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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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Characterization of depolarization-induced FM dye release. (A, B) FM dye release curves of all single synapses were fitted mono-exponentially. The monoexponential fitting parameters amplitude (A) and τ (B), were normalized to the mean and the median of the 60 mM [K+] condition, respectively, within each separate culture preparation. Box plots show median (horizontal line), interquartile range (box) and 10th and 90th percentiles (whiskers); mean is displayed as red triangle. (C) Boltzmann fit of normalized mean time constants (mean ± sem) plotted against the respective membrane potential as calculated by the Goldman–Hodgkin–Katz equation (half maximum activation = −39.8 mV). (D) Boltzmann fit of fractional release amplitudes (Rf; mean ± sem) relative to the release observed at τ of the 60 mM [K+] condition (τc, blue vertical line in Fig. 1B: 15.4 ± 8.9 s [mean ± SD]; half maximum activation = −37.6 mV). Data represent average values of 108, 113, 177, 313, 703 (20–60 mM; B, C) and 345, 199, 190, 316, 712 (20–60 mM; D) synapses. [ANOVA: F(4, 40) = 16.1, p < 0.001 (A), F(4, 40) = 3.7, p = 0.011 (B), F(4, 40) = 29.2, p < 0.001 (D); Holm–Sidak post hoc test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001].
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f0010: Characterization of depolarization-induced FM dye release. (A, B) FM dye release curves of all single synapses were fitted mono-exponentially. The monoexponential fitting parameters amplitude (A) and τ (B), were normalized to the mean and the median of the 60 mM [K+] condition, respectively, within each separate culture preparation. Box plots show median (horizontal line), interquartile range (box) and 10th and 90th percentiles (whiskers); mean is displayed as red triangle. (C) Boltzmann fit of normalized mean time constants (mean ± sem) plotted against the respective membrane potential as calculated by the Goldman–Hodgkin–Katz equation (half maximum activation = −39.8 mV). (D) Boltzmann fit of fractional release amplitudes (Rf; mean ± sem) relative to the release observed at τ of the 60 mM [K+] condition (τc, blue vertical line in Fig. 1B: 15.4 ± 8.9 s [mean ± SD]; half maximum activation = −37.6 mV). Data represent average values of 108, 113, 177, 313, 703 (20–60 mM; B, C) and 345, 199, 190, 316, 712 (20–60 mM; D) synapses. [ANOVA: F(4, 40) = 16.1, p < 0.001 (A), F(4, 40) = 3.7, p = 0.011 (B), F(4, 40) = 29.2, p < 0.001 (D); Holm–Sidak post hoc test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001].

Mentions: At all [K+] concentrations inducing synaptic release (30–60 mM) amplitudes were similar and significantly different from the 20 mM [K+] condition (Fig. 2A). This confirms that FM dye release at all [K+] concentrations depended on the same vesicle pool and that increasing the stimulus strength did not recruit additional pools within the observed time frame. In contrast, the rate of release sequentially increased (decreased time constants, τ) until saturation at 50 mM [K+] (Fig. 2B). This indicates that stronger depolarization of the membrane by increasing [K+] concentrations directly affected the release efficacy of the individual synapses. As evident in the mean release curves (see Fig. 1B) release was maximal at 50 mM [K+] and therefore time constants could not be further enhanced by stronger depolarization (Fig. 2B). Plotting the mean time constants against the corresponding membrane potential as calculated by the Goldman–Hodgkin–Katz equation (half maximum activation = −39.8 mV), revealed a sigmoid relationship of the release rate to the strength of depolarization (Fig. 2C).


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)

Characterization of depolarization-induced FM dye release. (A, B) FM dye release curves of all single synapses were fitted mono-exponentially. The monoexponential fitting parameters amplitude (A) and τ (B), were normalized to the mean and the median of the 60 mM [K+] condition, respectively, within each separate culture preparation. Box plots show median (horizontal line), interquartile range (box) and 10th and 90th percentiles (whiskers); mean is displayed as red triangle. (C) Boltzmann fit of normalized mean time constants (mean ± sem) plotted against the respective membrane potential as calculated by the Goldman–Hodgkin–Katz equation (half maximum activation = −39.8 mV). (D) Boltzmann fit of fractional release amplitudes (Rf; mean ± sem) relative to the release observed at τ of the 60 mM [K+] condition (τc, blue vertical line in Fig. 1B: 15.4 ± 8.9 s [mean ± SD]; half maximum activation = −37.6 mV). Data represent average values of 108, 113, 177, 313, 703 (20–60 mM; B, C) and 345, 199, 190, 316, 712 (20–60 mM; D) synapses. [ANOVA: F(4, 40) = 16.1, p < 0.001 (A), F(4, 40) = 3.7, p = 0.011 (B), F(4, 40) = 29.2, p < 0.001 (D); Holm–Sidak post hoc test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001].
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f0010: Characterization of depolarization-induced FM dye release. (A, B) FM dye release curves of all single synapses were fitted mono-exponentially. The monoexponential fitting parameters amplitude (A) and τ (B), were normalized to the mean and the median of the 60 mM [K+] condition, respectively, within each separate culture preparation. Box plots show median (horizontal line), interquartile range (box) and 10th and 90th percentiles (whiskers); mean is displayed as red triangle. (C) Boltzmann fit of normalized mean time constants (mean ± sem) plotted against the respective membrane potential as calculated by the Goldman–Hodgkin–Katz equation (half maximum activation = −39.8 mV). (D) Boltzmann fit of fractional release amplitudes (Rf; mean ± sem) relative to the release observed at τ of the 60 mM [K+] condition (τc, blue vertical line in Fig. 1B: 15.4 ± 8.9 s [mean ± SD]; half maximum activation = −37.6 mV). Data represent average values of 108, 113, 177, 313, 703 (20–60 mM; B, C) and 345, 199, 190, 316, 712 (20–60 mM; D) synapses. [ANOVA: F(4, 40) = 16.1, p < 0.001 (A), F(4, 40) = 3.7, p = 0.011 (B), F(4, 40) = 29.2, p < 0.001 (D); Holm–Sidak post hoc test: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001].
Mentions: At all [K+] concentrations inducing synaptic release (30–60 mM) amplitudes were similar and significantly different from the 20 mM [K+] condition (Fig. 2A). This confirms that FM dye release at all [K+] concentrations depended on the same vesicle pool and that increasing the stimulus strength did not recruit additional pools within the observed time frame. In contrast, the rate of release sequentially increased (decreased time constants, τ) until saturation at 50 mM [K+] (Fig. 2B). This indicates that stronger depolarization of the membrane by increasing [K+] concentrations directly affected the release efficacy of the individual synapses. As evident in the mean release curves (see Fig. 1B) release was maximal at 50 mM [K+] and therefore time constants could not be further enhanced by stronger depolarization (Fig. 2B). Plotting the mean time constants against the corresponding membrane potential as calculated by the Goldman–Hodgkin–Katz equation (half maximum activation = −39.8 mV), revealed a sigmoid relationship of the release rate to the strength of depolarization (Fig. 2C).

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