cacna1  (Alomone Labs)


Bioz Verified Symbol Alomone Labs is a verified supplier
Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92

    Structured Review

    Alomone Labs cacna1
    Cacna1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cacna1/product/Alomone Labs
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cacna1 - by Bioz Stars, 2022-08
    92/100 stars

    Images

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Alomone Labs snx 482
    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 <t>SNX-482</t> (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).
    Snx 482, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snx 482/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snx 482 - by Bioz Stars, 2022-08
    94/100 stars
      Buy from Supplier

    93
    Alomone Labs anti cav2 3 cacna1e antibody
    KCTDs directly bind <t>Cav2.3</t> in vitro and KCTD8 enhances currents through <t>Cav2.3</t> A Co-immunoprecipitation from total cell lysates of HEK293 cells transfected with Flag-tagged KCTDs and Cav2.3. Immunoprecipitation of Cav2.3 co-precipitated KCTD8 and KCTD12b, but not KCTD12. Input lanes (bottom) indicate expression of the tagged proteins in the cell lysates. B Whole-cell recordings from HEK293 cells stably expressing Cav2.3. Ba 2+ current densities measured in response to a single depolarizing voltage step from −80 to 10 mV were significantly increased in KCTD8 co-transfected cells. * P
    Anti Cav2 3 Cacna1e Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cav2 3 cacna1e antibody/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cav2 3 cacna1e antibody - by Bioz Stars, 2022-08
    93/100 stars
      Buy from Supplier

    Image Search Results


    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).

    Journal: Neuroscience

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

    doi: 10.1016/j.neuroscience.2009.09.013

    Figure Lengend 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).

    Article Snippet: All chemicals and drugs were obtained from Sigma (Saint Louis, MO), except for SNX-482, ω-Conotoxin-GVIA (ω-CTx-GVIA) and tACPD, which were obtained from Peptide International (Louisville, KY), Alomone Labs (Jerusalem, Israel), and Tocris (Ballwin, MO), respectively.

    Techniques: Transmission Assay, Inhibition, Produced

    SNX-482 inhibition of non-L-type I Ca in cultured midbrain DA neurons. A. Representative traces illustrating the inhibition of non-L-type I Ca by 100 nM SNX-482 (red). Cells were initially perfused with a bath solution containing 3 μM isradipine (ISR, black). Full block was obtained using 2 μM Cd 2+ (blue). Square pulses (50 ms) were applied to 0 mV from a holding potential of −70 mV (top) B. Current amplitude values plotted as a function of time. After stabilization of I Ca with ISR (black circles), 100 nM SNX-482 was applied. The remaining currents was blocked by 2 μM Cd 2+ . C. SNX-482 inhibition expressed as % of control I Ca after LTCC block using 3 μM ISR. D. Mean current amplitude at the end of ISR application and at the end of SNX-482 application. Data represent the means ± SEM for the indicated number of experiments (N=4). Statistical significance was determined using paired Student’s t-test: *** p

    Journal: bioRxiv

    Article Title: Alternative splicing of auxiliary β2-subunits stabilizes Cav2.3 Ca2+ channel activity in continuously active midbrain dopamine neurons

    doi: 10.1101/2021.02.10.430224

    Figure Lengend Snippet: SNX-482 inhibition of non-L-type I Ca in cultured midbrain DA neurons. A. Representative traces illustrating the inhibition of non-L-type I Ca by 100 nM SNX-482 (red). Cells were initially perfused with a bath solution containing 3 μM isradipine (ISR, black). Full block was obtained using 2 μM Cd 2+ (blue). Square pulses (50 ms) were applied to 0 mV from a holding potential of −70 mV (top) B. Current amplitude values plotted as a function of time. After stabilization of I Ca with ISR (black circles), 100 nM SNX-482 was applied. The remaining currents was blocked by 2 μM Cd 2+ . C. SNX-482 inhibition expressed as % of control I Ca after LTCC block using 3 μM ISR. D. Mean current amplitude at the end of ISR application and at the end of SNX-482 application. Data represent the means ± SEM for the indicated number of experiments (N=4). Statistical significance was determined using paired Student’s t-test: *** p

    Article Snippet: For inhibition experiments, 100 nM SNX-482 (Alomone, Cat # RTS-500 dissolved in ACSF) or 10 µM nifedipine (Alomone, Cat # N-120 diluted into ACSF from a freshly prepared 10 mM stock solution in DMSO) was bath applied (in ACSF).

    Techniques: Inhibition, Cell Culture, Blocking Assay

    SNX-482 effects on pacemaking of cultured mouse midbrain DA neurons. A. Representative recording of spontaneous firing activity of cultured midbrain dopaminergic neurons before, during and after the application (wash-out) of 100 nM SNX-482. Inset (bottom right): overlay of one single AP before (control) and during the application of 100 nM SNX-482. B. Firing frequency [Hz], coefficient of variation of the interspike interval [%], and AHP peak [mV] before (control) and during the application of 100 nM SNX-482. Data represent the means ± SEM for the indicated number of experiments (N=3). Statistical significance was determined using paired Student’s t-test.: *** p

    Journal: bioRxiv

    Article Title: Alternative splicing of auxiliary β2-subunits stabilizes Cav2.3 Ca2+ channel activity in continuously active midbrain dopamine neurons

    doi: 10.1101/2021.02.10.430224

    Figure Lengend Snippet: SNX-482 effects on pacemaking of cultured mouse midbrain DA neurons. A. Representative recording of spontaneous firing activity of cultured midbrain dopaminergic neurons before, during and after the application (wash-out) of 100 nM SNX-482. Inset (bottom right): overlay of one single AP before (control) and during the application of 100 nM SNX-482. B. Firing frequency [Hz], coefficient of variation of the interspike interval [%], and AHP peak [mV] before (control) and during the application of 100 nM SNX-482. Data represent the means ± SEM for the indicated number of experiments (N=3). Statistical significance was determined using paired Student’s t-test.: *** p

    Article Snippet: For inhibition experiments, 100 nM SNX-482 (Alomone, Cat # RTS-500 dissolved in ACSF) or 10 µM nifedipine (Alomone, Cat # N-120 diluted into ACSF from a freshly prepared 10 mM stock solution in DMSO) was bath applied (in ACSF).

    Techniques: Cell Culture, Activity Assay

    KCTDs directly bind Cav2.3 in vitro and KCTD8 enhances currents through Cav2.3 A Co-immunoprecipitation from total cell lysates of HEK293 cells transfected with Flag-tagged KCTDs and Cav2.3. Immunoprecipitation of Cav2.3 co-precipitated KCTD8 and KCTD12b, but not KCTD12. Input lanes (bottom) indicate expression of the tagged proteins in the cell lysates. B Whole-cell recordings from HEK293 cells stably expressing Cav2.3. Ba 2+ current densities measured in response to a single depolarizing voltage step from −80 to 10 mV were significantly increased in KCTD8 co-transfected cells. * P

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: KCTDs directly bind Cav2.3 in vitro and KCTD8 enhances currents through Cav2.3 A Co-immunoprecipitation from total cell lysates of HEK293 cells transfected with Flag-tagged KCTDs and Cav2.3. Immunoprecipitation of Cav2.3 co-precipitated KCTD8 and KCTD12b, but not KCTD12. Input lanes (bottom) indicate expression of the tagged proteins in the cell lysates. B Whole-cell recordings from HEK293 cells stably expressing Cav2.3. Ba 2+ current densities measured in response to a single depolarizing voltage step from −80 to 10 mV were significantly increased in KCTD8 co-transfected cells. * P

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: In Vitro, Immunoprecipitation, Transfection, Expressing, Stable Transfection

    Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals A Active zones double-labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle) or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN, bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. B Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8 or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. C Nearest neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. P values calculated via Kolmogorov-Smirnov test.

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals A Active zones double-labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle) or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN, bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. B Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8 or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. C Nearest neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. P values calculated via Kolmogorov-Smirnov test.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Labeling, Immunolabeling

    Absence of KCTD12b leads to a compensatory increase of KCTD8 inside the active zone of ventral MHb terminals A Example images of active zones containing Cav2.3 and either GABA B1 (left panels) or KCTD8 (right panels) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm B Quantification of relative densities for Cav2.3, KCTD8 and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicate the number of replicas used for quantification. ** indicate P

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: Absence of KCTD12b leads to a compensatory increase of KCTD8 inside the active zone of ventral MHb terminals A Example images of active zones containing Cav2.3 and either GABA B1 (left panels) or KCTD8 (right panels) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm B Quantification of relative densities for Cav2.3, KCTD8 and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicate the number of replicas used for quantification. ** indicate P

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay

    Expression and function of Cav2.3 and GABA B receptors at two parallel MHb-IPN pathways A Schematic drawing of the two MHb-IPN pathways. In red: the dorsal part of the MHb projects to the lateral subnuclei of the IPN. In blue: the ventral part of the MHb projects to the rostral/central subnuclei of the IPN. B Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. C Pharmacological inhibition of Cav2.3 with SNX-482 in whole-cell recordings of rostral IPN neurons. Left: example traces before and after the application of SNX-482; middle: example time course of EPSC amplitude reduction by SNX-482; right: averaged time course of relative EPSC amplitude reduction by SNX-482. EPSC amplitudes were reduced by 83% on average (n=9 cells/9 mice). D In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n=8 cells/4 mice). E Confocal image of GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. F In whole-cell recordings of rostral IPN neurons, activation of GBRs by baclofen (1 µM) produced a potentiation of electrically evoked EPSC amplitudes. Left: example EPSC traces before (black) and during the application of baclofen (red) and after washout of baclofen (blue); middle: example time course of EPSC amplitudes in one cell; right: averaged time course of relative EPSC amplitude change after baclofen (n=13 cells/9 mice). G Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n=10 cells/5 mice). Scale bars in panels ( B ) and ( E ) are 100 µm. Averaged data is presented as mean ± SEM.

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: Expression and function of Cav2.3 and GABA B receptors at two parallel MHb-IPN pathways A Schematic drawing of the two MHb-IPN pathways. In red: the dorsal part of the MHb projects to the lateral subnuclei of the IPN. In blue: the ventral part of the MHb projects to the rostral/central subnuclei of the IPN. B Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. C Pharmacological inhibition of Cav2.3 with SNX-482 in whole-cell recordings of rostral IPN neurons. Left: example traces before and after the application of SNX-482; middle: example time course of EPSC amplitude reduction by SNX-482; right: averaged time course of relative EPSC amplitude reduction by SNX-482. EPSC amplitudes were reduced by 83% on average (n=9 cells/9 mice). D In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n=8 cells/4 mice). E Confocal image of GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. F In whole-cell recordings of rostral IPN neurons, activation of GBRs by baclofen (1 µM) produced a potentiation of electrically evoked EPSC amplitudes. Left: example EPSC traces before (black) and during the application of baclofen (red) and after washout of baclofen (blue); middle: example time course of EPSC amplitudes in one cell; right: averaged time course of relative EPSC amplitude change after baclofen (n=13 cells/9 mice). G Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n=10 cells/5 mice). Scale bars in panels ( B ) and ( E ) are 100 µm. Averaged data is presented as mean ± SEM.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing, Immunofluorescence, Inhibition, Mouse Assay, Activation Assay, Produced

    SDS-digested freeze-fracture replica labeling confirms Cav2.3 in the active zone of medial habenula terminals in the IPN A Example image of a grid-glued replica containing the whole IPN. White line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm B Example image of a presynaptic P face and a postsynaptic E face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. C Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. D Left panel: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST and neurexin. Right panel: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone marker labeling. Scale bars: 100 nm. F Left panel: quantification of Cav2.3 labeling densities in the presynaptic P face in WT and Cav2.3 KO mice. *** indicates P

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: SDS-digested freeze-fracture replica labeling confirms Cav2.3 in the active zone of medial habenula terminals in the IPN A Example image of a grid-glued replica containing the whole IPN. White line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm B Example image of a presynaptic P face and a postsynaptic E face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. C Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. D Left panel: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST and neurexin. Right panel: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone marker labeling. Scale bars: 100 nm. F Left panel: quantification of Cav2.3 labeling densities in the presynaptic P face in WT and Cav2.3 KO mice. *** indicates P

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Labeling, Marker, Mouse Assay

    Quantification of the sub-synaptic localization of presynaptic Cav2.3, GBRs and KCTDs along both MHb-IPN pathways Transmission electron microscopy images of 70 nm−thick sections following pre-embedding immunolabeled IPN slices for Cav2.3 ( A ), GABA B1 ( B ), KCTD8 ( C ), KCTD12 ( D ) and KCTD12b ( E ) from synapses in the rostral (left images) and lateral (right image) IPN subnuclei. Scale bars: 200 nm. Graph on the right displays quantification of relative and absolute silver-enhanced gold particle densities in the active zone and at distances of 50 − 200 nm from the edge of the active zone (50 nm bins). F Absolute labeling densities are summarized for synapses in the rostral (left panel) and lateral IPN (right panel). Note absence of KCTD12 and KCTD12b particles in presynaptic terminals inside the lateral IPN subnuclei. KCTD12 was not included in panel F because of predominantly postsynaptic localization inside the rostral IPN. Data was pooled from two animals, showing no significant difference in gold particle distribution patterns with Kolmogorov-Smirnov test (see Supplementary Figure S1).

    Journal: bioRxiv

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.1101/2020.04.16.045112

    Figure Lengend Snippet: Quantification of the sub-synaptic localization of presynaptic Cav2.3, GBRs and KCTDs along both MHb-IPN pathways Transmission electron microscopy images of 70 nm−thick sections following pre-embedding immunolabeled IPN slices for Cav2.3 ( A ), GABA B1 ( B ), KCTD8 ( C ), KCTD12 ( D ) and KCTD12b ( E ) from synapses in the rostral (left images) and lateral (right image) IPN subnuclei. Scale bars: 200 nm. Graph on the right displays quantification of relative and absolute silver-enhanced gold particle densities in the active zone and at distances of 50 − 200 nm from the edge of the active zone (50 nm bins). F Absolute labeling densities are summarized for synapses in the rostral (left panel) and lateral IPN (right panel). Note absence of KCTD12 and KCTD12b particles in presynaptic terminals inside the lateral IPN subnuclei. KCTD12 was not included in panel F because of predominantly postsynaptic localization inside the rostral IPN. Data was pooled from two animals, showing no significant difference in gold particle distribution patterns with Kolmogorov-Smirnov test (see Supplementary Figure S1).

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 h at 4 °C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Transmission Assay, Electron Microscopy, Immunolabeling, Labeling

    Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits. Fold increase of KCTD signal at the plasma membrane (PM) compared to the averaged KCTD signal in total membrane (TM). Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits.

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits. Fold increase of KCTD signal at the plasma membrane (PM) compared to the averaged KCTD signal in total membrane (TM). Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing

    Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals. ( A ) Active zones double labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle), or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN; bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. ( B ) Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8, or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. ( C ) Nearest-neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. p-values calculated via Kolmogorov–Smirnov test. Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals.

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals. ( A ) Active zones double labeled for Cav2.3 and either GABA B1 (left), KCTD8 (middle), or KCTD12b (right) in IPN replicas. Top row images are from presynaptic terminals in the rostral IPN; bottom row images are from presynaptic terminals in the lateral IPN. Scale bar: 100 nm. ( B ) Quantification of active zone immunolabeling in the rostral and lateral IPN. With the exception of the absence of KCTD12b in lateral IPN terminals, absolute particle numbers per active zone (left graph) and particle densities (middle graph) are comparable between MHb terminals in the rostral and lateral IPN. Right graph: Over 97% of active zones positive for Cav2.3 labeling also show labeling for one of the other molecules (GABA B1 , KCTD8, or KCTD12b), suggesting co-localization of all presynaptic molecules inside the same active zone. Numbers inside the bars indicate the number of replicas used for each quantification. ( C ) Nearest-neighbor distance (NND) for all presynaptic molecules in MHb terminals inside the rostral and lateral IPN based on the real (black line) and simulated random distribution (blue line). Smaller NND values in real distributions compared to simulation suggest clustering of all presynaptic molecules. p-values calculated via Kolmogorov–Smirnov test. Co-localization of Cav2.3 with GBR and KCTDs in the active zone of medial habenula terminals.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Labeling, Immunolabeling

    Allen Brain Atlas images showing lack of expression of Cav2.1 ( http://mouse.brain-map.org/gene/show/12071 ) and Cav2.2 ( http://mouse.brain-map.org/gene/show/12072 ) mRNA in MHb neurons, whereas Cav2.3 shows strong expression in MHb neurons ( http://mouse.brain-map.org/gene/show/12075 ).

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Allen Brain Atlas images showing lack of expression of Cav2.1 ( http://mouse.brain-map.org/gene/show/12071 ) and Cav2.2 ( http://mouse.brain-map.org/gene/show/12072 ) mRNA in MHb neurons, whereas Cav2.3 shows strong expression in MHb neurons ( http://mouse.brain-map.org/gene/show/12075 ).

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing

    SDS-digested freeze-fracture replica labeling confirms Cav2.3 in the active zone of medial habenula terminals in the IPN. ( A ) Example image of a grid-glued replica containing the whole IPN. White dashed line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm. ( B ) Example image of a presynaptic P-face and a postsynaptic E-face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. ( C ) Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. ( D ) Left: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST, and neurexin. Right: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone-marker labeling. Scale bars: 100 nm. ( F ) Left: quantification of Cav2.3 labeling densities in the presynaptic P-face in WT and Cav2.3 KO mice. ***p

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: SDS-digested freeze-fracture replica labeling confirms Cav2.3 in the active zone of medial habenula terminals in the IPN. ( A ) Example image of a grid-glued replica containing the whole IPN. White dashed line indicates demarcation of rostral/central and lateral subnuclei. Scale bar: 20 µm. ( B ) Example image of a presynaptic P-face and a postsynaptic E-face of a habenular synapse in the rostral IPN that was double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold). Scale bar: 100 nm. ( C ) Example image of a similar synaptic profile double labeled with antibodies against AMPA receptors (10 nm gold) and Cav2.3 (5 nm gold) in the rostral IPN of a Cav2.3 KO mouse. Scale bar: 100 nm. ( D ) Left: double labeling of a WT carbon-only replica with antibodies against Cav2.3 (5 nm gold) and a mixture of active zone proteins (2 nm gold), including RIM1/2, CAST, and neurexin. Right: the same image with additional coloring of 2 nm (red) and 5 nm (blue) particles and demarcation of the active zone area based on active zone-marker labeling. Scale bars: 100 nm. ( F ) Left: quantification of Cav2.3 labeling densities in the presynaptic P-face in WT and Cav2.3 KO mice. ***p

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Labeling, Marker, Mouse Assay

    Comparison of distribution of silver-enhanced immunogold particles outside the active zone for Cav2.3, KCTD8, KCTD12, and KCTD12b in MHb terminals inside the rostral and lateral IPN. Distribution of particles in samples from two mice was not significantly different. p-values above graph resulted from Kolmogorov–Smirnov test. M1 = mouse 1, M2 = mouse 2, n indicates the total number of particles analyzed. Comparison of distribution of silver-enhanced immunogold particles outside the active zone for Cav2.3, KCTD8, KCTD12, and KCTD12b in MHb terminals inside the rostral and lateral IPN.

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Comparison of distribution of silver-enhanced immunogold particles outside the active zone for Cav2.3, KCTD8, KCTD12, and KCTD12b in MHb terminals inside the rostral and lateral IPN. Distribution of particles in samples from two mice was not significantly different. p-values above graph resulted from Kolmogorov–Smirnov test. M1 = mouse 1, M2 = mouse 2, n indicates the total number of particles analyzed. Comparison of distribution of silver-enhanced immunogold particles outside the active zone for Cav2.3, KCTD8, KCTD12, and KCTD12b in MHb terminals inside the rostral and lateral IPN.

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay

    Absence of KCTD12b leads to a compensatory increase of KCTD8 inside the active zone of ventral MHb terminals. ( A ) Example images of active zones containing Cav2.3 and either GABA B1 (left) or KCTD8 (right) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm. ( B ) Quantification of relative densities for Cav2.3, KCTD8, and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicates the number of replicas used for quantification. **p

    Journal: eLife

    Article Title: GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Absence of KCTD12b leads to a compensatory increase of KCTD8 inside the active zone of ventral MHb terminals. ( A ) Example images of active zones containing Cav2.3 and either GABA B1 (left) or KCTD8 (right) in replicas of WT (upper row) and KCTD12b KO IPN tissue (lower row). Scale bars: 100 nm. ( B ) Quantification of relative densities for Cav2.3, KCTD8, and GABA B1 in active zones located in the rostral IPN of WT and KCTD12b KO mice. Densities were normalized to the average density in MHb terminals inside the lateral IPN of the same replica. The number inside the bars indicates the number of replicas used for quantification. **p

    Article Snippet: Thereafter, lysates were incubated by rotating for 16 hr at 4°C in the presence of 2.5 μl of 0.3 μg/μl anti-Cav2.3 (CACNA1E) antibody (ACC-006, Alomone Labs, Jerusalem, Israel).

    Techniques: Mouse Assay