Drosophila NMJ Morphometrics

Here, we present the image analysis algorithm ‘Drosophila_NMJ_Morphometrics’, available as a Fiji-compatible macro, for quantitative, accurate and objective synapse morphometry of the Drosophila larval neuromuscular junction (NMJ), a well-established glutamatergic model synapse. We developed this methodology for semi-automated multiparametric analyses of NMJ terminals immunolabeled for the commonly used markers Dlg1 and Brp and showed that it also works for Hrp, Csp and Syt.<br><div><br></div

The influence of the abdominal body segments on NMJ features.

<p>The plots display the mean (dot) and 95% confidence interval (blue error bars) of the NMJ values for each feature over the abdominal body segments A2 (n = 148), A3 (n = 356), A4 (n = 399) and A5 (n = 198). Four different categories are indicated by color. The blue category includes the features active zones (A), branches (B), branching points (C) and islands (D) and show a relative decrease from anterior to posterior with, except for the islands, A2 being significantly different. The second, orange, category includes the length (E) and longest branch length (F), showing an opposite pattern, in which A2 is significantly smaller than the other segments. The third category (green) includes the muscle area (G), that shows a significant increase in values from A2 to A3 and a significant decrease from A3 to A5. The last category (red) includes the features NMJ area (H) and perimeter (I), which shows a similarity between A2 and A5 versus A3-A4, only significant for NMJ area.</p

A Fiji macro to systematically quantify NMJ features.

<p>Representation of the Fiji-based macro for NMJ systems analysis (A). The macro was developed using Dlg1 and Brp image stacks as input (left to the arrow). For each NMJ, the indicated nine NMJ features are quantified. The NMJ area, perimeter and boutons are deduced from the NMJ outline and inner segmentations (indicated in yellow). The skeleton (blue) provides the five features total length, longest branch length (sub branches are excluded), the number of Dlg1-based islands (unconnected Dlg1 compartments, in blue and yellow), the number of branches (in different colors), the number of branching points (red dot). The Brp channel is used to count the number of active zones within the NMJ outline (white dots within the yellow outline). The NMJ muscle area is measured at lower magnification by the Fiji freehand selection (yellow box rectangle). NMJs are oriented anterior left, dorsal up. Schematic representation of the NMJ workflow (B). Larvae are dissected and NMJs are stained for Dlg1 and Brp and captured in images at the two magnifications 10x and 63x. Lower magnification snapshots are used to measure the muscle4 area and are simultaneously checked to ensure that the full NMJ is captured at high magnification (= checkpoint 1). Images at high magnification are progressed to stacks and projections in which the region of interest (ROI) is defined to exclusively analyze type 1b NMJs. Images are excluded in checkpoint 2 if the specificity of type 1b cannot be guaranteed or if the quality of the immunohistochemistry is poor. The first two bars of the bar graph (C) represent the amount of NMJs and the percentage that passed the two input checkpoints (93.15%). Sub macro 3 processes the NMJ towards quantitative output and the macro-annotated images are evaluated (= checkpoint 3). Bars three to five represent the percentage of NMJs per threshold that passed the output quality checkpoint (87.75% for the NMJ skeleton; 82.17% for the NMJ outline and 79.89% for Active zones). The final bar represents the percentage of investigated NMJs for which all features were of high quality (73.79%).</p

Macro validation on high-content microscopy images.

<p>Macro validation on high-content microscopy images.</p

Evaluation of reproducibility.

<p>Macro assessment and quantification of NMJs on muscle 4. Ankyrin2 knockdown (<i>w;UAS-Dicer-2/UAS-KK107238;elav-Gal4/+</i>, n = 18 and <i>w;UAS-Dicer-2/UAS-KK107369;elav-Gal4/+</i>, n = 19) was confirmed to result in smaller NMJ size compared to genetic background controls (<i>w;UAS-Dicer-2/+;elav-Gal4/+</i>, n = 500) when using the macro (A-B). The macro-annotated outline is indicated in yellow. Highwire knockdown (<i>w;UAS-Dicer-2/UAS-GD28163;elav-Gal4/+</i>, n = 15 and <i>w;UAS-Dicer-2/UAS-GD36085;elav-Gal4/+</i>, n = 26) resulted in longer and more branched NMJs compared to genetic background control NMJs (<i>w;UAS-Dicer-2/+;elav-Gal4/+</i>, n = 402) (C-D). The macro-annotated skeleton is indicated in blue. Rab3 knockdown (<i>w;UAS-Dicer-2/UAS-KK100787;elav-Gal4/+</i>, n = 18) resulted in NMJs with a lower number of Brp-positive active zones compared to genetic background controls (<i>w;UAS-Dicer-2/+;elav-Gal4/+</i>, n = 476) (E-F). The macro-annotated Brp-positive active zones are indicated as white foci. Error bars indicate 95% confidence interval and asterisks represent significance (p_adj < 0.05).</p

Macro validation on confocal microscopy images.

<p>Macro validation on confocal microscopy images.</p

The influence of gender on NMJ features.

<p>The plots display the mean value (black dot) and 95% confidence interval for each indicated feature. Males are represented in blue, females in red. The features are significantly different between sexes for the size-related NMJ features (upper panel), whereas they largely overlap for structural NMJ features (lower panel).</p

The different layers of <i>Drosophila</i> Neuromuscular Junction morphometry.

<p>The five principal components NMJ size, Muscle area, Active zones, Islands and NMJ geometry are indicated by the green boxes in the front layer. The second (turquoise) layer represents the different patterns in which the NMJ features behave over the four investigated body segments (A2-A5). The colored line in the larvae sketch shows the tendency of the parameters from anterior (A2, left) to posterior (A5, right). The third (orange) layer distinguishes the gender-dependent NMJ features from the gender independent ones. Variation by genetic background is not depicted since it depends on the identity of the specific genotypes investigated.</p

The influence of the genetic background on NMJ features.

<p>The plots display the mean value (black dot) and 95% confidence interval for the genetic background of the VDRC GD library (blue) versus the genetic background of the KK library (red) for each feature. The upper panel represents the features that are significantly different between the two genetic backgrounds and the lower panel represents the features for which the genetic background does not significantly influence the outcome.</p

Principal components of NMJ morphometry.

<p>The correlation-matrix represents the pair wise correlations between all possible feature pairs, displayed as scatter plots (bottom left side) or as correlation coefficients (top right side) (A). The matrix is ordered so that those with the highest correlation are closer together. Correlation strength is color coded, ranging from light reddish (no-weak correlation) to an intense red color (for strongly correlating features). Five principle components were determined and the contribution of each feature to a certain component is summarized, together with the percentage of variance that is explained by that principal component and the cumulative percentage of total variance (B). The most contributing features per principal component (PC) are indicated in red. The first two components, which together make up for 59.83% of total variance are plotted with the first principal component (PC1) on the x-axis and the second principal component (PC2) on the y-axis (C). The arrow length reflects the contribution of each feature to the first two principal components: features showing a major contribution to PC1 or PC2 are indicated in black, and NMJ features with a minor contribution are grey.</p