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. 2019 Mar 29;10:1414. doi: 10.1038/s41467-019-09216-8

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© The Author(s) 2019

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. 3 — Multiplex imaging of quantal glutamate release and presynaptic Ca²⁺ dynamics. a CA3 pyramidal cell. Left: SF-iGluSnFR.A184V channel⁷ (Methods); arrows, two main axonal branches; ~60 µm z-stack, λ_x^2p = 910 nm. Right, Cal-590 channel (~20 µm z-stack fragment), whole-cell (300 µM Cal-590); patch pipette seen; scale bars, 20 µm. b Axonal bouton traced into CA1 from CA3 pyramid^9,49 : left, SF-iGluSnFR.A184V channel; tornado scan position shown; right, tornado linescan examples (rotation angle 0–5π rad reflects 2.5 spiral circles) during four evoked action potentials (APs; 20 Hz, arrows) shown in SF-iGluSnFR.A184V (green, note release failures) and Cal-590 (magenta) channels; scale bars, 2 µm (left), 200 ms (right). c Four characteristic sequential one-sweep recordings (SF-iGluSnFR channel) depicting individual quantal releases and failures. d Summary, glutamate release kinetics in bouton shown in b (SF-iGluSnFR.A184V channel; green, 20 trials 1 min apart; black, average); scale bars, 50% ΔF/F (vertical) and 100 ms (horizontal). e Summary, Cal-590 intensity signal ΔF/F (red channel) recorded as in d (magenta, individual trials; black, average); arrow, spontaneous Ca²⁺ entry with no glutamate release (see d); scale bar, 30% ΔF/F. f Dynamics of free presynaptic [Ca²⁺] averaged over 20 trials: Cal-590 fluorescence lifetime imaging microscopic readout (normalised photon count), converted to [Ca²⁺]. Note: data reflect free ion concentration volume-equilibrated and time-averaged over 5–10 ms. g Amplitude histograms (SF-iGluSnFR.A184V ΔF/F signal, first–to-fourth response counts combined; 8 ms pre-AP baseline subtracted); blue line, semi-constrained multi-Gaussian fit (Methods); peaks correspond to quantal amplitudes; leftmost peak, zero signal (release failure noise; yellow shade); false-positive cut-off at ~0.12 ΔF/F. h Trial-to-trial glutamate release signal amplitude (bouton in b) shown as ΔF/F SF-iGluSnFR.A184V readout (grey dots) and as quantal content (magenta bars, right ordinate; calculated from histogram in g) plotted against evoked Ca²⁺ entry signal (ΔF/F Cal-590, as in e) for all recorded APs (8 ms pre-AP baseline subtracted). Black and magenta lines, linear regressions (r, Pearson’s correlation; p, regression slope significance) for ΔF/F and quantal content data, respectively; yellow shade, failure response cut-off (as in g)