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. 2019 Dec 19;143(2):441–451. doi: 10.1093/brain/awz391

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© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Patch-clamp electrophysiological characterization of Munc18-1 null neurons expressing Munc18^L446F. (A–C) Spontaneous release of synaptic vesicles is not significantly altered by expression of Munc18^L446F. (A) Example traces of spontaneous release of single synaptic vesicles (mEPSCs) of Munc18-1 null neurons expressing Munc18^WT or Munc18^L446F. (B and C) No differences are observed in spontaneous mEPSC frequency (B: Munc18^WT median = 3.253 Hz, IQR = 0.584–7.49; Munc18^L446F median = 6.473 Hz, IQR = 1.375–13.25; P = 0.165, Mann-Whitney U-test) and amplitude (C: Munc18^WT median = −20.76 pA, IQR = −24.19–18.24; Munc18^L446F median = −21.28 pA, IQR = −24.29–18.03; P = 0.943, unpaired t-test) between Munc18^WT and Munc18^L446F. (D–F) Evoked synaptic responses are increased in neurons expressing Munc18^L446F. (D) Typical examples of a single evoked EPSC. Stimulus artefact has been blanked out for visualization purposes. (E) Average EPSC amplitude is significantly larger in neurons expressing Munc18^L446F compared Munc18^WT (Munc18^WT median = 3.332 nA, IQR = 2.213–5.798; Munc18^L446F median = 7.716 nA, IQR = 3.838–13.81; P = 0.0018, Mann-Whitney U-test) and in parallel, the average charge (F) transferred during the EPSC response is increased (Munc18^WT median = 37.36 pC, IQR = 27.65–77.65; Munc18^L446F median = 117.0 pC, IQR = 43.69–164.7; P = 0.0012, Mann-Whitney U-test). (G and H) Paired-pulse recordings at various intervals were performed to quantify release probability. (G) Example traces showing an overlay of paired-pulse recordings at several time intervals (20–50–100–200–500 ms interpulse interval) for a neuron expressing Munc18^WT or Munc18^L446F. (H) Paired-pulse ratios (calculated as the ratio of the second EPSC to the first EPSC) are significantly higher for neurons expressing Munc18^L446F at the stimulus intervals between 0.05 and 0.5 s (0.02 s: Munc18^WT median = 0.488, IQR = 0.363–0.880; Munc18^L446F median = 0.479, IQR = 0.317–0.657, P = 0.109, unpaired t-test with Welch’s correction; 0.05 s: Munc18^WT median = 0.872, IQR = 0.689–1.198; Munc18^L446F median = 0.701, IQR = 0.544–0.849, P = 0.0103; 0.1 s: Munc18^WT median = 0.913, IQR = 0.743–1.157; Munc18^L446F median = 0.732, IQR = 0.589–0.891, P = 0.0045; 0.2 s: Munc18^WT median = 0.873, IQR = 0.754–0.955, Munc18^L446F median = 0.777, IQR = 0.625–0.866, P = 0.0207; 0.5 s: Munc18^WT median = 0.833, IQR = 0.775–0.905, Munc18^L446F median = 0.751, IQR = 0.614–0.839, P = 0.019, Mann-Whitney U-tests). (I) Estimate of the RRP by back-extrapolation indicates no difference in RRP size between Munc18^WT or Munc18^L446F (Munc18^WT median = 432.7 pC, IQR = 220.1–803.6, Munc18^L446F median = 455.4 pC, IQR = 300.3–1219, P = 0.511, Mann-Whitney U-test). (J) Total charge transferred during the 40-Hz train stimulation is not significantly different between Munc18^WT or Munc18^L446F (Munc18^WT median = 1488 pC, IQR = 724.4–2424, Munc18^L446F median = 1296 pC, IQR = 939.0–2239, P = 0.594, Mann-Whitney U-test). (K) Run-down kinetics and steady-state responses to 100 action potentials at 10 Hz are similar between Munc18^WT and Munc18^L446F, except for the first few pulses (inset) at which neurons expressing Munc18^L446F display more pronounced depression compared to Munc18^WT. (L) EPSC rundown in response to a stimulation of 100 action potentials at 40 Hz are also similar between Munc18^WT and Munc18^L446F. Inset: first five pulses of the train. (M) EPSC amplitude in response to single action potential stimulations 2 and 30 s after the 10-Hz train stimulation expressed as a percentage of the amplitude of the first EPSC of the train stimulation. Neurons expressing Munc18^L446F have a smaller EPSC response at 2 s (Munc18^WT median = 65.96%, IQR = 53.26–82.32, Munc18^L446F median = 43.27%, IQR = 28.32–56.44, P = 0.0003, Mann-Whitney U-test) and 30 s (Munc18^WT median = 81.15%, IQR = 62.58–100.6, Munc18^L446F median = 70.29%, IQR = 53.31–77.36, P = 0.021, Mann-Whitney U-test) after a 10-Hz train. (N) Recovery of the RRP was measured at 2 and 60 s after the train, respectively. After 2 s, recovery is not significantly different but shows a trend towards a reduction in Munc18^L446F expressing neurons (Munc18^WT median = 84.22%, IQR = 50.45–104.0, Munc18^L446F median = 63.92%, IQR = 46.39–76.59, P = 0.068, Mann-Whitney U-test). Sixty seconds after 40-Hz stimulation, neurons expressing Munc18^WT show full recovery whereas recovery is impaired in neurons expressing Munc18^L446F (Munc18^WT median = 94.90%, IQR = 83.97–107.5, Munc18^L446F median = 83.93%, IQR = 73.73–91.31, P = 0.0396, unpaired t-test with Welch’s correction). (O) Recruitment rate of vesicles at steady-state during 40 Hz stimulation as estimated by the back-extrapolation procedure (C) is not different between Munc18^WT or Munc18^L446F (Munc18^WT median = 388.5 s⁻¹, IQR = 160.7–755.8, Munc18^L446F median = 377.3 s⁻¹, IQR = 243.4–484.4, P = 0.778, Mann-Whitney U-test). The number of analysed cells and number of independent cultures tested is indicated below the graphs. *P < 0.05, **P < 0.01.