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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or <t>α1D</t> (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).
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( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or α1D (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).

Journal: Science Advances

Article Title: α-Synuclein expression is required for somatodendritic dopamine release and immediate early gene induction

doi: 10.1126/sciadv.ady6978

Figure Lengend Snippet: ( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or α1D (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).

Article Snippet: Primary antibodies include anti-actin (mouse monoclonal, Sigma-Aldrich, #A5441, RRID: AB_476744; 1:1000), anti–glyceraldehyde-3-phosphate dehydrogenase (mouse monoclonal, Proteintech, #60004-1-Ig, RRID: AB_2107436; 1:1000), anti–pCREB (Ser 133 , rabbit monoclonal, Cell Signaling Technology, #9198S; 1:500), anti-CREB1 (rabbit polyclonal, ABclonal, #A11064, RRID: AB_2758389; 1:500), anti–c-Fos (rabbit monoclonal, Cell Signaling Technology, #2250S, RRID: AB_2247211; 1:1000), anti-Ca v 1.2 α1C (rabbit polyclonal, Proteintech, #21774-1-AP, RRID: AB_2878918; 1:500), and anti-Ca v 1.3 α1D (rabbit polyclonal, Alomone Labs, #ACC-005, RRID: AB_2039775; 1:100).

Techniques: Cell Culture, Staining, Membrane