t type voltage gated calcium channel 3 3  (Alomone Labs)


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    Alomone Labs t type voltage gated calcium channel 3 3
    Summary of primary antibodies and dilutions for the immunolabeling
    T Type Voltage Gated Calcium Channel 3 3, 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/t type voltage gated calcium channel 3 3/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    t type voltage gated calcium channel 3 3 - by Bioz Stars, 2023-06
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    1) Product Images from "Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei"

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    Journal: Brain Structure & Function

    doi: 10.1007/s00429-021-02315-7


    Figure Legend Snippet: Summary of primary antibodies and dilutions for the immunolabeling

    Techniques Used: Marker

    anti cav2 3 antibody  (Alomone Labs)


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    Alomone Labs anti cav2 3 antibody
    Anti Cav2 3 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t type voltage gated calcium channel 3 3  (Alomone Labs)


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    Alomone Labs t type voltage gated calcium channel 3 3
    Summary of primary antibodies and dilutions for the immunolabeling
    T Type Voltage Gated Calcium Channel 3 3, 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
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    Average 93 stars, based on 1 article reviews
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    1) Product Images from "Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei"

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    Journal: Brain Structure & Function

    doi: 10.1007/s00429-021-02315-7


    Figure Legend Snippet: Summary of primary antibodies and dilutions for the immunolabeling

    Techniques Used: Marker

    t type voltage gated calcium channel 3 2  (Alomone Labs)


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    Alomone Labs t type voltage gated calcium channel 3 2
    Summary of primary antibodies and dilutions for the immunolabeling
    T Type Voltage Gated Calcium Channel 3 2, 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/t type voltage gated calcium channel 3 2/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    1) Product Images from "Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei"

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    Journal: Brain Structure & Function

    doi: 10.1007/s00429-021-02315-7


    Figure Legend Snippet: Summary of primary antibodies and dilutions for the immunolabeling

    Techniques Used: Marker

    anti cav2 3  (Alomone Labs)


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    Alomone Labs anti cav2 3
    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of <t>Cav2.3</t> immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
    Anti Cav2 3, 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/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cav2 3 - by Bioz Stars, 2023-06
    93/100 stars

    Images

    1) Product Images from "GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals"

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

    Journal: eLife

    doi: 10.7554/eLife.68274

    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
    Figure Legend Snippet: ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.

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

    ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.
    Figure Legend Snippet: ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

    Techniques Used: Immunofluorescence, Immunoprecipitation, Transfection, Expressing, Stable Transfection, Activation Assay, In Vitro

    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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.
    Figure Legend Snippet: 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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.

    Techniques Used: Transmission Assay, Electron Microscopy, Immunolabeling, Labeling

    Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .
    Figure Legend Snippet: Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .

    Techniques Used:

    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 ).
    Figure Legend 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 ).

    Techniques Used: Expressing


    Figure Legend Snippet:

    Techniques Used: Stable Transfection, Expressing, Concentration Assay, Recombinant, Software

    anti cav2 3 cacna1e antibody  (Alomone Labs)


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    Alomone Labs anti cav2 3 cacna1e antibody
    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image <t>of</t> <t>Cav2.3</t> immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
    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, 2023-06
    93/100 stars

    Images

    1) Product Images from "GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals"

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

    Journal: eLife

    doi: 10.7554/eLife.68274

    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
    Figure Legend Snippet: ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.

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

    ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.
    Figure Legend Snippet: ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

    Techniques Used: Immunofluorescence, Immunoprecipitation, Transfection, Expressing, Stable Transfection, Activation Assay, In Vitro

    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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.
    Figure Legend Snippet: 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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.

    Techniques Used: Transmission Assay, Electron Microscopy, Immunolabeling, Labeling

    Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .
    Figure Legend Snippet: Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .

    Techniques Used:

    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 ).
    Figure Legend 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 ).

    Techniques Used: Expressing


    Figure Legend Snippet:

    Techniques Used: Stable Transfection, Expressing, Concentration Assay, Recombinant, Software

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    Alomone Labs anti cav2 3 cacna1e antibody
    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image <t>of</t> <t>Cav2.3</t> immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
    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
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    1) Product Images from "GABA B receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals"

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

    Journal: eLife

    doi: 10.7554/eLife.68274

    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
    Figure Legend Snippet: ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.

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

    ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.
    Figure Legend Snippet: ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

    Techniques Used: Immunofluorescence, Immunoprecipitation, Transfection, Expressing, Stable Transfection, Activation Assay, In Vitro

    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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.
    Figure Legend Snippet: 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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.

    Techniques Used: Transmission Assay, Electron Microscopy, Immunolabeling, Labeling

    Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .
    Figure Legend Snippet: Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .

    Techniques Used:

    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 ).
    Figure Legend 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 ).

    Techniques Used: Expressing


    Figure Legend Snippet:

    Techniques Used: Stable Transfection, Expressing, Concentration Assay, Recombinant, Software

    cav2 3  (Alomone Labs)


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    Alomone Labs cav2 3
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    anti cav2 3  (Alomone Labs)


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    immunoblotting  (Alomone Labs)


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    Alomone Labs immunoblotting
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    cav2 3  (Alomone Labs)


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    Structured Review

    Alomone Labs cav2 3
    Antibodies used in this study
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    1) Product Images from "Phosphorylated CRMP2 Regulates Spinal Nociceptive Neurotransmission"

    Article Title: Phosphorylated CRMP2 Regulates Spinal Nociceptive Neurotransmission

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-018-1445-6

    Antibodies used in this study
    Figure Legend Snippet: Antibodies used in this study

    Techniques Used:

    (A) Immunoblots showing the integrity of the synaptic fractionation from lumbar dorsal horn of the spinal cord. The non-post synaptic density (PSD) fraction was enriched in the pre-synaptic marker Synaptophysin and the PSD fraction was enriched in the post-synaptic marker PSD95. The membrane-associated protein flotillin was used as a loading control. (B) Immunoblots showing the presynaptic CRMP2 expression, CRMP2 p522, CaV2.2 and NaV1.7 levels in the lumbar dorsal horn of the spinal cord of animals having received (S)-Lacosamide (20μg in 5 μl, i.th.) compared to vehicle (0.1% DMSO in saline). Spinal cords were harvested from rats 1 hour following treatment. Flotilin is used as a loading control. (C) Bar graph showing decreased CRMP2 p522 concomitant with decreased CaV2.2 and NaV1.7 levels at the presynaptic sites of lumbar dorsal horn of the spinal cord in (S)-lacosamide treated animals. Mean ± SEM, *p<0.05, MannWhitney compared to the contralateral side.
    Figure Legend Snippet: (A) Immunoblots showing the integrity of the synaptic fractionation from lumbar dorsal horn of the spinal cord. The non-post synaptic density (PSD) fraction was enriched in the pre-synaptic marker Synaptophysin and the PSD fraction was enriched in the post-synaptic marker PSD95. The membrane-associated protein flotillin was used as a loading control. (B) Immunoblots showing the presynaptic CRMP2 expression, CRMP2 p522, CaV2.2 and NaV1.7 levels in the lumbar dorsal horn of the spinal cord of animals having received (S)-Lacosamide (20μg in 5 μl, i.th.) compared to vehicle (0.1% DMSO in saline). Spinal cords were harvested from rats 1 hour following treatment. Flotilin is used as a loading control. (C) Bar graph showing decreased CRMP2 p522 concomitant with decreased CaV2.2 and NaV1.7 levels at the presynaptic sites of lumbar dorsal horn of the spinal cord in (S)-lacosamide treated animals. Mean ± SEM, *p<0.05, MannWhitney compared to the contralateral side.

    Techniques Used: Western Blot, Fractionation, Marker, Expressing

    Adult rats (n=6 per group) were used for these experiments 10 days after the SNI. (A) Representative immunoblots showing the presynaptic expression of CRMP2, CRMP2 pS522, CaV2.2, NaV1.7, CaV2.3 and cannabinoid receptor 1 (CB1R) in the lumbar dorsal horn of the spinal cord of SNI rats injected with (S)-Lacosamide (20μg in 5 μl, i.th.) compared to vehicle (0.1% DMSO in saline). Spinal cords (ipsilateral (i.e., injured) and contralateral (i.e., non-injured) side)) were harvested from rats 1 hour following treatment. Flotilin is used as a loading control. (B) Bar graph showing decreased CRMP2 p522 concomitant with decreased CaV2.2 and NaV1.7 levels at the presynaptic sites of lumbar dorsal horn of the spinal cord in (S)-lacosamide treated animals. Mean ± SEM, *p<0.05, Kruskal-Wallis compared to the DMSO-treated contralateral side. #p<0.05, Kruskal-Wallis compared to the DMSOtreated ipsilateral side.
    Figure Legend Snippet: Adult rats (n=6 per group) were used for these experiments 10 days after the SNI. (A) Representative immunoblots showing the presynaptic expression of CRMP2, CRMP2 pS522, CaV2.2, NaV1.7, CaV2.3 and cannabinoid receptor 1 (CB1R) in the lumbar dorsal horn of the spinal cord of SNI rats injected with (S)-Lacosamide (20μg in 5 μl, i.th.) compared to vehicle (0.1% DMSO in saline). Spinal cords (ipsilateral (i.e., injured) and contralateral (i.e., non-injured) side)) were harvested from rats 1 hour following treatment. Flotilin is used as a loading control. (B) Bar graph showing decreased CRMP2 p522 concomitant with decreased CaV2.2 and NaV1.7 levels at the presynaptic sites of lumbar dorsal horn of the spinal cord in (S)-lacosamide treated animals. Mean ± SEM, *p<0.05, Kruskal-Wallis compared to the DMSO-treated contralateral side. #p<0.05, Kruskal-Wallis compared to the DMSOtreated ipsilateral side.

    Techniques Used: Western Blot, Expressing, Injection

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    Alomone Labs t type voltage gated calcium channel 3 3
    Summary of primary antibodies and dilutions for the immunolabeling
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    Alomone Labs anti cav2 3 antibody
    Summary of primary antibodies and dilutions for the immunolabeling
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    Alomone Labs anti cav2 3
    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of <t>Cav2.3</t> immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
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    Alomone Labs anti cav2 3 cacna1e antibody
    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image <t>of</t> <t>Cav2.3</t> immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
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    Alomone Labs cav2 3
    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image <t>of</t> <t>Cav2.3</t> immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
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    Alomone Labs immunoblotting
    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image <t>of</t> <t>Cav2.3</t> immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.
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    Image Search Results


    Journal: Brain Structure & Function

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    doi: 10.1007/s00429-021-02315-7

    Figure Lengend Snippet: Summary of primary antibodies and dilutions for the immunolabeling

    Article Snippet: Cav3.3 , Rabbit/polyclonal , T-type voltage-gated calcium channel 3.3 , Alomone Labs Jerusalem BioPark (JBP) , AB_2039783 , 1:1000 (IHC).

    Techniques: Marker

    Journal: Brain Structure & Function

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    doi: 10.1007/s00429-021-02315-7

    Figure Lengend Snippet: Summary of primary antibodies and dilutions for the immunolabeling

    Article Snippet: Cav3.2 , Rabbit/polyclonal , T-type voltage-gated calcium channel 3.2 , Alomone Labs Jerusalem BioPark (JBP) , AB_2039781 , 1:1000 (IHC).

    Techniques: Marker

    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.

    Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA) for 2 hr at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10,000).

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

    ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

    Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA) for 2 hr at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10,000).

    Techniques: Immunofluorescence, Immunoprecipitation, Transfection, Expressing, Stable Transfection, Activation Assay, In Vitro

    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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: 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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.

    Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA) for 2 hr at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10,000).

    Techniques: Transmission Assay, Electron Microscopy, Immunolabeling, Labeling

    Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .

    Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA) for 2 hr at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10,000).

    Techniques:

    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: GABA B 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: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA) for 2 hr at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10,000).

    Techniques: Expressing

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet:

    Article Snippet: For immunoblotting analysis, proteins were transferred to 0.45 μM polivinylidene fluoride membranes (Milipore, Burlington, MA) for 2 hr at 200 mA and probed with the primary antibodies rabbit anti-Flag (F7425, Sigma) and anti-Cav2.3 (ACC-006, Alomone Labs) in combination with peroxidase-coupled secondary donkey anti-rabbit antibodies (NA934, GE Healthcare, 1:10,000).

    Techniques: Stable Transfection, Expressing, Concentration Assay, Recombinant, Software

    ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: ( 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 GABA B1 immunofluorescence signal indicates the presence of GABA B receptors (GBRs) in all IPN subnuclei. ( C ) 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). ( D ) Baclofen reduced the amplitude of light-evoked glutamatergic EPSCs in lateral IPN neurons (n = 10 cells/5 mice). ( E ) Confocal image of Cav2.3 immunofluorescence signal indicates Cav2.3 presence in MHb axonal projections of both MHb-IPN pathways. ( F ) 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). ( G ) In Tac1-ChR2-EYFP mice, SNX-482 reduced light-evoked glutamatergic EPSC amplitudes on average by 52% (n = 8 cells/4 mice). ( H, I ) Left: Positions of recording and stimulating electrodes in acute thick-slice preparations from Cav2.3 KO mice. The stimulating electrode was placed on the fasciculus retroflexus just below the MHb, 2–3 mm from the recording sites. The position of the stimulating electrode and the stimulation intensity remained unchanged between rostral and lateral IPN recordings. Right: 10 Hz EPSC traces of all recorded neurons (9 cells/3 mice). ( J ) Cumulative EPSC amplitude plot shows significantly higher EPSC amplitudes in lateral compared to rostral IPN neurons. ***p<0.0001 two-way ANOVA; ( K ) FR stimulation and lateral IPN recording in 1 mm thick slice of wild-type mice. Sequential application of SNX-482, ω-conotoxin GVIA, and CdCl 2 . ( L ) Time course overlay of ( F ), ( G ), and ( K ) to compare SNX-482 time courses between minutes 0 and 25. For simplicity, application of ω-conotoxin GVIA starting from minute 15 in ( K ) not indicated in the graph. ***p<0.0001 two-way ANOVA with Tukey’s post hoc test. Scale bars in( B ), ( E ), ( H ), and ( I ) are 100 µm. Averaged data is presented as mean ± SEM. See also . Figure 1—source data 1. Expression and function of GABA B receptors and Cav2.3 at two parallel MHb-IPN pathways.

    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: Immunofluorescence, Activation Assay, Produced, Inhibition, Expressing

    ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: ( A–C ) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. ( D ) Co-immunoprecipitation from total cell lysates of HEK293T 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. ( E ) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. ( F ) 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<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. ( G ) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. ( H ) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also . Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

    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: Immunofluorescence, Immunoprecipitation, Transfection, Expressing, Stable Transfection, Activation Assay, In Vitro

    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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: 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) and lateral IPN (right). Note the 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 and ). Figure 3—source data 1. Quantification of the presynaptic localization of Cav2.3, GBRs, and KCTDs along the ventral and dorsal MHb-IPN pathways.

    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: Transmission Assay, Electron Microscopy, Immunolabeling, Labeling

    Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet: Schematic representation of the distribution and function of Cav2.3, GBRs, and KCTDs in ventral MHb terminals of WT and KCTD12b KO mice. Left: In WT terminals, the active zone contains Cav2.3 and hetero-pentameric rings comprising KCTD12b in excess over KCTD8, whereas KCTD8 and GBRs are located peri-synaptically. Right: In absence of KCTD12b, KCTD8 invades the active zone and compensates for the loss of KCTD12b, resulting in increased release probability, potentially via increased Ca 2+ influx. See .

    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:

    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: GABA B 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

    Journal: eLife

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

    doi: 10.7554/eLife.68274

    Figure Lengend Snippet:

    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: Stable Transfection, Expressing, Concentration Assay, Recombinant, Software