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. 2021 Jun 2;4:665. doi: 10.1038/s42003-021-02192-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a–c Bath application of acetylcholine (ACh, 1 mM) increased intracellular free calcium concentration in Fura-2-loaded Kis (a) and AcerKis (b) neurons. Pretreatment with the voltage-gated calcium channel blocker CdCl₂, (50 µM) significantly reduced ACh-induced [Ca²⁺]_i rise in AcerKis neurons (b, c) but did not produce any effect on [Ca²⁺]_i elevation in Kis neurons (a, c). Inset shows images of Fura-2 fluorescence of a single neuron cell body isolated from Kis and AcerKis strains after application of 1 mM ACh. Bars represent mean ± S.E.M. (n = 10–27 for Kis neurons and n = 10–12 for AcerKis neurons). The statistical test used was Student unpaired t-test, *p < 0.05; ns, non-significant. d, e Comparative histograms illustrating the relative voltage-gated calcium channel mRNA expression in Kis and AcerKis strains. Note that there is no significant difference in the relative mRNA expression of calcium channels between Kis and AcerKis strains. Bars represent mean ± S.E.M. (n = 4). The statistical analysis was made by using Analysis of Variance (one-way ANOVA), ns, non-significant. f Effects of ACh on Kis and AcerKis neuronal nAChRs studied under current-clamp condition. Pulse application of ACh (1 mM, 3 s in duration) produced a membrane depolarization in AcerKis neurons greater than in Kis neurons. g Histogram illustrating the effects of the ACh-induced membrane depolarization in Kis and AcerKis neurons. Bars represent mean ± S.E.M. (n = 6), The statistical test used was Student unpaired t-test, *p < 0.05. h Characterization of the voltage-gated calcium current studied under voltage-clamp condition according to the protocol indicated above each current trace. Inset represents typical examples of the voltage-dependent inward calcium current recorded in Kis and AcerKis neurons. Currents are capacity- and leak-corrected. Voltage dependence of the normalized conductance of the inward calcium current, in Kis (n = 3–5) and AcerKis (n = 3–7) neurons, calculated according to Eq. 6. Bars represent mean ± S.E.M. The statistical test used was Student unpaired t-test, ns, non-significant. In all cases, the number of experiments (n) are biologically independent samples.