Comparison of its asynchronous event amplitudes (black fill) and the spontaneous synaptic current amplitudes (gray fill). Events during the last 5 s of stimulation were included in the analysis. The mean value from a Gaussian fit for the mutant was 601 pA. Red circles in ( A ), ( D ), and ( G ) mark the peaks of synchronous events. All experiments were performed with 5 mM EGTA in the intracellular solution. DOI: http://dx.doi.org/10.7554/eLife.01206.003
    Figure Legend Snippet: Asynchronous synaptic transmission remains intact in the P/Q calcium channel mutant tb204a and following treatment of wild-type fish with ω-conotoxin GVIA. ( A – C ) A representative paired recording from untreated wild-type fish. ( A ) Voltage clamp traces of EPCs in response to 20 s, 100 Hz stimulation of the motor neuron. Expanded views with both action potentials and associated postsynaptic EPCs showing early synchronous ( A1 ) and mixed synchronous and asynchronous release at the peak of release ( A2 ). ( B ) Quantitation of the time-dependence of synchronous (blue), asynchronous (red) and total (black) synaptic charge integrals determined using the methods described in Wen et al. (2010) . ( C ) Comparison of the stimulus evoked asynchronous event amplitudes recorded during the last 10 s of stimulation (black fill) and spontaneous synaptic current amplitudes (gray fill, 402 events from 17 cells). The distributions are fit by a Gaussian function with means corresponding to 637 pA and 556 pA. ( D – F ) A representative paired recording from fish treated with 1 µM ω-conotoxin GVIA. ( D ) Traces of EPCs with expanded views showing near elimination of synchronous release ( D1 ) and intact asynchronous release ( D2 ) in ω-conotoxin GVIA-treated fish. ( E ) Time course of release for the recording shown in D . ( F ) Comparison of its asynchronous event amplitude (black fill) and the same spontaneous synaptic current amplitudes used for 1C and 1I (gray fill). Events during the last 5 s of stimulation were included in the analysis. The mean value from a Gaussian fit for ω-conotoxin GVIA-treated fish was 620 pA. ( G – I ) A representative paired recording from the mutant line tb204a . ( G ) Traces of action potentials and EPCs from a homozygous tb204a mutant showing greatly reduced synchronous release ( G1 ) and intact late asynchronous release ( G2 ). ( H ) The time course of release for the recording shown in G . ( I ) Comparison of its asynchronous event amplitudes (black fill) and the spontaneous synaptic current amplitudes (gray fill). Events during the last 5 s of stimulation were included in the analysis. The mean value from a Gaussian fit for the mutant was 601 pA. Red circles in ( A ), ( D ), and ( G ) mark the peaks of synchronous events. All experiments were performed with 5 mM EGTA in the intracellular solution. DOI: http://dx.doi.org/10.7554/eLife.01206.003

    Techniques Used: Transmission Assay, Mutagenesis, Fluorescence In Situ Hybridization, Quantitation Assay

    Increasing the intracellular concentration of the calcium buffer EGTA further delays both the calcium signal in boutons and the onset of asynchronous release. ( A ) Sample traces comparing the time-dependent Fluo-5F fluorescence increases during stimulation for selected boutons of CaP motor neurons dialyzed with the indicated EGTA concentrations. ( B ) Cumulative data comparing the effects of 0.5 mM (blue; 61 boutons from 4 fish) and 5 mM (red; 44 boutons from 3 fish) EGTA on the time required to reach 20% maximal fluorescence change in boutons of ω-conotoxin GVIA-treated fish. The distances from the reference point were determined using Imaris filament reconstruction and binned (30 μm bin size). Control neuron dialyzed with 0.5 mM EGTA (black; 47 boutons from 3 fish) is shown for comparison. ( C ) Sample patch clamp recordings of muscle EPCs performed at three different EGTA concentrations in ω-conotoxin GVIA-treated fish. The action potentials are not shown but the stimulus lasted 35 s for the bottom trace and 20 s for the other traces. ( D ) The associated integrated synaptic currents as a function of time with 100 Hz stimulation beginning at time 0. Only a single example is shown for 25 mM EGTA because this concentration blocked most transmission in other trials. ( E ) Comparisons of time to reach peak release for all recordings made using 0.5 mM (4.8 ± 0.4 s, n = 8) and 5 mM (10.5 ± 2.4 s, n = 13) EGTA in ω-conotoxin GVIA-treated fish. Data set for 5 mM EGTA was duplicated from Figure 2 for comparison. DOI: http://dx.doi.org/10.7554/eLife.01206.014
    Figure Legend Snippet: Increasing the intracellular concentration of the calcium buffer EGTA further delays both the calcium signal in boutons and the onset of asynchronous release. ( A ) Sample traces comparing the time-dependent Fluo-5F fluorescence increases during stimulation for selected boutons of CaP motor neurons dialyzed with the indicated EGTA concentrations. ( B ) Cumulative data comparing the effects of 0.5 mM (blue; 61 boutons from 4 fish) and 5 mM (red; 44 boutons from 3 fish) EGTA on the time required to reach 20% maximal fluorescence change in boutons of ω-conotoxin GVIA-treated fish. The distances from the reference point were determined using Imaris filament reconstruction and binned (30 μm bin size). Control neuron dialyzed with 0.5 mM EGTA (black; 47 boutons from 3 fish) is shown for comparison. ( C ) Sample patch clamp recordings of muscle EPCs performed at three different EGTA concentrations in ω-conotoxin GVIA-treated fish. The action potentials are not shown but the stimulus lasted 35 s for the bottom trace and 20 s for the other traces. ( D ) The associated integrated synaptic currents as a function of time with 100 Hz stimulation beginning at time 0. Only a single example is shown for 25 mM EGTA because this concentration blocked most transmission in other trials. ( E ) Comparisons of time to reach peak release for all recordings made using 0.5 mM (4.8 ± 0.4 s, n = 8) and 5 mM (10.5 ± 2.4 s, n = 13) EGTA in ω-conotoxin GVIA-treated fish. Data set for 5 mM EGTA was duplicated from Figure 2 for comparison. DOI: http://dx.doi.org/10.7554/eLife.01206.014

    Techniques Used: Concentration Assay, Fluorescence, Fluorescence In Situ Hybridization, Patch Clamp, Transmission Assay

    The delayed rise of the calcium signal is similar for two different affinity calcium indicators Fluo-4 and Fluo-5F. Comparisons of the time to reach 20% peak stimulated fluorescence in ω-conotoxin GVIA-treated (shaded fill) and control CaP (no fill) motor neurons. In control fish, Fluo-4 onset was 0.18 ± 0.16 s (n = 64 boutons from 4 fish), and Fluo-5F onset was 0.47 ± 0.22 s (n = 91 boutons from 6 fish). For ω-conotoxin GVIA-treated fish, Fluo-4 onset was 1.98 ± 0.84 s (n = 69 boutons from 6 fish), and Fluo-5F onset was 2.14 ± 0.42 (n = 85 boutons from 7 fish). Experiments were performed with 0.5 mM EGTA in the intracellular solution. Asterisks indicate p
    Figure Legend Snippet: The delayed rise of the calcium signal is similar for two different affinity calcium indicators Fluo-4 and Fluo-5F. Comparisons of the time to reach 20% peak stimulated fluorescence in ω-conotoxin GVIA-treated (shaded fill) and control CaP (no fill) motor neurons. In control fish, Fluo-4 onset was 0.18 ± 0.16 s (n = 64 boutons from 4 fish), and Fluo-5F onset was 0.47 ± 0.22 s (n = 91 boutons from 6 fish). For ω-conotoxin GVIA-treated fish, Fluo-4 onset was 1.98 ± 0.84 s (n = 69 boutons from 6 fish), and Fluo-5F onset was 2.14 ± 0.42 (n = 85 boutons from 7 fish). Experiments were performed with 0.5 mM EGTA in the intracellular solution. Asterisks indicate p

    Techniques Used: Fluorescence, Fluorescence In Situ Hybridization

    16) Product Images from "Distinct mechanisms of CB1 and GABAB receptor presynaptic modulation of striatal indirect pathway projections to mouse Globus Pallidus"

    Article Title: Distinct mechanisms of CB1 and GABAB receptor presynaptic modulation of striatal indirect pathway projections to mouse Globus Pallidus

    Journal: bioRxiv

    doi: 10.1101/2022.07.21.500979

    P/Q-type are the predominant VGCC controlling presynaptic Ca 2+ and GABAergic transmission at the indirect pathway projections to the GPe. A: Timecourse of VGCC blocker application effects on PreCaTs and raw photometry traces. B: Summary of drug effects: baseline, last 2 minutes of drug, last 2 minutes of washout. Blockade of P/Q -type VGGCs (Agatoxin, n = 6 slices) significantly decreased PreCaTs from baseline, whereas no significant effect on PreCaTs was observed by blocking N-type VGCCs (Conotoxin, n= 8 slices) or L-type VGCCs (Nifedipine, n = 5 slices). C: Timecourse of VGCC blocker application effects on oIPSCs and raw electrophysiological traces. D: Comparison of drug effects: baseline, last 2 minutes of drug, last 2 minutes of washout. Blockade of P/Q -type VGGCs (Agatoxin, n = 5 cells) significantly decreased PreCaTs from baseline, and smaller but significant effects were induced by blocking N-type VGCCs (Conotoxin, n = 5 cells) or L-type VGCCs (Nifedipine, n = 5 cells). * p
    Figure Legend Snippet: P/Q-type are the predominant VGCC controlling presynaptic Ca 2+ and GABAergic transmission at the indirect pathway projections to the GPe. A: Timecourse of VGCC blocker application effects on PreCaTs and raw photometry traces. B: Summary of drug effects: baseline, last 2 minutes of drug, last 2 minutes of washout. Blockade of P/Q -type VGGCs (Agatoxin, n = 6 slices) significantly decreased PreCaTs from baseline, whereas no significant effect on PreCaTs was observed by blocking N-type VGCCs (Conotoxin, n= 8 slices) or L-type VGCCs (Nifedipine, n = 5 slices). C: Timecourse of VGCC blocker application effects on oIPSCs and raw electrophysiological traces. D: Comparison of drug effects: baseline, last 2 minutes of drug, last 2 minutes of washout. Blockade of P/Q -type VGGCs (Agatoxin, n = 5 cells) significantly decreased PreCaTs from baseline, and smaller but significant effects were induced by blocking N-type VGCCs (Conotoxin, n = 5 cells) or L-type VGCCs (Nifedipine, n = 5 cells). * p

    Techniques Used: Transmission Assay, Blocking Assay

    17) Product Images from "Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A2A receptors in striatal projection neurons"

    Article Title: Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A2A receptors in striatal projection neurons

    Journal: Purinergic Signalling

    doi: 10.1007/s11302-013-9386-z

    Activation of adenosine A 1 -receptor mostly reduces current through Ca V 2.2 (N) channels. a Time course illustrates that 1 μM ω-conotoxin GVIA almost completely occluded the action of 100 nM adenosine. b Representative I-V
    Figure Legend Snippet: Activation of adenosine A 1 -receptor mostly reduces current through Ca V 2.2 (N) channels. a Time course illustrates that 1 μM ω-conotoxin GVIA almost completely occluded the action of 100 nM adenosine. b Representative I-V

    Techniques Used: Activation Assay

    18) Product Images from "Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons"

    Article Title: Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201511383

    GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P
    Figure Legend Snippet: GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P

    Techniques Used: Activity Assay, Mouse Assay, Activation Assay

    GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P
    Figure Legend Snippet: GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P

    Techniques Used: Activity Assay, Transfection, Inhibition

    19) Product Images from "Adenosine A1 Receptor-Mediated Attenuation of Reciprocal Dendro-Dendritic Inhibition in the Mouse Olfactory Bulb"

    Article Title: Adenosine A1 Receptor-Mediated Attenuation of Reciprocal Dendro-Dendritic Inhibition in the Mouse Olfactory Bulb

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00435

    Adenosine inhibits N-type and P/Q-type calcium currents. (A) Effect of the L-type calcium channel blocker nifedipine (Nif, 10 μM) on calcium currents. (B) IV relationship of nifedipine-sensitive calcium currents (green graph). (C) Effect of the N-type calcium channel blocker conotoxin GVIA (CTX, 100 nM) on calcium currents. (D) IV relationship of CTX-sensitive calcium currents (yellow graph). (E) P/Q-type calcium currents were isolated by blocking N-type and L-type calcium currents with CTX + Nif. (F) IV relationship of isolated P/Q-type calcium currents (red graph). (G) Adenosine strongly reduces calcium currents in the presence of Nif. (H) In the presence of CTX and (I) CTX plus Nif, adenosine only weakly reduces calcium currents. (J) Effect of adenosine on calcium currents in the absence of calcium channel blockers (Ctrl) and in the presence of Nif, CTX and CTX plus Nif. Incubation with CTX as well as CTX plus Nif significantly reduced the adenosine-mediated attenuation on calcium currents, while Nif alone had no effect on the adenosine-mediated attenuation. n.s., not significant. ∗∗∗ p
    Figure Legend Snippet: Adenosine inhibits N-type and P/Q-type calcium currents. (A) Effect of the L-type calcium channel blocker nifedipine (Nif, 10 μM) on calcium currents. (B) IV relationship of nifedipine-sensitive calcium currents (green graph). (C) Effect of the N-type calcium channel blocker conotoxin GVIA (CTX, 100 nM) on calcium currents. (D) IV relationship of CTX-sensitive calcium currents (yellow graph). (E) P/Q-type calcium currents were isolated by blocking N-type and L-type calcium currents with CTX + Nif. (F) IV relationship of isolated P/Q-type calcium currents (red graph). (G) Adenosine strongly reduces calcium currents in the presence of Nif. (H) In the presence of CTX and (I) CTX plus Nif, adenosine only weakly reduces calcium currents. (J) Effect of adenosine on calcium currents in the absence of calcium channel blockers (Ctrl) and in the presence of Nif, CTX and CTX plus Nif. Incubation with CTX as well as CTX plus Nif significantly reduced the adenosine-mediated attenuation on calcium currents, while Nif alone had no effect on the adenosine-mediated attenuation. n.s., not significant. ∗∗∗ p

    Techniques Used: Isolation, Blocking Assay, Incubation

    20) Product Images from "Isoflurane Inhibits Dopaminergic Synaptic Vesicle Exocytosis Coupled to CaV2.1 and CaV2.2 in Rat Midbrain Neurons"

    Article Title: Isoflurane Inhibits Dopaminergic Synaptic Vesicle Exocytosis Coupled to CaV2.1 and CaV2.2 in Rat Midbrain Neurons

    Journal: eNeuro

    doi: 10.1523/ENEURO.0278-18.2018

    Ca V 2.1 and Ca V 2.2 contribute to SV exocytosis in VTA neurons. A , Schematic of protocol using trains of 100 APs in the absence (black) or presence of ω-conotoxin GVIA (cono, 1 μM, purple bar) alone, ω-agatoxin IVA (aga, 400 nM, orange bar) alone, or both toxins together. B , Comparison of the effect of conotoxin and agatoxin on dopaminergic and non-dopaminergic neurons. The combination of conotoxin and agatoxin abolished exocytosis in both dopaminergic (DA) and non-dopaminergic (non-DA) neurons; **** p
    Figure Legend Snippet: Ca V 2.1 and Ca V 2.2 contribute to SV exocytosis in VTA neurons. A , Schematic of protocol using trains of 100 APs in the absence (black) or presence of ω-conotoxin GVIA (cono, 1 μM, purple bar) alone, ω-agatoxin IVA (aga, 400 nM, orange bar) alone, or both toxins together. B , Comparison of the effect of conotoxin and agatoxin on dopaminergic and non-dopaminergic neurons. The combination of conotoxin and agatoxin abolished exocytosis in both dopaminergic (DA) and non-dopaminergic (non-DA) neurons; **** p

    Techniques Used:

    21) Product Images from "Identification and Modulation of Voltage-Gated Ca2+ Currents in Zebrafish Rohon-Beard Neurons"

    Article Title: Identification and Modulation of Voltage-Gated Ca2+ Currents in Zebrafish Rohon-Beard Neurons

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.00625.2010

    Pharmacological dissection of high-voltage-activated (HVA)- I Ca in R-B neurons. A and B : time courses of I Ca amplitude during serial application of 10 μM nifedipine, 0.5 μM ω-agatoxin IVA, 3 μM ω-conotoxin GVIA,
    Figure Legend Snippet: Pharmacological dissection of high-voltage-activated (HVA)- I Ca in R-B neurons. A and B : time courses of I Ca amplitude during serial application of 10 μM nifedipine, 0.5 μM ω-agatoxin IVA, 3 μM ω-conotoxin GVIA,

    Techniques Used: Dissection

    22) Product Images from "Isoflurane Inhibits Dopaminergic Synaptic Vesicle Exocytosis Coupled to CaV2.1 and CaV2.2 in Rat Midbrain Neurons"

    Article Title: Isoflurane Inhibits Dopaminergic Synaptic Vesicle Exocytosis Coupled to CaV2.1 and CaV2.2 in Rat Midbrain Neurons

    Journal: eNeuro

    doi: 10.1523/ENEURO.0278-18.2018

    Ca V 2.1 and Ca V 2.2 contribute to SV exocytosis in VTA neurons. A , Schematic of protocol using trains of 100 APs in the absence (black) or presence of ω-conotoxin GVIA (cono, 1 μM, purple bar) alone, ω-agatoxin IVA (aga, 400 nM, orange bar) alone, or both toxins together. B , Comparison of the effect of conotoxin and agatoxin on dopaminergic and non-dopaminergic neurons. The combination of conotoxin and agatoxin abolished exocytosis in both dopaminergic (DA) and non-dopaminergic (non-DA) neurons; **** p
    Figure Legend Snippet: Ca V 2.1 and Ca V 2.2 contribute to SV exocytosis in VTA neurons. A , Schematic of protocol using trains of 100 APs in the absence (black) or presence of ω-conotoxin GVIA (cono, 1 μM, purple bar) alone, ω-agatoxin IVA (aga, 400 nM, orange bar) alone, or both toxins together. B , Comparison of the effect of conotoxin and agatoxin on dopaminergic and non-dopaminergic neurons. The combination of conotoxin and agatoxin abolished exocytosis in both dopaminergic (DA) and non-dopaminergic (non-DA) neurons; **** p

    Techniques Used:

    23) Product Images from "Differential regulation of nimodipine-sensitive and -insensitive Ca2+ influx by the Na+/Ca2+ exchanger and mitochondria in the rat suprachiasmatic nucleus neurons"

    Article Title: Differential regulation of nimodipine-sensitive and -insensitive Ca2+ influx by the Na+/Ca2+ exchanger and mitochondria in the rat suprachiasmatic nucleus neurons

    Journal: Journal of Biomedical Science

    doi: 10.1186/s12929-018-0447-z

    Effects of CaV2 channel blockers on nimodipine-insensitive Ca 2+ transients. a A representative experiment showing the effect of FCCP on nimodipine-insensitive Ca 2+ transients (an average of 11 cells). b Two representative experiments showing the effects of CaV2 channel blockers, applied additively in order of 0.2 μM SNX-482, 0.2 μM ω-agatoxin IVA, and 2 μM ω-conotoxin GVIA, on the nimodipine-insensitive Ca 2+ transients in the absence (dark grey trace) and presence (black trace) of FCCP. c Summary of experiments showing the amplitude of nimodipine-insensitive Ca 2+ transients reduced by each drug and the amplitude of Ca 2+ transients resistant to the blocker cocktail in the absence (dark grey bars) and presence (black bars) of FCCP. Note that FCCP induced a 3- to 4-hold increase (indicated by the number on top of black bars) in the Ca 2+ transient sensitive to ω-agatoxin IVA or ω-conotoxin GVIA or resistant to the cocktail of blockers. *** P
    Figure Legend Snippet: Effects of CaV2 channel blockers on nimodipine-insensitive Ca 2+ transients. a A representative experiment showing the effect of FCCP on nimodipine-insensitive Ca 2+ transients (an average of 11 cells). b Two representative experiments showing the effects of CaV2 channel blockers, applied additively in order of 0.2 μM SNX-482, 0.2 μM ω-agatoxin IVA, and 2 μM ω-conotoxin GVIA, on the nimodipine-insensitive Ca 2+ transients in the absence (dark grey trace) and presence (black trace) of FCCP. c Summary of experiments showing the amplitude of nimodipine-insensitive Ca 2+ transients reduced by each drug and the amplitude of Ca 2+ transients resistant to the blocker cocktail in the absence (dark grey bars) and presence (black bars) of FCCP. Note that FCCP induced a 3- to 4-hold increase (indicated by the number on top of black bars) in the Ca 2+ transient sensitive to ω-agatoxin IVA or ω-conotoxin GVIA or resistant to the cocktail of blockers. *** P

    Techniques Used:

    24) Product Images from "CB1 receptor-dependent and -independent inhibition of excitatory postsynaptic currents in the hippocampus by WIN 55,212-2"

    Article Title: CB1 receptor-dependent and -independent inhibition of excitatory postsynaptic currents in the hippocampus by WIN 55,212-2

    Journal: Neuropharmacology

    doi: 10.1016/j.neuropharm.2007.07.003

    CB 1 R-independent effect of WIN 55,212-2 at excitatory synapses is mediated via inhibition of N-type Ca 2+ channels. Rat slices were pre-treated with 250 nM ω-agatoxin IVA or with ω-conotoxin GVIA at least for an hour before the experiments. To block CB 1 Rs, 2 μM AM251 was included in the solution. a , In slices pre-incubated with ω-agatoxin IVA, 10 μM WIN 55,212-2 effectively reduced the amplitude of evoked EPSCs as shown on the averaged recordings of 8–10 consecutive events before (right) and after (left) drug application. The bottom graph calculated from 4 experiments indicates that wash-in of 10 μM WIN 55,212-2 significantly suppressed the EPSC amplitude. b , In contrast, when 10 μM WIN 55,212-2 was applied onto slices that were pre-incubated in ω-conotoxin GVIA, no change in the amplitude of EPSC was observed. Averaged traces before (right) and after (left) drug application are shown. The stimulus artefacts were removed from the traces. Scale bars are 20 pA and 5 ms. The bottom plot obtained from 6 experiments shows that WIN 55,212-2 could not alter the glutamatergic transmission, indicating that, independent of CB 1 Rs, N-type voltage-gated Ca 2+ channels are required for presynaptic inhibition by this cannabinoid compound applied in high doses.
    Figure Legend Snippet: CB 1 R-independent effect of WIN 55,212-2 at excitatory synapses is mediated via inhibition of N-type Ca 2+ channels. Rat slices were pre-treated with 250 nM ω-agatoxin IVA or with ω-conotoxin GVIA at least for an hour before the experiments. To block CB 1 Rs, 2 μM AM251 was included in the solution. a , In slices pre-incubated with ω-agatoxin IVA, 10 μM WIN 55,212-2 effectively reduced the amplitude of evoked EPSCs as shown on the averaged recordings of 8–10 consecutive events before (right) and after (left) drug application. The bottom graph calculated from 4 experiments indicates that wash-in of 10 μM WIN 55,212-2 significantly suppressed the EPSC amplitude. b , In contrast, when 10 μM WIN 55,212-2 was applied onto slices that were pre-incubated in ω-conotoxin GVIA, no change in the amplitude of EPSC was observed. Averaged traces before (right) and after (left) drug application are shown. The stimulus artefacts were removed from the traces. Scale bars are 20 pA and 5 ms. The bottom plot obtained from 6 experiments shows that WIN 55,212-2 could not alter the glutamatergic transmission, indicating that, independent of CB 1 Rs, N-type voltage-gated Ca 2+ channels are required for presynaptic inhibition by this cannabinoid compound applied in high doses.

    Techniques Used: Inhibition, Blocking Assay, Incubation, Mass Spectrometry, Transmission Assay

    25) Product Images from "Balance of calcineurin A? and CDK5 activities sets release probability at nerve terminals"

    Article Title: Balance of calcineurin A? and CDK5 activities sets release probability at nerve terminals

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

    doi: 10.1523/JNEUROSCI.4288-12.2013

    Calcineurin Aα and CDK5 regulate single action potential stimulated vesicle release at nerve terminals (A) Ensemble average traces of single AP stimulated exocytosis reported by vG-pH in WT, CNAαKD, and CDK5KD synapses. (B) Mean values of single AP vG-pH response amplitudes normalized to WT in WT, CNAαKD, CDK5KD, and WT neurons treated with Roscovitine (Ros). WT 1AP =1.00 ± 0.12 (n= 20), CNAαKD 1AP = 0.23 ± 0.08 (n=8), CDK5KD 1AP = 1.61 ± 0.13 (n=8), WT+Ros 1AP =1.65 ± 0.25 (n= 10). (C) Ensemble average of single AP stimulated exocytosis recorded by vG-pH in WT and WT with Ros and cyclosporine A (CSA) simultaneously. (D) Mean values of single AP responses of vG-pH in WT and WT with Ros and CSA simultaneously. WT 1AP = 1.00 ±0.12, WT+Ros+CSA 1AP = 1.05 ± 0.09 (n= 9). (E) Ensemble average of single AP stimulated exocytosis recorded by vG-pH in CNAα KD and CNAα KD with Ros. (F) Mean values of single AP vG-pH responses in CNAα KD and CNAα KD treated with Ros. CNAα KD 1AP = 0.17 ±0.06, CNAα KD+CSA 1AP = 0.19 ± 0.06 (n= 8). (G) Mean amplitudes of single-AP vG-pH response in WT neurons normalized to control WT condition for ω-agatoxin IVA (Aga) or ω-Conotoxin GVIA (Cono) treatments: WT 1AP = 1 ± 0.18 (n= 17), WT+Aga 1AP = 0.52 ± 0.13 (n= 7), WT+Cono 1AP = 0.28 ± 0.04 (n= 10). (H) Mean amplitudes of single-AP vG-pH responses in CNAαKD neurons normalized to control WT conditions for ω-agatoxin IVA (Aga) and ω-Conotoxin GVIA (Cono) treatments: CNAαKD only 1AP =0.17 ± 0.09 (n= 16), CNAαKD+Aga 1AP = 0.06 ± 0.06 (n= 10), CNAαKD+ Cono 1AP = 0.16 ± 0.04 (n= 6). (I) Mean amplitudes of single-AP vG-pH response in WT neurons normalized to WT control condition for ω-agatoxin IVA (Aga) and ω-agatoxin IVA (Aga) + Ros treatments: WT 1AP = 1 ± 0.24 (n= 5), WT+Aga 1AP = 0.50 ± 0.12 (n= 5), WT+Aga+Ros 1AP = 1.17 ± 0.15 (n= 5). (J) Mean amplitudes of single-AP vG-pH response in WT neurons normalized to WT control condition for ω-Conotoxin GVIA (Cono), ω-Conotoxin GVIA (Cono) + Ros treatments: WT 1AP = 1 ± 0.13 (n= 9), WT+Cono 1AP = 0.30 ± 0.1 (n= 9), WT+Cono+Ros 1AP = 0.35 ± 0.07 (n= 9). *P
    Figure Legend Snippet: Calcineurin Aα and CDK5 regulate single action potential stimulated vesicle release at nerve terminals (A) Ensemble average traces of single AP stimulated exocytosis reported by vG-pH in WT, CNAαKD, and CDK5KD synapses. (B) Mean values of single AP vG-pH response amplitudes normalized to WT in WT, CNAαKD, CDK5KD, and WT neurons treated with Roscovitine (Ros). WT 1AP =1.00 ± 0.12 (n= 20), CNAαKD 1AP = 0.23 ± 0.08 (n=8), CDK5KD 1AP = 1.61 ± 0.13 (n=8), WT+Ros 1AP =1.65 ± 0.25 (n= 10). (C) Ensemble average of single AP stimulated exocytosis recorded by vG-pH in WT and WT with Ros and cyclosporine A (CSA) simultaneously. (D) Mean values of single AP responses of vG-pH in WT and WT with Ros and CSA simultaneously. WT 1AP = 1.00 ±0.12, WT+Ros+CSA 1AP = 1.05 ± 0.09 (n= 9). (E) Ensemble average of single AP stimulated exocytosis recorded by vG-pH in CNAα KD and CNAα KD with Ros. (F) Mean values of single AP vG-pH responses in CNAα KD and CNAα KD treated with Ros. CNAα KD 1AP = 0.17 ±0.06, CNAα KD+CSA 1AP = 0.19 ± 0.06 (n= 8). (G) Mean amplitudes of single-AP vG-pH response in WT neurons normalized to control WT condition for ω-agatoxin IVA (Aga) or ω-Conotoxin GVIA (Cono) treatments: WT 1AP = 1 ± 0.18 (n= 17), WT+Aga 1AP = 0.52 ± 0.13 (n= 7), WT+Cono 1AP = 0.28 ± 0.04 (n= 10). (H) Mean amplitudes of single-AP vG-pH responses in CNAαKD neurons normalized to control WT conditions for ω-agatoxin IVA (Aga) and ω-Conotoxin GVIA (Cono) treatments: CNAαKD only 1AP =0.17 ± 0.09 (n= 16), CNAαKD+Aga 1AP = 0.06 ± 0.06 (n= 10), CNAαKD+ Cono 1AP = 0.16 ± 0.04 (n= 6). (I) Mean amplitudes of single-AP vG-pH response in WT neurons normalized to WT control condition for ω-agatoxin IVA (Aga) and ω-agatoxin IVA (Aga) + Ros treatments: WT 1AP = 1 ± 0.24 (n= 5), WT+Aga 1AP = 0.50 ± 0.12 (n= 5), WT+Aga+Ros 1AP = 1.17 ± 0.15 (n= 5). (J) Mean amplitudes of single-AP vG-pH response in WT neurons normalized to WT control condition for ω-Conotoxin GVIA (Cono), ω-Conotoxin GVIA (Cono) + Ros treatments: WT 1AP = 1 ± 0.13 (n= 9), WT+Cono 1AP = 0.30 ± 0.1 (n= 9), WT+Cono+Ros 1AP = 0.35 ± 0.07 (n= 9). *P

    Techniques Used:

    CNAα and CDK5 regulate N-type but not P/Q-type Ca 2+ channels at hippocampal synapses and soma (A-D) Measurement of Ca 2+ influx with synaptophysin-GCaMP3 at presynaptic terminal with/without ω-agatoxin IVA or ω-Conotoxin GVIA in WT and CNAαKD neurons. (A) Mean linearized single-AP GCaMP3 response amplitudes at boutons in WT neurons normalized to control condition for ω-agatoxin IVA (Aga) or ω-Conotoxin GVIA (Cono) treatments: WT boutons 1AP =1.00 ± 0.07 (n=24), WT+Aga boutons 1AP =0.80 ± 0.07 (n=16), WT+Cono boutons 1AP =0.50 ± 0.07 (n=8). (B) Mean linearized single-AP GCaMP3 responses in CNAαKD neurons normalized to control WT conditions for ω-agatoxin IVA (Aga) and ω-Conotoxin GVIA (Cono) treatments: CNAαKD boutons 1AP =0.49 ± 0.08 (n=14), CNAαKD+Aga boutons 1AP = 0.35 ± 0.03 (n=6), CNAαKD+ Cono boutons 1AP = 0.44 ± 0.04 (n=8). (C) Mean linearized single-AP GCaMP3 response amplitudes in WT neurons normalized to WT control condition for ω-agatoxin IVA (Aga) and ω-agatoxin IVA (Aga) + Ros treatments: WT boutons 1AP =1.00 ± 0.03, WT+Aga boutons 1AP =0.75 ± 0.07, WT+Aga+Ros boutons 1AP =1.05 ± 0.10 (n=7). (D) Mean linearized single-AP GCaMP3 response amplitudes in WT neurons normalized to WT control condition for ω-Conotoxin GVIA (Cono), ω-Conotoxin GVIA (Cono) + Ros treatments: WT boutons 1AP =1.00 ±0.10, WT+Cono boutons 1AP =0.63 ± 0.12, WT+Cono+Ros boutons 1AP = 0.66 ± 0.11 (n=7). (E-H) Measurement of somatic Ca 2+ influx with Fluo5F with/without ω-agatoxin IVA or ω-Conotoxin GVIA and Roscovitine in WT neurons. (E) Mean single-AP response amplitudes of Ca 2+ influx at cell somas in WT neurons normalized to control condition for ω-agatoxin IVA (Aga) or ω-Conotoxin GVIA (Cono) treatments: WT soma 1AP =1.00 ± 0.03 (n=23), WT+Aga soma 1AP = 0.66 ± 0.03 (n= 9), WT+Cono soma 1AP =0.85 ± 0.02 (n=14). (F) Mean single-AP response amplitudes of Ca 2+ influx at cell body with/without Roscovitine or Cyclosporin A: WT soma 1AP = 1.00 ± 0.09 (n=x), WT+Ros soma 1AP =1.15 ± 0.14 (n= 9), WT+CSA soma 1AP = 0.77 ± 0.06 (n= 10). (G) Mean single-AP response amplitudes of Ca 2+ influx at cell body with ω-agatoxin IVA (Aga) and ω-agatoxin IVA (Aga) + Ros treatments: WT soma 1AP = 1.00 ± 0.04, WT+Aga soma 1AP = 0.66 ± 0.04, WT+Aga+Ros soma 1AP = 0.76 ± 0.06 (n= 7). (H) Mean single-AP response amplitudes of Ca 2+ influx at cell body with ω-Conotoxin GVIA (Cono) and ω-Conotoxin GVIA (Cono)+ Ros treatments: WT soma 1AP = 1.00 ± 0.13, WT+Cono soma 1AP = 0.79 ± 0.09, WT+Cono+Ros soma 1AP = 0.84 ± 0.08. (n= 6).**P
    Figure Legend Snippet: CNAα and CDK5 regulate N-type but not P/Q-type Ca 2+ channels at hippocampal synapses and soma (A-D) Measurement of Ca 2+ influx with synaptophysin-GCaMP3 at presynaptic terminal with/without ω-agatoxin IVA or ω-Conotoxin GVIA in WT and CNAαKD neurons. (A) Mean linearized single-AP GCaMP3 response amplitudes at boutons in WT neurons normalized to control condition for ω-agatoxin IVA (Aga) or ω-Conotoxin GVIA (Cono) treatments: WT boutons 1AP =1.00 ± 0.07 (n=24), WT+Aga boutons 1AP =0.80 ± 0.07 (n=16), WT+Cono boutons 1AP =0.50 ± 0.07 (n=8). (B) Mean linearized single-AP GCaMP3 responses in CNAαKD neurons normalized to control WT conditions for ω-agatoxin IVA (Aga) and ω-Conotoxin GVIA (Cono) treatments: CNAαKD boutons 1AP =0.49 ± 0.08 (n=14), CNAαKD+Aga boutons 1AP = 0.35 ± 0.03 (n=6), CNAαKD+ Cono boutons 1AP = 0.44 ± 0.04 (n=8). (C) Mean linearized single-AP GCaMP3 response amplitudes in WT neurons normalized to WT control condition for ω-agatoxin IVA (Aga) and ω-agatoxin IVA (Aga) + Ros treatments: WT boutons 1AP =1.00 ± 0.03, WT+Aga boutons 1AP =0.75 ± 0.07, WT+Aga+Ros boutons 1AP =1.05 ± 0.10 (n=7). (D) Mean linearized single-AP GCaMP3 response amplitudes in WT neurons normalized to WT control condition for ω-Conotoxin GVIA (Cono), ω-Conotoxin GVIA (Cono) + Ros treatments: WT boutons 1AP =1.00 ±0.10, WT+Cono boutons 1AP =0.63 ± 0.12, WT+Cono+Ros boutons 1AP = 0.66 ± 0.11 (n=7). (E-H) Measurement of somatic Ca 2+ influx with Fluo5F with/without ω-agatoxin IVA or ω-Conotoxin GVIA and Roscovitine in WT neurons. (E) Mean single-AP response amplitudes of Ca 2+ influx at cell somas in WT neurons normalized to control condition for ω-agatoxin IVA (Aga) or ω-Conotoxin GVIA (Cono) treatments: WT soma 1AP =1.00 ± 0.03 (n=23), WT+Aga soma 1AP = 0.66 ± 0.03 (n= 9), WT+Cono soma 1AP =0.85 ± 0.02 (n=14). (F) Mean single-AP response amplitudes of Ca 2+ influx at cell body with/without Roscovitine or Cyclosporin A: WT soma 1AP = 1.00 ± 0.09 (n=x), WT+Ros soma 1AP =1.15 ± 0.14 (n= 9), WT+CSA soma 1AP = 0.77 ± 0.06 (n= 10). (G) Mean single-AP response amplitudes of Ca 2+ influx at cell body with ω-agatoxin IVA (Aga) and ω-agatoxin IVA (Aga) + Ros treatments: WT soma 1AP = 1.00 ± 0.04, WT+Aga soma 1AP = 0.66 ± 0.04, WT+Aga+Ros soma 1AP = 0.76 ± 0.06 (n= 7). (H) Mean single-AP response amplitudes of Ca 2+ influx at cell body with ω-Conotoxin GVIA (Cono) and ω-Conotoxin GVIA (Cono)+ Ros treatments: WT soma 1AP = 1.00 ± 0.13, WT+Cono soma 1AP = 0.79 ± 0.09, WT+Cono+Ros soma 1AP = 0.84 ± 0.08. (n= 6).**P

    Techniques Used:

    26) Product Images from "Midbrain dopaminergic neurons generate calcium and sodium currents and release dopamine in the striatum of pups"

    Article Title: Midbrain dopaminergic neurons generate calcium and sodium currents and release dopamine in the striatum of pups

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2012.00007

    Pharmacology of the spontaneous Ca 2+ activities of embryonic and early postnatal mDA neurons. Representative calcium fluorescence traces from mDA neurons and corresponding quantitative data at the indicated ages showing the effect of TTX (1 μ M)—nifedipin (10 μ M) (A) nifedipin (3 μ M) (B) ω-conotoxin GVIA (1 μ M) (C) and blockers of ionotropic glutamate and GABA receptors [APV (40 μ M)—CNQX (10 μ M)—Gabazine (5 μ M), D and E ].
    Figure Legend Snippet: Pharmacology of the spontaneous Ca 2+ activities of embryonic and early postnatal mDA neurons. Representative calcium fluorescence traces from mDA neurons and corresponding quantitative data at the indicated ages showing the effect of TTX (1 μ M)—nifedipin (10 μ M) (A) nifedipin (3 μ M) (B) ω-conotoxin GVIA (1 μ M) (C) and blockers of ionotropic glutamate and GABA receptors [APV (40 μ M)—CNQX (10 μ M)—Gabazine (5 μ M), D and E ].

    Techniques Used: Multiple Displacement Amplification, Fluorescence

    27) Product Images from "Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons"

    Article Title: Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201511383

    GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P
    Figure Legend Snippet: GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P

    Techniques Used: Activity Assay, Mouse Assay, Activation Assay

    GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P
    Figure Legend Snippet: GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P

    Techniques Used: Activity Assay, Transfection, Inhibition

    28) Product Images from "Ion Channel Expression in the Developing Enteric Nervous System"

    Article Title: Ion Channel Expression in the Developing Enteric Nervous System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123436

    Expression of Ca 2+ channels and the lack of effect of Ca 2+ channel blockers on ENCC migration and neuritogenesis. A. RT-PCR confirming expression of transcripts encoding 8 calcium channels in purified (FACS-sorted) ENCCs from E11.5 gut. Adult mouse brain (Br) was used as a positive control and-RT was a negative control (-). B, C. Immunohistochemistry using antisera to Ca v 2.1 ( B ) and Ca v 2.2 ( C ) of dissociated E14.5 gut cultured for 48 hours revealed Ca v 2.1 and Ca v 2.2 immunostaining of Tuj1+ neurites ( arrows ). Tuj1+ cell bodies show some Ca v 2.1 staining, but little, if any, Ca v 2.2 immunostaining. D. There was no significant difference in the distance migrated by ENCCs in explants grown in the presence of the N-type blocker, ω-conotoxin GVIA (n = 9), the P/Q-type blocker, ω-agatoxin IVA (n = 10), the L-type blocker, nicardipine (n = 9) or the T-type blocker, mibefradil (n = 10) compared to controls (n = 11) (mean ± SEM; one way ANOVA; minimum of 2 experiments). E. Effects of calcium channel blockers on neuritogenesis. There was no significant difference in the percentage of Tuj1+ cells that extended neurites between control and drug-treated cultures of E14.5 dissociated gut (for controls and each drug, a minimum of 950 Tuj1+ cells was examined from 5 or 6 coverslips from 2 experiments).
    Figure Legend Snippet: Expression of Ca 2+ channels and the lack of effect of Ca 2+ channel blockers on ENCC migration and neuritogenesis. A. RT-PCR confirming expression of transcripts encoding 8 calcium channels in purified (FACS-sorted) ENCCs from E11.5 gut. Adult mouse brain (Br) was used as a positive control and-RT was a negative control (-). B, C. Immunohistochemistry using antisera to Ca v 2.1 ( B ) and Ca v 2.2 ( C ) of dissociated E14.5 gut cultured for 48 hours revealed Ca v 2.1 and Ca v 2.2 immunostaining of Tuj1+ neurites ( arrows ). Tuj1+ cell bodies show some Ca v 2.1 staining, but little, if any, Ca v 2.2 immunostaining. D. There was no significant difference in the distance migrated by ENCCs in explants grown in the presence of the N-type blocker, ω-conotoxin GVIA (n = 9), the P/Q-type blocker, ω-agatoxin IVA (n = 10), the L-type blocker, nicardipine (n = 9) or the T-type blocker, mibefradil (n = 10) compared to controls (n = 11) (mean ± SEM; one way ANOVA; minimum of 2 experiments). E. Effects of calcium channel blockers on neuritogenesis. There was no significant difference in the percentage of Tuj1+ cells that extended neurites between control and drug-treated cultures of E14.5 dissociated gut (for controls and each drug, a minimum of 950 Tuj1+ cells was examined from 5 or 6 coverslips from 2 experiments).

    Techniques Used: Expressing, Migration, Reverse Transcription Polymerase Chain Reaction, Purification, FACS, Positive Control, Negative Control, Immunohistochemistry, Cell Culture, Immunostaining, Staining

    29) Product Images from "Characterization of Na+ and Ca2+ Channels in Zebrafish Dorsal Root Ganglion Neurons"

    Article Title: Characterization of Na+ and Ca2+ Channels in Zebrafish Dorsal Root Ganglion Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042602

    Pharmacological dissection of HVA- I Ca in zebrafish DRG neurons. A , Left , Time courses of I Ca amplitude during serial application of nifedipine (10 µM), ω-agatoxin IVA (0.5 µM), ω-conotoxin GVIA (3 µM), SNX-482 (300 nM) and CdCl 2 (100 µM). I Ca was evoked every 10 s by 70 ms test pulses to 0 mV from a holding potential of −80 mV. The horizontal bars indicate the duration of drug application. Right , superimposed current traces obtained at different time points during drug application (labeled as a–f). B , Bar graph representing the mean I Ca inhibition (%) produced by application of the indicated antagonists or toxins. Error bars represent s . e . m . The number of neurons tested is indicated in parentheses. C , Effect of non-dihydropyridine Ca 2+ channel agonist FPL 64176 (FPL) on I Ca in DRG neurons. FPL (1 µM) was applied to rat superior cervical ganglion (SCG) neurons as a positive control (left panel). Note that FPL applied to zebrafish DRG I Ca display neither an increase in macroscopic inward currents nor greatly prolonged trajectory of the tail currents (right panel).
    Figure Legend Snippet: Pharmacological dissection of HVA- I Ca in zebrafish DRG neurons. A , Left , Time courses of I Ca amplitude during serial application of nifedipine (10 µM), ω-agatoxin IVA (0.5 µM), ω-conotoxin GVIA (3 µM), SNX-482 (300 nM) and CdCl 2 (100 µM). I Ca was evoked every 10 s by 70 ms test pulses to 0 mV from a holding potential of −80 mV. The horizontal bars indicate the duration of drug application. Right , superimposed current traces obtained at different time points during drug application (labeled as a–f). B , Bar graph representing the mean I Ca inhibition (%) produced by application of the indicated antagonists or toxins. Error bars represent s . e . m . The number of neurons tested is indicated in parentheses. C , Effect of non-dihydropyridine Ca 2+ channel agonist FPL 64176 (FPL) on I Ca in DRG neurons. FPL (1 µM) was applied to rat superior cervical ganglion (SCG) neurons as a positive control (left panel). Note that FPL applied to zebrafish DRG I Ca display neither an increase in macroscopic inward currents nor greatly prolonged trajectory of the tail currents (right panel).

    Techniques Used: Dissection, Mass Spectrometry, Labeling, Inhibition, Produced, Positive Control

    30) Product Images from "Effects of IgG anti-GM1 monoclonal antibodies on neuromuscular transmission and calcium channel binding in rat neuromuscular junctions"

    Article Title: Effects of IgG anti-GM1 monoclonal antibodies on neuromuscular transmission and calcium channel binding in rat neuromuscular junctions

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2015.2575

    Effects of pretreatment with the N-type calcium channel blocker, ω-conotoxin GVIA, on the inhibitory effect of IgG anti-GM1 mAb (1:100) on spontaneous muscle action potentials (SMAPs) in the spinal cord-muscle co-culture system. (A) Inhibition
    Figure Legend Snippet: Effects of pretreatment with the N-type calcium channel blocker, ω-conotoxin GVIA, on the inhibitory effect of IgG anti-GM1 mAb (1:100) on spontaneous muscle action potentials (SMAPs) in the spinal cord-muscle co-culture system. (A) Inhibition

    Techniques Used: Co-Culture Assay, Inhibition

    31) Product Images from "CaV2.1 α1 Subunit Expression Regulates Presynaptic CaV2.1 Abundance and Synaptic Strength at a Central Synapse"

    Article Title: CaV2.1 α1 Subunit Expression Regulates Presynaptic CaV2.1 Abundance and Synaptic Strength at a Central Synapse

    Journal: Neuron

    doi: 10.1016/j.neuron.2018.11.028

    Ca V 2.1 α 1 OE Results in Increased Ca V 2.1 Currents and Almost Complete Loss of Ca V 2.2 Currents at the P7 Calyx (A) Schematic of auditory brainstem. Globular bushy cells (GBC) which give rise to the calyx of Held are depicted for clarity. (B) (Top) Developmental transition of calyx of Held from multiple Ca V 2 subtype synapse to Ca V 2 exclusive at onset of hearing (P12). (Bottom) Experimental timeline from virus injection into VCN at P1 to electrophysiological recordings at P7. (C) Schematic of HdAd constructs expressing either Ca V 2.1 or Ca V 2.2 cDNAs (light blue) driven by the Punisher overexpression cassette and mEGFP marker (green) driven by a 470 bp human synapsin promoter; arrows indicate viral inverted terminal repeat sequences; J indicates the viral genome packaging signal sequence. (D) Pharmacological isolation of Ca V 2 isoforms expressed in the presynaptic terminal at P7 in control (n = 5), Ca V 2.1 α 1 OE (n = 4), or Ca V 2.2 α 1 OE (n = 6). Average traces before application of blockers (black), after applying 200 nM ω-agatoxin IVA to specifically block Ca V 2.1 (Aga, brown), after 2 μM ω-conotoxin GVIA to block Ca V 2.2 (Cono, blue) and 50 μM Cd 2+ to block the remaining Ca 2+ currents (gray). (E–H) Ca 2+ current amplitudes before blocker application (Ca V 2.1 α 1 OE versus control, p = 0.0328 Mood’s median test and post hoc Bonferroni test), Aga-sensitive Ca 2+ current amplitudes (Ca V 2.1 α 1 OE versus control, p = 0.0328), Cono-sensitive Ca 2+ current amplitudes (Ca V 2.1 α 1 OE versus control, p = 0.0054), and Cd 2+ -sensitive Ca 2+ current amplitudes (n.s., Kruskal Wallis and post hoc Dunn’s test, n = 5/4/6 for control, Ca V 2.1 α 1 OE, and Ca V 2.2 α 1 OE, respectively). (I) Relative Ca 2+ current fractions sensitive to respective blockers. (J and K) Average Ca 2+ current traces to 10 ms step depolarizations from −80 mV holding to voltages between −50 and +40 mV for control (J and K, left, black) and Ca V 2.1 α 1 OE (J, right, brown) or Ca V 2.2 α 1 OE (K, right, blue). (L–O) Current-voltage relationships of either steady-state Ca 2+ currents (L and N) or tail Ca 2+ currents (M and O, n = 10 for control, Ca V 2.1 α 1 OE and Ca V 2.2 α 1 .
    Figure Legend Snippet: Ca V 2.1 α 1 OE Results in Increased Ca V 2.1 Currents and Almost Complete Loss of Ca V 2.2 Currents at the P7 Calyx (A) Schematic of auditory brainstem. Globular bushy cells (GBC) which give rise to the calyx of Held are depicted for clarity. (B) (Top) Developmental transition of calyx of Held from multiple Ca V 2 subtype synapse to Ca V 2 exclusive at onset of hearing (P12). (Bottom) Experimental timeline from virus injection into VCN at P1 to electrophysiological recordings at P7. (C) Schematic of HdAd constructs expressing either Ca V 2.1 or Ca V 2.2 cDNAs (light blue) driven by the Punisher overexpression cassette and mEGFP marker (green) driven by a 470 bp human synapsin promoter; arrows indicate viral inverted terminal repeat sequences; J indicates the viral genome packaging signal sequence. (D) Pharmacological isolation of Ca V 2 isoforms expressed in the presynaptic terminal at P7 in control (n = 5), Ca V 2.1 α 1 OE (n = 4), or Ca V 2.2 α 1 OE (n = 6). Average traces before application of blockers (black), after applying 200 nM ω-agatoxin IVA to specifically block Ca V 2.1 (Aga, brown), after 2 μM ω-conotoxin GVIA to block Ca V 2.2 (Cono, blue) and 50 μM Cd 2+ to block the remaining Ca 2+ currents (gray). (E–H) Ca 2+ current amplitudes before blocker application (Ca V 2.1 α 1 OE versus control, p = 0.0328 Mood’s median test and post hoc Bonferroni test), Aga-sensitive Ca 2+ current amplitudes (Ca V 2.1 α 1 OE versus control, p = 0.0328), Cono-sensitive Ca 2+ current amplitudes (Ca V 2.1 α 1 OE versus control, p = 0.0054), and Cd 2+ -sensitive Ca 2+ current amplitudes (n.s., Kruskal Wallis and post hoc Dunn’s test, n = 5/4/6 for control, Ca V 2.1 α 1 OE, and Ca V 2.2 α 1 OE, respectively). (I) Relative Ca 2+ current fractions sensitive to respective blockers. (J and K) Average Ca 2+ current traces to 10 ms step depolarizations from −80 mV holding to voltages between −50 and +40 mV for control (J and K, left, black) and Ca V 2.1 α 1 OE (J, right, brown) or Ca V 2.2 α 1 OE (K, right, blue). (L–O) Current-voltage relationships of either steady-state Ca 2+ currents (L and N) or tail Ca 2+ currents (M and O, n = 10 for control, Ca V 2.1 α 1 OE and Ca V 2.2 α 1 .

    Techniques Used: Injection, Construct, Expressing, Over Expression, Marker, Sequencing, Isolation, Blocking Assay, Mass Spectrometry

    Ca V 2.2 α 1 OE Results in Slight Loss of Ca V 2.1 Currents, while Ca V 2.1 α 1 OE Results in an Increase in Ca V 2.1 Currents at P20/21 Calyx (A) Experimental timeline from virus injection into CN at P14 to electrophysiological recordings at P20/21. (B) Confocal images of brainstem slices injected with Ca V 2.1 α 1 OE construct. (Left) CN injection site. (Right) Contralateral MNTB with mEGFP-expressing calyx of Held terminals. (C) Pharmacological isolation of Ca V 2 isoforms expressed in the presynaptic terminal at P21 in control (n = 3) and Ca V 2.2 α 1 OE (n = 3). Average current traces before application of blockers (black), after applying 200 nM ω-agatoxin IVA to specifically block Ca V 2.1 (Aga, brown) and after applying 2 μM ω-conotoxin GVIA to specifically block Ca V 2.2 (Cono, blue). (D) Ca 2+ current amplitudes before blocker application (black, n.s., two-tailed t test), Aga-sensitive Ca 2+ current amplitudes (brown, n.s., one-tailed t test), and Cono-sensitive Ca 2+ current amplitudes (blue, 0.016, one-tailed t test). (E) Relative Ca 2+ current fractions sensitive to blockers. (F) Average Ca 2+ -current traces to 10 ms step depolarizations from −80 mV holding to voltages between −50 and +40 mV for control (left, n = 9) and Ca V 2.1 α 1 OE (right, n = 10). (G and H) Current-voltage relationships of either peak Ca 2+ currents (G) or tail Ca 2+ .
    Figure Legend Snippet: Ca V 2.2 α 1 OE Results in Slight Loss of Ca V 2.1 Currents, while Ca V 2.1 α 1 OE Results in an Increase in Ca V 2.1 Currents at P20/21 Calyx (A) Experimental timeline from virus injection into CN at P14 to electrophysiological recordings at P20/21. (B) Confocal images of brainstem slices injected with Ca V 2.1 α 1 OE construct. (Left) CN injection site. (Right) Contralateral MNTB with mEGFP-expressing calyx of Held terminals. (C) Pharmacological isolation of Ca V 2 isoforms expressed in the presynaptic terminal at P21 in control (n = 3) and Ca V 2.2 α 1 OE (n = 3). Average current traces before application of blockers (black), after applying 200 nM ω-agatoxin IVA to specifically block Ca V 2.1 (Aga, brown) and after applying 2 μM ω-conotoxin GVIA to specifically block Ca V 2.2 (Cono, blue). (D) Ca 2+ current amplitudes before blocker application (black, n.s., two-tailed t test), Aga-sensitive Ca 2+ current amplitudes (brown, n.s., one-tailed t test), and Cono-sensitive Ca 2+ current amplitudes (blue, 0.016, one-tailed t test). (E) Relative Ca 2+ current fractions sensitive to blockers. (F) Average Ca 2+ -current traces to 10 ms step depolarizations from −80 mV holding to voltages between −50 and +40 mV for control (left, n = 9) and Ca V 2.1 α 1 OE (right, n = 10). (G and H) Current-voltage relationships of either peak Ca 2+ currents (G) or tail Ca 2+ .

    Techniques Used: Injection, Construct, Expressing, Isolation, Blocking Assay, Two Tailed Test, One-tailed Test, Mass Spectrometry

    32) Product Images from "Sumatriptan inhibition of N-type calcium channel mediated signaling in dural CGRP terminal fibres"

    Article Title: Sumatriptan inhibition of N-type calcium channel mediated signaling in dural CGRP terminal fibres

    Journal: Neuropharmacology

    doi: 10.1016/j.neuropharm.2012.04.016

    N-type Ca 2+ channels mediate Sumatriptan inhibition of Ca 2+ signaling (A–D) Graphs of timecourse experiments showing the effects of Ca 2+ channel blockers on the single action potential evoked Ca 2+ transient amplitudes. Individual example transients taken at the relative timepoints on the graphs are shown below. (A + B) Neither the P/Q Ca 2+ channel blocker Agatoxin-IVA (200 nm) nor the L-type Ca 2+ channel blocker Nifedipine (10 μM) decreased the amplitude of the Ca 2+ transient. (C + D) The T-type Ca 2+ channel blocker NNC 55-0936 (20 μM) as well as the N-type Ca 2+ channel blocker Conotoxin-GVIA (1μM) significantly inhibited the Ca 2+ transient amplitude. C) The amplitude of the T-type Ca 2+ channel mediated inhibition was further reduced by the application of sumatriptan, while the amplitude of the N-type Ca 2+ channel mediated inhibition remained unaffected with sumatriptan application (D). (E) Bar graph summaries under the experimental conditions seen in (A – D) showing the inhibition of Ca 2+ transient amplitudes ( F/F; % reduction) at the timepoints shown on the graphs (a 1 − d 1 ). The magnitude of the sumatriptan mediated inhibition alone was found to be equal to the magnitude of the sumatriptan mediated inhibition in the presence of the T-type Ca 2+ channel blocker (c 2 ), while in the presence of the N-type Ca 2+ channel blocker, the sumatriptan mediated inhibition was completely occluded (d 2 ).
    Figure Legend Snippet: N-type Ca 2+ channels mediate Sumatriptan inhibition of Ca 2+ signaling (A–D) Graphs of timecourse experiments showing the effects of Ca 2+ channel blockers on the single action potential evoked Ca 2+ transient amplitudes. Individual example transients taken at the relative timepoints on the graphs are shown below. (A + B) Neither the P/Q Ca 2+ channel blocker Agatoxin-IVA (200 nm) nor the L-type Ca 2+ channel blocker Nifedipine (10 μM) decreased the amplitude of the Ca 2+ transient. (C + D) The T-type Ca 2+ channel blocker NNC 55-0936 (20 μM) as well as the N-type Ca 2+ channel blocker Conotoxin-GVIA (1μM) significantly inhibited the Ca 2+ transient amplitude. C) The amplitude of the T-type Ca 2+ channel mediated inhibition was further reduced by the application of sumatriptan, while the amplitude of the N-type Ca 2+ channel mediated inhibition remained unaffected with sumatriptan application (D). (E) Bar graph summaries under the experimental conditions seen in (A – D) showing the inhibition of Ca 2+ transient amplitudes ( F/F; % reduction) at the timepoints shown on the graphs (a 1 − d 1 ). The magnitude of the sumatriptan mediated inhibition alone was found to be equal to the magnitude of the sumatriptan mediated inhibition in the presence of the T-type Ca 2+ channel blocker (c 2 ), while in the presence of the N-type Ca 2+ channel blocker, the sumatriptan mediated inhibition was completely occluded (d 2 ).

    Techniques Used: Inhibition

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    Alomone Labs ω conotoxin gvia
    GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM <t>ω-conotoxin-GVIA</t> (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P
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    GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P

    Journal: The Journal of General Physiology

    Article Title: Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

    doi: 10.1085/jgp.201511383

    Figure Lengend Snippet: GHSR1a activity modulates native Ca V 2 currents in hypothalamic neurons from GHSR-eGFP reporter mice. (A) Representative I Ba traces and averaged I Ba before (control) and after (+ghrelin) 500-nM ghrelin application in hypothalamic GHSR1a− and GHSR1a+ neurons. (B) Averaged peak I Ba –voltage (I-V) relationships (evoked from a holding of −80 mV), reversal (V rev ), and activation (V 1/2 ) potential midpoints (calculated by Boltzmann linear function) obtained from GHSR1a− and GHSR1a+ neurons. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from hypothalamic GHSR1a− (top) and GHSR1a+ neurons (middle and bottom; left). Averaged percentage of I Ba sensitive to agaTx and conoTx from GHSR1a− and GHSR1a+ neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from GHSR1a− and GHSR1a+ neurons. Paired (A) or two-sample (B and D) Student’s t test and ANOVA with Dunnett’s post-test (C). *, P

    Article Snippet: We used ghrelin esterified with n -octanoic acid (Global Peptide); a GHSR1a inverse agonist, [d -Arg1,d -Phe5,d -Trp7,9,Leu11]–substance P (SPA; Santa Cruz Biotechnology, Inc.); the inhibitor of Gs protein, cholera toxin (ChTx; Sigma Aldrich); a specific inhibitor of Gi/o protein, pertussis toxin (PTx; Sigma-Aldrich); the CaV 2.1 blocker, ω-agatoxin-IVA (Peptides International); and the CaV2.2 blocker, ω-conotoxin-GVIA (Alomone Labs).

    Techniques: Activity Assay, Mouse Assay, Activation Assay

    GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P

    Journal: The Journal of General Physiology

    Article Title: Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

    doi: 10.1085/jgp.201511383

    Figure Lengend Snippet: GHSR1a activity inhibits native Ca V 2 currents from rat hypothalamic neurons. (A) Representative and averaged I Ba from nontransfected (nt) and GFP-, GHSR1a-YFP–, and GHSR1a-A204E-YFP–transfected neurons. (B) Normalized I Ba traces before (control) and after (+ghrelin) 500-nM ghrelin application, and averaged percentage of I Ba inhibition by ghrelin in each condition. (C) I Ba time courses of application of 1 µM ω-conotoxin-GVIA (conoTx) and 0.2 µM ω-agatoxin-IVA (agaTx) with or without previous 500-nM ghrelin application from GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons (left). Averaged percentage of I Ba sensitive to agaTx and conoTx from nontransfected (nt), GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons, with (+ghrelin) or without 500-nM ghrelin application (right). (D) Representative and averaged I Na from nontransfected (nt) and GFP-, GHSR1a-, and GHSR1a-A204E–transfected neurons. ANOVA with Dunnett’s post-test (A–D). *, P

    Article Snippet: We used ghrelin esterified with n -octanoic acid (Global Peptide); a GHSR1a inverse agonist, [d -Arg1,d -Phe5,d -Trp7,9,Leu11]–substance P (SPA; Santa Cruz Biotechnology, Inc.); the inhibitor of Gs protein, cholera toxin (ChTx; Sigma Aldrich); a specific inhibitor of Gi/o protein, pertussis toxin (PTx; Sigma-Aldrich); the CaV 2.1 blocker, ω-agatoxin-IVA (Peptides International); and the CaV2.2 blocker, ω-conotoxin-GVIA (Alomone Labs).

    Techniques: Activity Assay, Transfection, Inhibition

    Rb1 inhibited the I Ba in hippocampal neurons, and this inhibitory effect was eliminated after the application of nifedipine (A). Neither ω-conotoxin-GVIA nor ω-agatoxin IVA diminished the Rb1-sensitive I Ba (B and C, respectively). Upper panel, pairs of the inward currents evoked by pulses from −60 to +0 mV (0–+20 mV) at the times indicated in the lower panel. Lower panel, time course of the effects of 10 μmol/L Rb1 on the I Ba amplitude before and after application of the Ca 2+ channel antagonists (10 μmol/L nifedipine, 1 μmol/L ω-conotoxin GVIA and 30 nmol/L ω-agatoxin IVA). The bar graphs for Rb1 inhibition (mean±SEM, n =5 for Rb1) on the I Ba in cells untreated or treated with Ca 2+ channel antagonists. c P

    Journal: Acta Pharmacologica Sinica

    Article Title: Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons

    doi: 10.1038/aps.2011.181

    Figure Lengend Snippet: Rb1 inhibited the I Ba in hippocampal neurons, and this inhibitory effect was eliminated after the application of nifedipine (A). Neither ω-conotoxin-GVIA nor ω-agatoxin IVA diminished the Rb1-sensitive I Ba (B and C, respectively). Upper panel, pairs of the inward currents evoked by pulses from −60 to +0 mV (0–+20 mV) at the times indicated in the lower panel. Lower panel, time course of the effects of 10 μmol/L Rb1 on the I Ba amplitude before and after application of the Ca 2+ channel antagonists (10 μmol/L nifedipine, 1 μmol/L ω-conotoxin GVIA and 30 nmol/L ω-agatoxin IVA). The bar graphs for Rb1 inhibition (mean±SEM, n =5 for Rb1) on the I Ba in cells untreated or treated with Ca 2+ channel antagonists. c P

    Article Snippet: Stock solutions of the Ca2+ channel antagonists, ω-conotoxin GVIA (Alomone Labs, UK), nifedipine (Sigma, UK) ω-agatoxin IVA (Alomone Labs, UK) and adenylyl cyclase agonist Forskolin (Sigma, UK), were prepared with the appropriate amounts of deionized water or dimethyl sulfoxide (DMSO) and frozen at −20 °C before appropriate dilution in the recording medium.

    Techniques: Inhibition

    (A) Phase-contrast image showing a single patch recording from 7-d cultured hippocampal neurons for the recording of the VGCCs. Scale bar, 10 μm. (B) Pharmacological separation of the VGCC subtypes in hippocampal neurons. Upper panel, inward Ca 2+ channel Ba 2+ currents evoked by pulses from −60 mV to 0 mV at the times indicated in the lower panel. Lower panel, time course of effects of ω-conotoxin GVIA (1 μmol/L), ω-agatoxin IVA (30 nmol/L) and nifedipine (10 μmol/L) on the Ba 2+ current amplitude.

    Journal: Acta Pharmacologica Sinica

    Article Title: Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons

    doi: 10.1038/aps.2011.181

    Figure Lengend Snippet: (A) Phase-contrast image showing a single patch recording from 7-d cultured hippocampal neurons for the recording of the VGCCs. Scale bar, 10 μm. (B) Pharmacological separation of the VGCC subtypes in hippocampal neurons. Upper panel, inward Ca 2+ channel Ba 2+ currents evoked by pulses from −60 mV to 0 mV at the times indicated in the lower panel. Lower panel, time course of effects of ω-conotoxin GVIA (1 μmol/L), ω-agatoxin IVA (30 nmol/L) and nifedipine (10 μmol/L) on the Ba 2+ current amplitude.

    Article Snippet: Stock solutions of the Ca2+ channel antagonists, ω-conotoxin GVIA (Alomone Labs, UK), nifedipine (Sigma, UK) ω-agatoxin IVA (Alomone Labs, UK) and adenylyl cyclase agonist Forskolin (Sigma, UK), were prepared with the appropriate amounts of deionized water or dimethyl sulfoxide (DMSO) and frozen at −20 °C before appropriate dilution in the recording medium.

    Techniques: Cell Culture

    The effect of L-type voltage-dependent Ca 2+ channels (VDCCs) on branching morphogenesis. ( A ) Morphological changes of SMG cultures (E13.5) upon 500 μM LaCl 3 treatment. ( B ) Bud numbers of SMG cultures upon 500 μM LaCl 3 (La) and 1 M EGTA treatment. n = 7, Data are represented as mean ± SEM. ( C ) Representative images of SMG cultures treated with various Ca 2+ channel inhibitors. ( D ) Bud numbers of SMG cultures (E12) upon treatment with various Ca 2+ channel inhibitors. Nif: 100 μM nifedipine; Gd: 500 μM GdCl3; SKF: 10 μM SKF 96365, n = 7, Data are represented as mean ±SEM. ( E ) Bud numbers of SMG cultures (E13) upon different concentrations of nifedipine treatment for 48 h. n = 5. Data are represented as mean ± SEM. ( F ) Relative acinar size of SMGs (E13) upon different concentrations of nifedipine treatment. n = 5. Data are represented as mean ±SEM. ( G ) Epithelial bud numbers of SMGs (E13.5) upon treatment with antagonists for different types of VDCCs: 2 μM w-Agatoxin IVA (Aga, P-type); 2 μM SNX 482 (SNX, R-type); 10 μM w-Conotoxin GVIA (Cono, N-type). n = 6. Data are represented as mean ±SEM. ( H ) Time-course changes of bud outline of developing SMG cultures. Arrowheads indicate the cleft initiation points. ( I ) Time-lapse images of epithelial rudiment cultures (E13) upon 100 μM nifedipine treatment. Arrowheads indicate cleft sites. Panels below indicate the bud numbers. Scale bars: 200 ( A , C ), 100 μm ( H , I ).

    Journal: Scientific Reports

    Article Title: Voltage-dependent Ca2+ channels promote branching morphogenesis of salivary glands by patterning differential growth

    doi: 10.1038/s41598-018-25957-w

    Figure Lengend Snippet: The effect of L-type voltage-dependent Ca 2+ channels (VDCCs) on branching morphogenesis. ( A ) Morphological changes of SMG cultures (E13.5) upon 500 μM LaCl 3 treatment. ( B ) Bud numbers of SMG cultures upon 500 μM LaCl 3 (La) and 1 M EGTA treatment. n = 7, Data are represented as mean ± SEM. ( C ) Representative images of SMG cultures treated with various Ca 2+ channel inhibitors. ( D ) Bud numbers of SMG cultures (E12) upon treatment with various Ca 2+ channel inhibitors. Nif: 100 μM nifedipine; Gd: 500 μM GdCl3; SKF: 10 μM SKF 96365, n = 7, Data are represented as mean ±SEM. ( E ) Bud numbers of SMG cultures (E13) upon different concentrations of nifedipine treatment for 48 h. n = 5. Data are represented as mean ± SEM. ( F ) Relative acinar size of SMGs (E13) upon different concentrations of nifedipine treatment. n = 5. Data are represented as mean ±SEM. ( G ) Epithelial bud numbers of SMGs (E13.5) upon treatment with antagonists for different types of VDCCs: 2 μM w-Agatoxin IVA (Aga, P-type); 2 μM SNX 482 (SNX, R-type); 10 μM w-Conotoxin GVIA (Cono, N-type). n = 6. Data are represented as mean ±SEM. ( H ) Time-course changes of bud outline of developing SMG cultures. Arrowheads indicate the cleft initiation points. ( I ) Time-lapse images of epithelial rudiment cultures (E13) upon 100 μM nifedipine treatment. Arrowheads indicate cleft sites. Panels below indicate the bud numbers. Scale bars: 200 ( A , C ), 100 μm ( H , I ).

    Article Snippet: The chemical reagents used in this study are as follows: 100 μM nifedipine (Sigma-Aldrich, St. Louis, MO; N7634); 500 μM gadolinium chloride (Sigma-Aldrich, G7532); 10 μM (Sigma-Aldrich, S7809); 1 M EGTA (Sigma-Aldrich, E4378); 500 μM lanthanum chloride (Sigma-Aldrich, L4131); 2 μM ω-Agatoxin IVA (Tocris, Bristol, UK; 2799); 2 μM SNX 482 (Tocris, 2799); 10 μM ω-Conotoxin GVIA (Alomone Labs, C-300); 10 μM U0126 (Sigma-Aldrich, U120); 50 mM potassium chloride (Sigma-Aldrich, P3911); 100 nM AP24534 (Tocris, 4274); 25 μM trifluoperazine dihydrochloride (Sigma Aldrich, T8516).

    Techniques:

    Inhibition of presynaptic voltage-gated Ca 2+ channels prevents axonal but not dendritic BDNF transport defects. (A) Representative images of MAP2 and AβO immunocytochemistry. Pretreatment of tau −/− neurons with 50 μM ω-agatoxin IVA (P/Q-type channel blocker), 100 μM ω-conotoxin GVIA (N-type channel blocker), 10 μM nimodipine, or 1.5 mM EGTA did not prevent AβO binding. (B) Inhibition of P/Q- and N-type VGCCs prevented axonal BDNF transport defects independent of tau. Consistent with the absence of L-type Ca 2+ channels in axons, nimodipine pretreatment did not prevent AβO-induced transport defects. Extracellular Ca 2+ chelation with EGTA precluded FAT disruption. (C) By contrast, in dendrites, inhibition of P/Q- and N-type VGCCs failed to prevent AβO-induced transport defects. Pretreatment with nimodipine or EGTA also did not prevent AβO-induced transport defects. Graphs show means ± SEM. A minimum of 15 cells from three different cultures were analyzed per condition; *** p

    Journal: Molecular Biology of the Cell

    Article Title: Dendritic and axonal mechanisms of Ca2+ elevation impair BDNF transport in Aβ oligomer–treated hippocampal neurons

    doi: 10.1091/mbc.E14-12-1612

    Figure Lengend Snippet: Inhibition of presynaptic voltage-gated Ca 2+ channels prevents axonal but not dendritic BDNF transport defects. (A) Representative images of MAP2 and AβO immunocytochemistry. Pretreatment of tau −/− neurons with 50 μM ω-agatoxin IVA (P/Q-type channel blocker), 100 μM ω-conotoxin GVIA (N-type channel blocker), 10 μM nimodipine, or 1.5 mM EGTA did not prevent AβO binding. (B) Inhibition of P/Q- and N-type VGCCs prevented axonal BDNF transport defects independent of tau. Consistent with the absence of L-type Ca 2+ channels in axons, nimodipine pretreatment did not prevent AβO-induced transport defects. Extracellular Ca 2+ chelation with EGTA precluded FAT disruption. (C) By contrast, in dendrites, inhibition of P/Q- and N-type VGCCs failed to prevent AβO-induced transport defects. Pretreatment with nimodipine or EGTA also did not prevent AβO-induced transport defects. Graphs show means ± SEM. A minimum of 15 cells from three different cultures were analyzed per condition; *** p

    Article Snippet: For all VGCC inhibition experiments, cells were incubated with 100 μM conotoxin GVIA (Alomone Labs, Jerusalem, Israel), 50 μM agatoxin IVA (Alomone Labs), or 10 μM nimodipine (Tocris Bioscience, Bristol, United Kingdom) for 30 min before AβO treatments.

    Techniques: Inhibition, Immunocytochemistry, Binding Assay