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Dominance of P/Q-type calcium channels in depolarization-induced presynaptic FM dye release in cultured hippocampal neurons.

Nimmervoll B, Flucher BE, Obermair GJ - Neuroscience (2013)

Bottom Line: Analysis of the release kinetics and the fractional release amplitude demonstrate that, whereas in only 15% of the synapses release depended exclusively on P/Q-type channels, the majority of synapses (85%) contained both N- and P/Q-type channels.Nevertheless, the kinetics of FM dye release in synapses containing both channel types was determined by the P/Q-type channels.Together, our data suggest a more direct coupling of P/Q-type channels to synaptic release compared to N-type channels, which may explain the high prevalence of neurological P/Q-type channelopathies.

View Article: PubMed Central - PubMed

Affiliation: Division of Physiology, Medical University Innsbruck, Fritz-Pregl-Str. 3, 6020 Innsbruck, Austria.

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Pharmacological characterization of FM dye release reveals its prime dependence on P/Q-type channels. (A) FM dye loading and unloading protocol. Respective toxins were applied to the bath solution after a 5 min washing period and also applied through the microperfusion pipette during unloading at 40 mM [K+]. (B) Normalized FM dye release over time in the presence of different calcium channel blockers: Cd2+ (purple; n = 4 experiments/1 culture preparation/157 synapses), ω-agatoxin IVA (Aga; green; n = 12/3/474), ω-conotoxin GVIA (CTx; red; n = 14/3/556), sham (blue; n = 18/4/707), Aga + CTx (orange; n = 10/3/398). (C–E) The monoexponential fitting parameters and the fractional release (Rf) derived from the release traces of individual synapses were normalized to mean amplitude (C), median τ (D), and mean Rf (E) of the parallel sham conditions. Box plots show median (horizontal line), interquartile range (box) and 10th and 90th percentiles (whiskers); mean is displayed as red triangle. Number of synapses (experiments) analyzed: 692 (18), 313 (14), 280 (12), 160 (10) (C, D, left to right) and 707 (18), 556 (14), 474 (12), 398 (10) (E) from 3 to 4 separate culture preparations. [ANOVA: F(3, 50) = 12.8, p < 0.001 (C), F(3, 50) = 2.8, p = 0.05 (D), F(3, 50) = 12.0, p < 0.001 (E), Holm–Sidak post hoc test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.]
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f0015: Pharmacological characterization of FM dye release reveals its prime dependence on P/Q-type channels. (A) FM dye loading and unloading protocol. Respective toxins were applied to the bath solution after a 5 min washing period and also applied through the microperfusion pipette during unloading at 40 mM [K+]. (B) Normalized FM dye release over time in the presence of different calcium channel blockers: Cd2+ (purple; n = 4 experiments/1 culture preparation/157 synapses), ω-agatoxin IVA (Aga; green; n = 12/3/474), ω-conotoxin GVIA (CTx; red; n = 14/3/556), sham (blue; n = 18/4/707), Aga + CTx (orange; n = 10/3/398). (C–E) The monoexponential fitting parameters and the fractional release (Rf) derived from the release traces of individual synapses were normalized to mean amplitude (C), median τ (D), and mean Rf (E) of the parallel sham conditions. Box plots show median (horizontal line), interquartile range (box) and 10th and 90th percentiles (whiskers); mean is displayed as red triangle. Number of synapses (experiments) analyzed: 692 (18), 313 (14), 280 (12), 160 (10) (C, D, left to right) and 707 (18), 556 (14), 474 (12), 398 (10) (E) from 3 to 4 separate culture preparations. [ANOVA: F(3, 50) = 12.8, p < 0.001 (C), F(3, 50) = 2.8, p = 0.05 (D), F(3, 50) = 12.0, p < 0.001 (E), Holm–Sidak post hoc test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.]

Mentions: Having established the basic properties of presynaptic FM dye release we next pharmacologically dissected the dependence of release at 40 mM [K+] on Ca2+ channels in order to reveal the contribution of the two major presynaptic channels P/Q- (CaV2.1) and N-type (CaV2.2). Dependence of FM dye release on CaVs was first confirmed by applying the universal Ca2+ channel pore blocker cadmium (Cd2+; 1 mM), which completely abolished the loss of FM dye fluorescence (Fig. 3B). In order to test which types of presynaptic Ca2+ channels mediate [K+]-induced FM dye release we used individual or combined application of CTx and Aga at concentrations known to be highly specific for blocking N- and P/Q-type channels, respectively (Catterall et al., 2005). Incubation with CTx did not affect release kinetics and Rf (Fig. 3), suggesting that FM dye release induced by 40 mM [K+] does not critically depend on N-type channels. In contrast, treatment of cultures with Aga resulted in reduced amplitude and prolonged release kinetics. The combined application of CTx and Aga further reduced amplitude to approximately 50% of control and increased time constants more than 3-fold (Fig. 3C, D). These effects were most strikingly revealed by a reduction of the fractional release amplitude (Rf) to 71% in Aga-only-treated neurons and to 29% in the combined CTx/Aga treatment. This synergistic effect of CTx and Aga indicates that the majority of synapses contain both N- and P/Q-type channels. Nevertheless, only P/Q-type channels can fully compensate the loss of N-type function, whereas N-type channels can compensate the loss of P/Q only with reduced efficacy.


Dominance of P/Q-type calcium channels in depolarization-induced presynaptic FM dye release in cultured hippocampal neurons.

Nimmervoll B, Flucher BE, Obermair GJ - Neuroscience (2013)

Pharmacological characterization of FM dye release reveals its prime dependence on P/Q-type channels. (A) FM dye loading and unloading protocol. Respective toxins were applied to the bath solution after a 5 min washing period and also applied through the microperfusion pipette during unloading at 40 mM [K+]. (B) Normalized FM dye release over time in the presence of different calcium channel blockers: Cd2+ (purple; n = 4 experiments/1 culture preparation/157 synapses), ω-agatoxin IVA (Aga; green; n = 12/3/474), ω-conotoxin GVIA (CTx; red; n = 14/3/556), sham (blue; n = 18/4/707), Aga + CTx (orange; n = 10/3/398). (C–E) The monoexponential fitting parameters and the fractional release (Rf) derived from the release traces of individual synapses were normalized to mean amplitude (C), median τ (D), and mean Rf (E) of the parallel sham conditions. Box plots show median (horizontal line), interquartile range (box) and 10th and 90th percentiles (whiskers); mean is displayed as red triangle. Number of synapses (experiments) analyzed: 692 (18), 313 (14), 280 (12), 160 (10) (C, D, left to right) and 707 (18), 556 (14), 474 (12), 398 (10) (E) from 3 to 4 separate culture preparations. [ANOVA: F(3, 50) = 12.8, p < 0.001 (C), F(3, 50) = 2.8, p = 0.05 (D), F(3, 50) = 12.0, p < 0.001 (E), Holm–Sidak post hoc test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.]
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f0015: Pharmacological characterization of FM dye release reveals its prime dependence on P/Q-type channels. (A) FM dye loading and unloading protocol. Respective toxins were applied to the bath solution after a 5 min washing period and also applied through the microperfusion pipette during unloading at 40 mM [K+]. (B) Normalized FM dye release over time in the presence of different calcium channel blockers: Cd2+ (purple; n = 4 experiments/1 culture preparation/157 synapses), ω-agatoxin IVA (Aga; green; n = 12/3/474), ω-conotoxin GVIA (CTx; red; n = 14/3/556), sham (blue; n = 18/4/707), Aga + CTx (orange; n = 10/3/398). (C–E) The monoexponential fitting parameters and the fractional release (Rf) derived from the release traces of individual synapses were normalized to mean amplitude (C), median τ (D), and mean Rf (E) of the parallel sham conditions. Box plots show median (horizontal line), interquartile range (box) and 10th and 90th percentiles (whiskers); mean is displayed as red triangle. Number of synapses (experiments) analyzed: 692 (18), 313 (14), 280 (12), 160 (10) (C, D, left to right) and 707 (18), 556 (14), 474 (12), 398 (10) (E) from 3 to 4 separate culture preparations. [ANOVA: F(3, 50) = 12.8, p < 0.001 (C), F(3, 50) = 2.8, p = 0.05 (D), F(3, 50) = 12.0, p < 0.001 (E), Holm–Sidak post hoc test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.]
Mentions: Having established the basic properties of presynaptic FM dye release we next pharmacologically dissected the dependence of release at 40 mM [K+] on Ca2+ channels in order to reveal the contribution of the two major presynaptic channels P/Q- (CaV2.1) and N-type (CaV2.2). Dependence of FM dye release on CaVs was first confirmed by applying the universal Ca2+ channel pore blocker cadmium (Cd2+; 1 mM), which completely abolished the loss of FM dye fluorescence (Fig. 3B). In order to test which types of presynaptic Ca2+ channels mediate [K+]-induced FM dye release we used individual or combined application of CTx and Aga at concentrations known to be highly specific for blocking N- and P/Q-type channels, respectively (Catterall et al., 2005). Incubation with CTx did not affect release kinetics and Rf (Fig. 3), suggesting that FM dye release induced by 40 mM [K+] does not critically depend on N-type channels. In contrast, treatment of cultures with Aga resulted in reduced amplitude and prolonged release kinetics. The combined application of CTx and Aga further reduced amplitude to approximately 50% of control and increased time constants more than 3-fold (Fig. 3C, D). These effects were most strikingly revealed by a reduction of the fractional release amplitude (Rf) to 71% in Aga-only-treated neurons and to 29% in the combined CTx/Aga treatment. This synergistic effect of CTx and Aga indicates that the majority of synapses contain both N- and P/Q-type channels. Nevertheless, only P/Q-type channels can fully compensate the loss of N-type function, whereas N-type channels can compensate the loss of P/Q only with reduced efficacy.

Bottom Line: Analysis of the release kinetics and the fractional release amplitude demonstrate that, whereas in only 15% of the synapses release depended exclusively on P/Q-type channels, the majority of synapses (85%) contained both N- and P/Q-type channels.Nevertheless, the kinetics of FM dye release in synapses containing both channel types was determined by the P/Q-type channels.Together, our data suggest a more direct coupling of P/Q-type channels to synaptic release compared to N-type channels, which may explain the high prevalence of neurological P/Q-type channelopathies.

View Article: PubMed Central - PubMed

Affiliation: Division of Physiology, Medical University Innsbruck, Fritz-Pregl-Str. 3, 6020 Innsbruck, Austria.

Show MeSH
Related in: MedlinePlus