MOESM2 of Multilevel analysis quantifies variation in the experimental effect while optimizing power and preventing false positives

Additional file 2. Calculating the optimal allocation of sample sizes and estimating statistical power to detect the overall experimental effect. Explanation on how to calculate the optimal allocation of sample sizes over clusters and within clusters given the available resources, and explanation on how to estimate power for a balanced (i.e., the number of observations per condition are equal both between conditions and between clusters) 2-level multilevel model without covariates

MOESM1 of Multilevel analysis quantifies variation in the experimental effect while optimizing power and preventing false positives

Additional file 1. Effect of neurite location (axon/dendrite) on traveling speed of intracellular vesicles: a worked example. An example of multilevel analysis of research design B data, including syntax to perform multilevel analysis in the statistical packages SPSS and R

Alternative splicing of Munc18-1 does not affect basal evoked release.

<p>Munc18-1a or -1b was reintroduced using lentivirus in autaptic hippocampal <i>munc18-1</i> null neurons cultured on glia islands. (A) Typical examples of paired pulse traces, traces from different intervals are plotted superimposed. (B) Both splice variants support evoked release to the same extent. Initial EPSC size (M18-1b: 3.21 ± 0.31 nA, n = 45; M18-1a: 3.50 ± 0.35 nA, n = 38, Unpaired t test, p = 0.54). (C) Quantification of the RRP estimate derived from back-extrapolation of the cumulative charge released during a RRP depleting train (100 pulses at 40 Hz) (M18-1b: 377.8 ± 52.7 pC, n = 35; M18-1a: 287.3 ± 33.3, n = 29, Unpaired t test with Welch correction, p = 0.15). (D) Quantification of the paired pulse ratio for different intervals (20 ms interval: M18-1b, 0.89 ± 0.04 EPSC^2nd/EPSC^1st; M18-1a, 0.79 ± 0.03 EPSC^2nd/EPSC^1st, Unpaired t test, p = 0.060).</p

Rate of EPSC depression during STP protocols depends on the splice variant expressed.

<p>Munc18-1a or -1b was reintroduced using lentivirus in autaptic hippocampal <i>munc18-1</i> null neurons cultured on glia islands. (A) Quantification of EPSC depression during 2.5 Hz stimulation normalized to the first EPSC amplitude (inset shows absolute value of the first EPSC). (B) Quantification of EPSC depression during 5 Hz stimulation normalized to the first EPSC amplitude (inset shows absolute value of the first EPSC). (C) Quantification of EPSC depression during 10 Hz stimulation normalized to the first EPSC amplitude. Left inset shows absolute value of the first EPSC. Right inset shows the relative depression at the end of the train normalized to the first EPSC (M18-1b, 0.26 ± 0.03 EPSC^#100/EPSC^#1; M18-1a, 0.20 ± 0.02 EPSC^#100/EPSC^#1, Unpaired t test with Welch correction, p = 0.042). (D) Quantification of the build-up of asynchronous charge during 10 Hz stimulation. (E-F) EPSC depression during two stimulation trains (100 pulses at 40Hz) given with a 2 seconds inter-train interval. (E) Shown are the responses to the first five pulses in the train, normalized to the first pulse. Inset shows the recovery of EPSC amplitude after two seconds (1^st EPSC of 2^nd train / 1^st EPSC of 1^st train). (F) Quantification of charge transferred within 25ms after each pulse relative to the first pulse in a train. Left inset shows the absolute EPSC charge at the start of the first train, right inset shows the first 5 pulses.</p

Morphology of hippocampal neurons expressing a single splice variant of Munc18-1.

<p>(A) Amino acid sequences of the C-terminus of mouse Munc18-1a and 1b starting at site 562 (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref010" target="_blank">10</a>]). Phosphorylation sites identified in high-throughput screens are highlighted (Fig 1b, M18-1a: S590, S594; M18-1b: T581, T588, S593, S594) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref016" target="_blank">16</a>]. Arrow points to a predicted CaMKII phosphorylation site in Munc18-1a identified using NetPhos 2.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref040" target="_blank">40</a>] and Phosida [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref038" target="_blank">38</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138950#pone.0138950.ref039" target="_blank">39</a>]. (B) Representative immunoblot of Munc18-1 protein levels in rescued <i>munc18-1</i> null neurons. Neurons were analysed at DIV13 with western blot analysis using double immuno-fluorescent labelling for Munc18-1 (red) and α-Tubulin (green). (C-E) Munc18-1a or -1b was reintroduced using lentivirus in hippocampal <i>munc18-1</i> null neurons cultured in the absence of glia. (C) Examples of MAP2 (dendritic marker) and VAMP2 (synaptic marker) immunostainings to visualize neuronal morphology. Scale bar = 100 μm. (D) Quantification of neuronal morphology. Number of synapses (M18-1b: 212.1 ± 8.9 synapses, n = 79; M18-1a: 241.3 ± 10.0 synapses, n = 78, Unpaired t test, p = 0.0309). Synapse density (M18-1b: 0.248 ± 0.009 synapses/μm dendrite, n = 79; M18-1a: 0.233 ± 0.006 synapses/μm dendrite, n = 78, Mann-Whitney Test, p = 0.1466). Total dendritic length (M18-1b: 0.895 ± 0.040 mm, n = 79; M18-1a: 1.057 ± 0.041 mm, n = 78, Mann-Whitney Test, p = 0.009). Number of branches (M18-1b: 127.2 ± 5.6 branches, n = 79; M18-1a: 147.0 ± 6.0 branches, n = 78, Mann-Whitney Test, p = 0.0043). (E) Sholl analysis suggests that dendritic branching is more elaborate in Munc18-1a expressing neurons. Shown are of the number of dendrite crossings per ring. Rings are placed with increasing radius around the soma (increments of 5 μm).</p

Alternative splicing of Munc18-1 does not affect spontaneous release.

<p>Munc18-1a or -1b was reintroduced using lentivirus in autaptic hippocampal <i>munc18-1</i> null neurons cultured on glia islands. (A) Example traces of spontaneous release of synaptic vesicles in the absence of stimulation. (B) Quantification of the mEPSC size and decay kinetics. mEPSC size (M18-1b: 18.11 ± 0.88 pA, n = 46; M18-1a: 16.29 ± 0.71 pA, n = 39, Unpaired t test with Welch correction, p = 0.11). mEPSC decay (M18-1b: 3.18 ± 0.15, n = 46; M18-1a: 2.84 ± 0.12, n = 39, Unpaired t test with Welch correction, p = 0.08). (C) Both splice variants support spontaneous release at similar rate. mEPSC frequency (M18-1b: 5.60 ± 1.29 Hz, n = 46; M18-1a: 6.21 ± 1.74 Hz, n = 39, Mann-Whitney Test, p = 0.5665.</p

Munc18-1a is expressed in the auditory brainstem of mice.

<p>(A) Munc18-1a immunostaining shows specific Munc18-1a expression in the Superior olivary complex (SOC), Facial motor nucleus (VII), and Lateral reticular nucleus (LRN) of the brainstem of wild-type (WT) mice. Scale bar 200 μm. (B) Munc18-1b-Venus is not expressed in nuclei of the brainstem of homozygous Munc18-1-Venus knock-in (M18V KI) mice. (C and D) Higher magnification of indicated regions in A and B, respectively, shows that Munc18-1a is highly expressed in the SOC, which lacks Munc18-1b-Venus expression. Scale bar 100 μm. (E) Bright field (bf) image of a coronal section of P21 wild-type mouse brain (images E-J all from same section), showing part of the brainstem including MNTB (white oval) and LSO. (F) Fluorescence image of immunostaining against Munc18-1a (M18-1a), showing relative high expression in the MNTB and LSO. Scale bar 100 μm. (G) Fluorescence image of immunostaining against Munc18-1b (M18-1b), showing relative low expression in the MNTB and LSO. (H) Composite image of E, F, G showing non-complementary expression of Munc18-1a (red) and Munc18-1b (green). (I and J) Confocal zoom from image in F and G of the MNTB region showing (I) Munc18-1a expression in presynaptic Calyces (arrowheads) and post-synaptic principal cells (*). In contrast, expression of Munc18-1b is punctate but unspecific for Calyces (J). Scale bar 10 μm.</p

Two different models of secretion. A. Sequential Pool Model (SPM).

<p>The Depot, Non-Releasable Pool (NRP), Readily-Releasable Pool (RRP) and Fused (F) states are represented by local minima of the free energy. Reactions between these states require the crossing of transition states that represent local maxima of the energy landscape. A Michaelis-Menten Ca²⁺-dependent step fills the NRP from the Depot Pool (recruitment, brown background). A Ca²⁺-dependent catalyst decreases the energy of the transition state for priming (transition to the RRP state) in a Ca²⁺-dependent manner, thus increasing the rates of both forward and backward reactions (priming, green background). A downstream clamp arrests release by imposing a high-energy transition state for fusion, which can be removed by Ca²⁺ binding to the associated sensor, thereby enabling fusion (fusion, blue background). The details of the cooperative Ca²⁺ sensor for fusion is given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003362#pcbi-1003362-g002" target="_blank">Fig. 2A</a>. <b>B. Parallel Pool Model (PPM).</b> In this model, the same Michaelis-Menten Ca²⁺-dependent step fills the SRP from the Depot Pool, as in the SPM (recruitment, brown background). Both the Slowly-Releasable Pool (SRP) and the Readily-Releasable Pool (RRP) are releasable, with different cooperative Ca²⁺ sensors (fusion, dark and light blue background, shown in detail in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003362#pcbi-1003362-g002" target="_blank">Fig. 2B</a>). The transition between SRP and RRP is Ca²⁺-independent (after <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003362#pcbi.1003362-Voets1" target="_blank">[16]</a> ).</p

Figure 6

<p>Deletion of syntaxin does not influence the intactness of the sub-membranous actin cytomatrix. Phalloidin-red staining of wild-type chromaffin cells infected with SFV-<i>egfp, BoNT/C</i>-ires-<i>egfp</i>, or <i>munc18-1</i>-ires-<i>egfp</i>. As a control Phalloidin-red staining of a <i>munc18-1</i> deficient chromaffin cell expressing EGFP is also shown. Merged pictures are shown in the right column. Scale bars represent 2 µm. The data in the lower half of the figures are similar to data published before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000126#pone.0000126-Toonen2" target="_blank">[24]</a> and are shown here for comparison.</p

Figure 3

<p>Spontaneous and evoked vesicle fusion is impaired in synapses lacking syntaxin. (A) Representative traces of mEPSC's in whole-cell voltage clamp recordings from control synapses showed frequent spontaneous miniature events, while syntaxin deleted synapses show a strong reduction of spontaneous release. (B) Frequency of spontaneous synaptic events. Numbers indicate mean±SEM for control (n = 5) and BoNT/C infected (n = 4) neurons from N = 2 different animals (***p<0.05, ANOVA and student's t-test). (C) Action potential triggered release is completely blocked by BoNT/C.</p