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The role of voltage-gated calcium channels in neurotransmitter phenotype specification: Coexpression and functional analysis in Xenopus laevis.

Lewis BB, Miller LE, Herbst WA, Saha MS - J. Comp. Neurol. (2014)

Bottom Line: Calcium activity was also analyzed on a single-cell level, and spike frequency was correlated with the expression of VGCC α1 subunits in cell culture.Cells expressing Cav 2.1 and Cav 2.2 displayed increased calcium spiking compared with cells not expressing this marker.The VGCC antagonist diltiazem and agonist (-)BayK 8644 were used to manipulate calcium activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of William and Mary, Williamsburg, Virginia, 23185.

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Pharmacological disruption of VGCC activity and neurotransmitter phenotype. Embryos were dissected at neural plate, neural fold, and neural tube stages, incubated in either a VGCC antagonist (diltiazem) or a VGCC agonist (BayK 8644), and then assayed for expression of the glutamatergic marker xVGlut1 or the GABAergic marker xGAD67. The number of cells expressing the gene of interest (termed “positives”) and the number of cells not expressing the gene of interest (termed “negatives”) for representative fields of view were recorded. A two-sample z-test was used to compare the percent positive between treatment groups and controls. P ≤ 0.05 was recorded as significant. A: xVGlut1 expression of diltiazem-exposed cells. B: xGAD67 expression of diltiazem-exposed cells. C: xVGlut1 expression of BayK 8644-exposed cells. D: xGAD67 expression of BayK 8644-exposed cells. For diltiazem exposures with xVGlut1 expression, n = 1,398 cells (0 μM, neural plate), 1,069 cells (10 μM, neural plate), 936 cells (100 μM, neural plate), 909 cells (0 μM, neural fold), 1,003 cells (10 μM, neural fold), 1,160 cells (100 μM, neural fold), 2,419 cells (0 μM, neural tube), 1,147 cells (10 μM, neural tube), and 160 cells (100 μM, neural tube). For diltizem exposures with xGAD67 expression, n = 1,259 cells (0 μM, neural plate), 843 cells (10 μM, neural plate), 658 cells (100 μM, neural plate), 576 cells (0 μM, neural fold), 1,523 cells (10 μM, neural fold), 1,441 cells (100 μM, neural fold), 1,486 cells (0 μM, neural tube), 1,509 cells (10 μM, neural tube), and 1,171 cells (100 μM, neural tube). For BayK 8644 exposures with xVGlut1 expression, n = 1,178 cells (0 μM, neural plate), 1,817 cells (10 μM, neural plate), 1,755 cells (100 μM, neural plate), 1,222 cells (0 μM, neural fold), 1,249 cells (10 μM, neural fold), 1,126 cells (100 μM, neural fold), 1,455 cells (0 μM, neural tube), 1,384 cells (10 μM, neural tube), and 1,519 cells (100 μM, neural tube). For BayK 8644 exposures with xGAD67 expression, n = 851 cells (0 μM, neural plate), 1,777 cells (10 μM, neural plate), 1,075 cells (100 μM, neural plate), 1,018 cells (0 μM, neural fold), 674 cells (10 μM, neural fold), 745 cells (100 μM, neural fold), 1,560 cells (0 μM, neural tube), 1,469 cells (10 μM, neural tube), and 1,001 cells (100 μM, neural tube).
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fig08: Pharmacological disruption of VGCC activity and neurotransmitter phenotype. Embryos were dissected at neural plate, neural fold, and neural tube stages, incubated in either a VGCC antagonist (diltiazem) or a VGCC agonist (BayK 8644), and then assayed for expression of the glutamatergic marker xVGlut1 or the GABAergic marker xGAD67. The number of cells expressing the gene of interest (termed “positives”) and the number of cells not expressing the gene of interest (termed “negatives”) for representative fields of view were recorded. A two-sample z-test was used to compare the percent positive between treatment groups and controls. P ≤ 0.05 was recorded as significant. A: xVGlut1 expression of diltiazem-exposed cells. B: xGAD67 expression of diltiazem-exposed cells. C: xVGlut1 expression of BayK 8644-exposed cells. D: xGAD67 expression of BayK 8644-exposed cells. For diltiazem exposures with xVGlut1 expression, n = 1,398 cells (0 μM, neural plate), 1,069 cells (10 μM, neural plate), 936 cells (100 μM, neural plate), 909 cells (0 μM, neural fold), 1,003 cells (10 μM, neural fold), 1,160 cells (100 μM, neural fold), 2,419 cells (0 μM, neural tube), 1,147 cells (10 μM, neural tube), and 160 cells (100 μM, neural tube). For diltizem exposures with xGAD67 expression, n = 1,259 cells (0 μM, neural plate), 843 cells (10 μM, neural plate), 658 cells (100 μM, neural plate), 576 cells (0 μM, neural fold), 1,523 cells (10 μM, neural fold), 1,441 cells (100 μM, neural fold), 1,486 cells (0 μM, neural tube), 1,509 cells (10 μM, neural tube), and 1,171 cells (100 μM, neural tube). For BayK 8644 exposures with xVGlut1 expression, n = 1,178 cells (0 μM, neural plate), 1,817 cells (10 μM, neural plate), 1,755 cells (100 μM, neural plate), 1,222 cells (0 μM, neural fold), 1,249 cells (10 μM, neural fold), 1,126 cells (100 μM, neural fold), 1,455 cells (0 μM, neural tube), 1,384 cells (10 μM, neural tube), and 1,519 cells (100 μM, neural tube). For BayK 8644 exposures with xGAD67 expression, n = 851 cells (0 μM, neural plate), 1,777 cells (10 μM, neural plate), 1,075 cells (100 μM, neural plate), 1,018 cells (0 μM, neural fold), 674 cells (10 μM, neural fold), 745 cells (100 μM, neural fold), 1,560 cells (0 μM, neural tube), 1,469 cells (10 μM, neural tube), and 1,001 cells (100 μM, neural tube).

Mentions: Because VGCCs are expressed in neural tissue during early development and certain VGCCs are correlated with specific spike frequencies in cell culture, the effect of the VGCC blocker diltiazem and agonist (−)Bayk 8644 on neurotransmitter phenotype specification was examined next. The percentage of cells expressing xVGlut1 or xGAD67 was assessed among treatment conditions, and a two-sample z-test was used to compare the percentage of positive cells in cultures exposed to diltiazem or (−)BayK 8644 with untreated controls. Exposure to the VGCC antagonist leads to an increase in glutamatergic neurons and a decrease in GABAergic neurons (Fig. 8A,B). Exposure to the VGCC agonist significantly decreases the percentage of cells expressing glutamatergic cells while having no effect on the number of GABAergic cells. (Fig. 8C,D)


The role of voltage-gated calcium channels in neurotransmitter phenotype specification: Coexpression and functional analysis in Xenopus laevis.

Lewis BB, Miller LE, Herbst WA, Saha MS - J. Comp. Neurol. (2014)

Pharmacological disruption of VGCC activity and neurotransmitter phenotype. Embryos were dissected at neural plate, neural fold, and neural tube stages, incubated in either a VGCC antagonist (diltiazem) or a VGCC agonist (BayK 8644), and then assayed for expression of the glutamatergic marker xVGlut1 or the GABAergic marker xGAD67. The number of cells expressing the gene of interest (termed “positives”) and the number of cells not expressing the gene of interest (termed “negatives”) for representative fields of view were recorded. A two-sample z-test was used to compare the percent positive between treatment groups and controls. P ≤ 0.05 was recorded as significant. A: xVGlut1 expression of diltiazem-exposed cells. B: xGAD67 expression of diltiazem-exposed cells. C: xVGlut1 expression of BayK 8644-exposed cells. D: xGAD67 expression of BayK 8644-exposed cells. For diltiazem exposures with xVGlut1 expression, n = 1,398 cells (0 μM, neural plate), 1,069 cells (10 μM, neural plate), 936 cells (100 μM, neural plate), 909 cells (0 μM, neural fold), 1,003 cells (10 μM, neural fold), 1,160 cells (100 μM, neural fold), 2,419 cells (0 μM, neural tube), 1,147 cells (10 μM, neural tube), and 160 cells (100 μM, neural tube). For diltizem exposures with xGAD67 expression, n = 1,259 cells (0 μM, neural plate), 843 cells (10 μM, neural plate), 658 cells (100 μM, neural plate), 576 cells (0 μM, neural fold), 1,523 cells (10 μM, neural fold), 1,441 cells (100 μM, neural fold), 1,486 cells (0 μM, neural tube), 1,509 cells (10 μM, neural tube), and 1,171 cells (100 μM, neural tube). For BayK 8644 exposures with xVGlut1 expression, n = 1,178 cells (0 μM, neural plate), 1,817 cells (10 μM, neural plate), 1,755 cells (100 μM, neural plate), 1,222 cells (0 μM, neural fold), 1,249 cells (10 μM, neural fold), 1,126 cells (100 μM, neural fold), 1,455 cells (0 μM, neural tube), 1,384 cells (10 μM, neural tube), and 1,519 cells (100 μM, neural tube). For BayK 8644 exposures with xGAD67 expression, n = 851 cells (0 μM, neural plate), 1,777 cells (10 μM, neural plate), 1,075 cells (100 μM, neural plate), 1,018 cells (0 μM, neural fold), 674 cells (10 μM, neural fold), 745 cells (100 μM, neural fold), 1,560 cells (0 μM, neural tube), 1,469 cells (10 μM, neural tube), and 1,001 cells (100 μM, neural tube).
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fig08: Pharmacological disruption of VGCC activity and neurotransmitter phenotype. Embryos were dissected at neural plate, neural fold, and neural tube stages, incubated in either a VGCC antagonist (diltiazem) or a VGCC agonist (BayK 8644), and then assayed for expression of the glutamatergic marker xVGlut1 or the GABAergic marker xGAD67. The number of cells expressing the gene of interest (termed “positives”) and the number of cells not expressing the gene of interest (termed “negatives”) for representative fields of view were recorded. A two-sample z-test was used to compare the percent positive between treatment groups and controls. P ≤ 0.05 was recorded as significant. A: xVGlut1 expression of diltiazem-exposed cells. B: xGAD67 expression of diltiazem-exposed cells. C: xVGlut1 expression of BayK 8644-exposed cells. D: xGAD67 expression of BayK 8644-exposed cells. For diltiazem exposures with xVGlut1 expression, n = 1,398 cells (0 μM, neural plate), 1,069 cells (10 μM, neural plate), 936 cells (100 μM, neural plate), 909 cells (0 μM, neural fold), 1,003 cells (10 μM, neural fold), 1,160 cells (100 μM, neural fold), 2,419 cells (0 μM, neural tube), 1,147 cells (10 μM, neural tube), and 160 cells (100 μM, neural tube). For diltizem exposures with xGAD67 expression, n = 1,259 cells (0 μM, neural plate), 843 cells (10 μM, neural plate), 658 cells (100 μM, neural plate), 576 cells (0 μM, neural fold), 1,523 cells (10 μM, neural fold), 1,441 cells (100 μM, neural fold), 1,486 cells (0 μM, neural tube), 1,509 cells (10 μM, neural tube), and 1,171 cells (100 μM, neural tube). For BayK 8644 exposures with xVGlut1 expression, n = 1,178 cells (0 μM, neural plate), 1,817 cells (10 μM, neural plate), 1,755 cells (100 μM, neural plate), 1,222 cells (0 μM, neural fold), 1,249 cells (10 μM, neural fold), 1,126 cells (100 μM, neural fold), 1,455 cells (0 μM, neural tube), 1,384 cells (10 μM, neural tube), and 1,519 cells (100 μM, neural tube). For BayK 8644 exposures with xGAD67 expression, n = 851 cells (0 μM, neural plate), 1,777 cells (10 μM, neural plate), 1,075 cells (100 μM, neural plate), 1,018 cells (0 μM, neural fold), 674 cells (10 μM, neural fold), 745 cells (100 μM, neural fold), 1,560 cells (0 μM, neural tube), 1,469 cells (10 μM, neural tube), and 1,001 cells (100 μM, neural tube).
Mentions: Because VGCCs are expressed in neural tissue during early development and certain VGCCs are correlated with specific spike frequencies in cell culture, the effect of the VGCC blocker diltiazem and agonist (−)Bayk 8644 on neurotransmitter phenotype specification was examined next. The percentage of cells expressing xVGlut1 or xGAD67 was assessed among treatment conditions, and a two-sample z-test was used to compare the percentage of positive cells in cultures exposed to diltiazem or (−)BayK 8644 with untreated controls. Exposure to the VGCC antagonist leads to an increase in glutamatergic neurons and a decrease in GABAergic neurons (Fig. 8A,B). Exposure to the VGCC agonist significantly decreases the percentage of cells expressing glutamatergic cells while having no effect on the number of GABAergic cells. (Fig. 8C,D)

Bottom Line: Calcium activity was also analyzed on a single-cell level, and spike frequency was correlated with the expression of VGCC α1 subunits in cell culture.Cells expressing Cav 2.1 and Cav 2.2 displayed increased calcium spiking compared with cells not expressing this marker.The VGCC antagonist diltiazem and agonist (-)BayK 8644 were used to manipulate calcium activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of William and Mary, Williamsburg, Virginia, 23185.

Show MeSH
Related in: MedlinePlus