We acknowledge the Vienna Drosophila Resource Center and Bloomington Drosophila stock center (NIH P40OD018537)for providing Drosophila strains. We thank Jack Fransen from the Microscopical Imaging Centre for expert support in imaging. This study was supported by VIDI and TOP grants (917-96-346, 912-12-109) from the Netherlands Organization for Scientific Research (NWO), by two DCN/Radboud University Medical Center PhD fellowships, by the German Mental Retardation Network funded by the NGFN+ program of the German Federal Ministry of Education and Research (BMBF) and by the European Union's FP7 large scale integrated network Gencodys (HEALTH-241995) to AS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Requirements Prior to Image Processing
<ol>
	<li>Perform Drosophila open book preparations of third instar wandering larvae (L3), as previously described19.</li>
	<li>Co-immunolabel Drosophila NMJ terminals using a combination of two markers: Dlg-1 or Hrp together with Brp for analysis with &#34;Drosophila NMJ Morphometrics&#34;, and Syt or Csp together with Brp for analysis with &#34;Drosophila NMJ Bouton Morphometrics&#34;20. 
	NOTE: Antibodies from the same species can be combined by pre-labeling one with an antibody-conjugation kit such as the Zenon Alexa Labeling Kits17.</li>
	<li>Image NMJ terminals using a microscope of choice, e.g., fluorescence (with or without ApoTome) or confocal microscopy.
	<ol>
		<li>Acquire a 2-channel image stack of the NMJ terminal.
		<ol>
			<li>Adjust the microscope settings in a manner that channel 1 acquires the NMJ terminal immunolabeled with Dlg-1 (or Hrp, Syt, Csp) and channel 2 the NMJ terminal immunolabeled with Brp.</li>
			<li>Optionally, analyze one-channel images (of synapses immunolabeled with a single antibody) with the macros. Image NMJs immunolabeled uniquely with Dlg-1 or Hrp for analysis with &#34;Drosophila NMJ Morphometrics&#34;, or Syt or Csp for &#34;Drosophila NMJ Bouton Morphometrics&#34;. 
			NOTE: It is not possible to analyze synapses immunostained with only anti-Brp.</li>
		</ol>
		</li>
		<li>Export the obtained images as individual &#39;.tiff files. Invert the channel order prior to running the macros if not acquired as indicated.</li>
	</ol>
	</li>
</ol>
2. Software Requirements and Installation
<ol>
	<li>Download the macros: &#34;Drosophila NMJ Morphometrics&#34; and &#34;Drosophila NMJ Bouton Morphometrics&#34; from the following website: <a href="https://doi.org/10.6084/m9.figshare.2077399.v1">https://doi.org/10.6084/m9.figshare.2077399.v1</a>21.</li>
	<li>Move the cursor to the folder &#34;Macros update 1&#34;, and click on the appearing option &#34;view&#34;. A list with the content of this folder will appear. The folder contains the macros &#34;Drosophila NMJ Morphometrics&#34; and &#34;Drosophila NMJ Bouton Morphometrics&#34;. 
	NOTE: Both macros are compatible with Fiji versions 1.4, which is also provided in the same folder. The macros may not run on recent versions. Please utilize the provided version 1.4. It is unproblematic to start this version, even on computers with a more recent Fiji version available.</li>
	<li>Click on &#34;Download all&#34;. The folder content will be downloaded to the computer as a .zip file. Unzip the downloaded file.</li>
	<li>Copy the Drosophila_NMJ_Morphometrics.ijm and Drosophila_NMJ_Bouton Morphometrics.ijm files to Fiji.app/plugins/ directory. When restarting the program, the macros will appear at the bottom of the Plugins dropdown menu.</li>
</ol>
3. Run Sub-macro &#34;Convert to Stack&#34; to Create Z-projections and Hyperstacks of the NMJ Images
<ol>
	<li>Start the graphical interface by selecting Plugins in the toolbar and choose &#34;Drosophila NMJ Morphometrics&#34; in the dropdown menu.</li>
	<li>Define the &#34;Unique File String&#34; setting in the macro&#39;s graphical interface. 
	NOTE: The microscope software uses an identification signature to organize planes and channels when storing stacks as individual &#39;.tiff files. The entered unique file string setting needs to specify the signature assigned by the software to the first plane of the first channel (important: lowest plane and channel number needs to be indicated).</li>
	<li>Select only the sub-macro &#34;Convert to stack&#34; and click &#34;ok&#34; and select the folder where the images are located. If a main directory with several subfolders is selected, all individual &#39;.tiff files within the main directory and subfolder matching the unique file string criteria will be processed.
	<ol>
		<li>If the z-stack only contains one channel, select the box &#34;Channel 1 only&#34;.</li>
	</ol>
	</li>
	<li>Notice that two new files per NMJ image, by default termed as stack_image_name and flatstack_image_name will appear. Store only these stack and flatstack for further analysis. The .tiff file series can be deleted at this point, minimizing the required storage capacities and avoiding potential error sources.</li>
</ol>
4. Run Sub-macro &#34;Define ROI&#34; to Delineate the NMJ Terminal of Interest
<ol>
	<li>Start the graphical interface of &#34;Drosophila NMJ Morphometrics&#34;.</li>
	<li>Select only the checkbox &#34;Define ROI&#34; and press &#34;OK&#34; and select the main directory where the images entitled flatstack_name are stored and press &#34;Select&#34;. The sub-macro &#34;Define ROI&#34; automatically searches through all subfolders within the selected main directory.</li>
	<li>As the first projection opens, select the &#34;Freehand selections&#34; tool in the toolbar.</li>
	<li>Using the mouse draw a selection that exclusively contains the complete NMJ terminal of interest and click &#34;OK&#34; in the window &#34;Define terminal&#34;. The macro will proceed with the next projection.</li>
	<li>Delineate the next ROI and repeat until all ROIs are defined. The ROI image file, named &#34;roi_image_name&#34;, will be stored in the same directory as the previously generated stack and projection images for each of the processed images. The output of this sub-macro is a binary image of the ROI in white on a black background.</li>
</ol>
5. Run Sub-macro &#34;Analyze&#34; to Quantify NMJ Terminal Features
<ol>
	<li>Go to the toolbar, select &#34;Plugins&#34; and use: 
	&#34;Drosophila_NMJ_Morphometrics&#34; when analyzing synapses immunolabeled with anti-Dlg-1 or anti-Hrp (channel 1) together with anti-Brp (channel 2), or &#34;Drosophila_NMJ_Bouton_Morphometrics&#34; when analyzing synapses immunolabeled with anti-Syt or anti-Csp (channel 1) together with Brp (channel 2).
	<ol>
		<li>When one channel image stacks are to be analyzed (the structural channel Dlg-1 or HRP for &#34;Drosophila_NMJ_Morphometrics&#34;, or Syt or Csp for &#34;Drosophila_NMJ__Bouton_Morphometrics&#34;), select the box &#34;Channel 1 only&#34;.</li>
	</ol>
	</li>
	<li>Adjust the scale corresponding to the images to be analyzed.
	<ol>
		<li>If one pixel in the image corresponds to 2.5 &#181;m, indicate Scale-Pixels = 1, Scale-Distance in &#181;m = 2.5. In case both settings are left at 0, the NMJ area, perimeter, length and longest branch length will be expressed in number of pixels.</li>
	</ol>
	</li>
</ol>
<ol start="3">
	<li>If required, adjust the default analysis settings of the macro. Perform adjustments only if the sub-macro &#8220;Analyze&#8221; has previously been run with unsatisfactory results (see end of this section, and section 6 for instructions how to optimize the settings).</li>
	<li>Select the checkboxes &#34;Analyze&#34; and &#34;Wait&#34; and press &#34;OK&#34;.
	<ol>
		<li>Select the &#34;Wait&#34; check box when running the sub-macro &#34;Analyze&#34; on 2 channel images. Otherwise errors in active zone counting can occur due to limited computer capacities .</li>
	</ol>
	</li>
	<li>As a new window &#34;Choose a Directory&#34; opens, select the directory where the images are located and press &#34;select&#34;. The macro will analyze all images stored in the main directory and, if applicable, subsequent folders (using the three files from executing the previous sub-macros: stack_image_name, flatstack_image_name and roi_image_name). The macro processes each image individually and consecutively. This can take several minutes per image stack (depending on the computer capacity).</li>
	<li>After running the macro, note that a new image file named res_image_name for each analyzed synapse stored will be created in the parental folder. The quantitative measurements will be stored as &#34;results.txt&#34; file.</li>
	<li>Inspect all result images to detect and exclude pictures with segmentation errors. Possible segmentation errors are described in Table 3, along with advice how to adjust settings to circumvent these errors. Result images with such segmentation errors are provided as examples in Figure 4. 
	NOTE: When running the macro with the default settings observed in the user interface, there was an accuracy of approximately 95% when macro assessment was compared to manual evaluation17.</li>
</ol>
6. Adjust the Macro Settings to the Images
<ol start="1">
	<li>When more than 5% of images show segmentation errors, explore the different algorithms to define/choose the most suitable macro settings for the images.</li>
	<li>Adjust rolling ball radius value 
	NOTE: The rolling ball radius function subtracts the background of the image. This function is of crucial importance when working with images acquired on fluorescence microscopes and/or when images have high background noise. The subtraction of the background will help the macro&#39;s auto-thresholding steps to produce adequate segmentation of the NMJ terminals.
	<ol>
		<li>Select three NMJ stack_image_name images generated by the sub-macro &#8220;Convert to stack&#8221;. Choose images which are representative for the image dataset.</li>
		<li>In the toolbar, select Image | Color | Split channels. Two image stacks will be created, one representing channel 1 and the other channel 2, respectively and save them.</li>
		<li>Open the image stack belonging to channel 1 open, corresponding to Dlg-1, Hrp, Syt or Csp immunolabeling.</li>
		<li>Run the filter &#34;Subtract background&#34; by selecting &#34;Process&#34; in the toolbar followed by &#34;Subtract Background&#8230;&#34; in the dropdown menu.</li>
		<li>Click on the preview checkbox in the pop-up window and adjust the rolling ball radius to the value most appropriate for the images. The &#8220;Rolling ball radius&#8221; setting should be adjusted to values that increase the contrast between synapse and background (see Figure 5A&#8217;).
		<ol>
			<li>See Figure 5 for an example. In panel A, parts of the synapse show the same grey levels as the background, whereas in Figure 5 panel A&#8217; a &#8220;Rolling ball radius&#8221; of 500 results in strong contrast between the synapse and the background.</li>
		</ol>
		</li>
		<li>Create a z-projection by selecting in the toolbar Image | Stack| Z-projection, chose Projection type = Max Intensity and save the resulting image. When the appropriate value for the rolling ball radius is defined, run the &#8220;Subtract background&#8221; algorithm on the remaining representative images with the same rolling ball radius value. Create the Z-projections and save them (in any directory). 
		NOTE: The rolling ball radius value for 8-bit or RGB images should be at least as large as the radius of the largest object in the image that is not part of the background. For 16-bit and 32-bit images the radius should be inversely proportional to the pixel value range22.</li>
	</ol>
	</li>
	<li>Determine the different auto-thresholds that will be used
	<ol>
		<li>Open the Z-projections saved in the previous step (6.2.6) and Select Image | Adjust | AutoThreshold | Try all.</li>
		<li>As a binary thresholded result image will appear with all different auto-threshold algorithms, determine the most suitable algorithm for the images.
		<ol>
			<li>When running the macro later on, change the threshold in the macro settings accordingly.</li>
			<li>Use more restrictive thresholds such as &#34;RenyiEntrophy&#34; or &#34;Moments&#34; as the NMJ outline threshold and more permissive thresholds such as &#34;Li&#34; to determine the NMJ skeleton, and &#34;Huang&#34; to determine active zones. When images are very sharp with little to no background, use &#34;Huang&#34; as&#34; NMJ outline threshold. Otherwise parts of the synapse might be missing after image segmentation.</li>
			<li>See Figure 5B for an example. Appropriate segmentation of the synapse is obtained with auto-thresholds highlighted by green boxes. Some examples of non-suitable thresholds are highlighted by red boxes (check synapses at high magnification). In the latter, either parts of the synapse are missing or parts of the background are included. See reference 23 for more information.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<ol start="4">
	<li>Determine the maximum size of the small particles 
	NOTE: This function will exclude all particles detected by the NMJ outline threshold and Skeleton threshold that are smaller than the defined value in the &#34;small particles setting&#34; from the analysis. This value is defined in pixels. This function serves as a noise filter and is very useful when high rates of non-uniform background (such as crystals/dust) are present in the obtained images.
	<ol>
		<li>Open the Z-projections saved in step 6.2.6. and set the scale to detect number of pixels via Analyze | Set Scale. Apply the following settings: distance in pixels=1, known distance=1, pixel aspect ratio=1, Unit of length=pixel and press &#34;Ok&#34;. Click on the &#34;Oval selection&#34; tool in the toolbar.</li>
		<li>Using the mouse draw a selection closely surrounding single particles that are present in the immunostaining but do not belong to the NMJ. Press Ctrl+m for a Windows user or cmd+m for Mac users. A result window will open, indicating the area of the particles selected in number of pixels.</li>
		<li>Repeat the previous step several times with several artifacts present in the images to determine the biggest contaminating particle/artifact area. This will be the value to set in the setting when running the macro later on. When running the macro set the &#34;Small Particles Size&#34; as the smallest particle size observed pus a margin of 25%.</li>
		<li>See Figure 5D for an example. The biggest crystal detected has an area of 112 pixels. The &#34;Small particles size&#34; setting, when processing this image with the macro, should be set to 125 - 150.</li>
	</ol>
	</li>
</ol>
<ol start="5">
	<li>Determine minimum bouton size 
	NOTE: This function will exclude all boutons detected by the NMJ outline threshold that are smaller than the defined value from the analysis. This value is defined in pixels.
	<ol>
		<li>Follow the same steps as described in section 6.4, but in this case draw a selection surrounding the smallest boutons present in the NMJ terminal. Choose the smallest area corresponding to the smallest bouton of the measured ones. This is the value to set in the minimum bouton size setting when running the macro later on.</li>
	</ol>
	</li>
</ol>
<ol start="6">
	<li>Define &#34;Maxima noise tolerance&#34; value
	<ol>
		<li>To define the &#8220;Find maxima noise tolerance&#8221; value for the macro, open the channel 2 Z-stack saved in section 6.2.2.</li>
		<li>Go to the plugins tab in the popup menu, select Process | Maximum(3D), and when the maximum_image_name appears (which can take up a few minutes), close the original image stack.</li>
		<li>Select the Maximum&#8230;_image_name (the newly obtained image stack) and select Plugins | Process | Minimum (3D), when the new image Minimum of Maximum&#8230;_image_name apears close the Maximum &#8230; _image_name stack.</li>
		<li>In the toolbar, select Process | Find maxima&#8230;. A new window &#34;Find maxima&#8230;&#34; will open. Click the checkbox &#34;Preview point selection&#8230;&#34; and fill the &#34;Noise tolerance&#34; box with the default macro setting 50. The maxima points will be indicated in the image as little crosses.
		<ol>
			<li>Increase the &#8220;Noise tolerance&#8221; value if observing an excess of annotated active zones, i.e. crosses that are not on top of active zones that are not in focus on the selected stack plane, or false active zones that are detected in the background.
			<ol>
				<li>On the other hand, if observing incompletely annotated active zones, i.e. active zones in focus not being recognized, decrease the &#8220;Maxima noise tolerance&#8221; value. Keep trying different values following this procedure till the crosses are appropriately labeling active zones in focus. Fill the &#8220;Find maxima noise tolerance&#8221; with this value.</li>
				<li>See Figure 5C for an example. Too many active zones are detected. In Figure 5C&#8217; only the active zones in focus are detected when increasing the &#8220;Maxima noise tolerance&#8221; value.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Run the sub-macro &#34;Analyze&#34; for the representative images selected in step 5.1, with the settings defined in the all the previous steps.</li>
	</ol>
	</li>
	<li>Adjust Brp-puncta lower and upper threshold
	<ol>
		<li>Notice that a new file will be appearing after running the macro according to step 6.6, called 2_active_zone_stack_image_name. In this image stack the active zones detected by the &#34;Find maxima&#34; function are indicated by white dots in each plane.</li>
		<li>Open this file by dragging and dropping it into the toolbar and select Image | Stack | Z-project | Projection type = Sum slices. A projection of the 2_active_zone_stack_image_name will be obtained.</li>
		<li>Select Image | Adjust | Threshold. A new window &#34;Threshold&#34; will open. Slide the upper bar to choose a threshold value where, all desired foci/Brp-positive spots are visualized in red. 
		NOTE: If the threshold is set too low, an excess of active zones will be counted. If set too high, a fraction of the active zones will be missed.
		<ol>
			<li>See Figure 5E for an example. When threshold is set to 400, most of the active zones (symbolized as a 1 pixel foci) are not included in the segmentation, since they are not highlighted in red (Figure 5E). When threshold is set to a value of 50 all the active zones are highlighted in red (Figure 5E&#8217;).</li>
		</ol>
		</li>
		<li>Define this value as minimum threshold. Leave &#8220;Upper puncta threshold&#8221; on the maximum value.</li>
		<li>Re-run the sub-macro &#8220;Analyze&#8221; for the representative images with the settings defined in all the previous steps of this section. Critically evaluate the resulting image files and make sure that the segmentation is done properly. If this is not the case readjust the settings according to the nature of the segmentation errors (Figure 4, Table 3).</li>
	</ol>
	</li>
</ol>

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R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. , (2009).</li><li>Dubos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+depletion+of+intellectual+disability+and+Parkinsonism+candidate+gene+ATP6AP2+in+fly+and+mouse+induces+cognitive+impairment+and+neurodegeneration.">Conditional depletion of intellectual disability and Parkinsonism candidate gene ATP6AP2 in fly and mouse induces cognitive impairment and neurodegeneration.</a> Hum Mol Genet. 24, 6736-6755 (2015).</li><li>Nijhof, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila NMJ Morphometrics. , (2017).</li><li>Ferreira, T., Rasband, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ User Guide IJ 1.46. , (2014).</li><li>Ferreira, T., Rasband, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ User Guide IJ 1.46. , (2014).</li><li>Pielage, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+presynaptic+giant+ankyrin+stabilizes+the+NMJ+through+regulation+of+presynaptic+microtubules+and+transsynaptic+cell+adhesion.">A presynaptic giant ankyrin stabilizes the NMJ through regulation of presynaptic microtubules and transsynaptic cell adhesion.</a> Neuron. 58, 195-209 (2008).</li><li>Koch, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+ankyrin+2+is+required+for+synaptic+stability.">Drosophila ankyrin 2 is required for synaptic stability.</a> Neuron. 58, 210-222 (2008).</li><li>Iqbal, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homozygous+and+heterozygous+disruptions+of+ANK3:+at+the+crossroads+of+neurodevelopmental+and+psychiatric+disorders.">Homozygous and heterozygous disruptions of ANK3: at the crossroads of neurodevelopmental and psychiatric disorders.</a> Hum Mol Genet. 22, 1960-1970 (2013).</li><li>Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+efferent+system+and+structure+of+neuromuscular+junctions+in+Drosophila.">Organization of the efferent system and structure of neuromuscular junctions in Drosophila.</a> Int Rev Neurobiol. 75, 71-90 (2006).</li><li>Wan, H. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highwire+regulates+synaptic+growth+in+Drosophila.">Highwire regulates synaptic growth in Drosophila.</a> Neuron. 26, 313-329 (2000).</li><li>Shi, L., Fu, A. K., Ip, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+underlying+maturation+and+maintenance+of+the+vertebrate+neuromuscular+junction.">Molecular mechanisms underlying maturation and maintenance of the vertebrate neuromuscular junction.</a> Trends Neurosci. 35, 441-453 (2012).</li><li>Sutcliffe, B., Forero, M. G., Zhu, B., Robinson, I. M., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-type+specific+functions+of+DNT1,+DNT2+and+Spz+at+the+Drosophila+neuromuscular+junction.">Neuron-type specific functions of DNT1, DNT2 and Spz at the Drosophila neuromuscular junction.</a> PLoS One. 8, e75902 (2013).</li><li>Carpenter, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7, R100 (2006).</li></ol>

The authors have no conflicts of interest to disclose.

&#34;Drosophila NMJ Morphometrics&#34; and &#34;Drosophila NMJ bouton Morphometrics&#34; are powerful tools for researchers interested in evaluating synapse morphology. Manual assessment of NMJ parameters is laborious; it is estimated that the macros would save an experienced researcher up to 15 min/NMJ spent on manual image segmentation. With one to two dozens of synapses evaluated per condition or genotype, this quickly sums up to considerable amounts of saved time, even in small scale studies. When performing large screens, the gain of using high throughput analysis, compared to manual assessment and quantification, can be immense. In addition to increased throughput, the macros readily provide objective analysis; they exclude personal biases that otherwise require blinded experiments as well as interpersonal differences that occur when multiple researchers are involved in the analysis. Finally, the macros provide a sensitive and accurate analysis of NMJ features, allowing the identification of synaptic regulators that cause rather subtle than dramatic NMJ defects and have so far remained unappreciated by the researcher&#39;s eye. Detailed information about validation procedures and the algorithms utilized in the macros are found in the publication Nijhof et al.17.
The functionality of the macros has been validated to appropriately measure morphological features of Drosophila melanogaster NMJs at muscle 4. Subsequently, it was demonstrated that the macros were also suitable to analyze synapses at other muscles in this organism. It is likely that the macros can also be used to measure morphological parameters of NMJ with similar structure in other species, including other Drosophila species and further insects. Even NMJs very distant in evolution, e.g., NMJs of mice, show a quite similar structural conformation29. The macros have not been tested on NMJ preparations from other species but potential users are encouraged to test the macros for such purposes.
It is very important that the user explores the different auto-thresholds and algorithms to define/choose the most suitable macro settings for the images. With these settings, an accuracy of approximately 95% is achieved when macro assessment was compared to manual evaluation. Adjusting the macro settings to properly segment 100% of the images can be a very laborious or even impossible procedure. Therefore, exclusion of images not properly segmented is recommended if their numbers are below 5%. Evidently, if the quality of the images is low, the macros will generate higher ratios of unsatisfactory image segmentations. Low quality images will similarly influence manual evaluation and can therefore not be linked to the performance of the macros. Nonetheless the macros are rather robust as they were designed for images generated on a high content microscope (an automated fluorescence microscope that allows imaging of large numbers of samples)17.
A critical point is that the user visually inspects all result images generated by the macros. This will allow to detect and exclude pictures with unsatisfying segmentation. In section 6 of this protocol, the user is guided how to adjust the settings for correct image segmentation when running the sub-macro &#34;Analyze&#34;. To quickly familiarize with the requirements of the macros and how to adjust the macro settings a folder called &#34;Examples_adjusting macro settings&#34; is included in the macro repository <a href="https://doi.org/10.6084/m9.figshare.2077399.v1">https://doi.org/10.6084/m9.figshare.2077399.v1</a>. Thirteen subfolders, each with examples images obtained at different microscope platforms (high content/confocal/fluorescence microscopes) and different immunostainings, are provided. A PDF entitled &#8220;Examples guide&#8221; is included in the same folder where the settings required for each example are provided, along with a text document providing the expected results and results images.
The macros were designed to process images saved as .tiff separated files, nonetheless some users might have saved their images in a different format. The following website <a href="https://doi.org/10.6084/m9.figshare.2077399.v1">https://doi.org/10.6084/m9.figshare.2077399.v1</a>21 contains a folder named &#34;Drosophila NMJ&#34; where three example files (Example 1 - 3) and the document &#34;Examples Guide&#34; with detailed instructions how to import images into the macro if not stored as .tiff separated files can also be found in the same folder.
Together, &#34;Drosophila NMJ Morphometrics&#34; and &#34;Drosophila NMJ bouton Morphometrics&#34; macros quantify ten different NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ bouton area, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones. This provides a great advantage over so far available tools that can assess only one or a few synaptic features30,31. Multiparametric quantitative analysis bears great potential for new discoveries, e.g., to identify novel regulators that control one up to numerous aspects of synapse biology. It also provides the required resolution to determine genes that coregulate exactly the same or overlapping NMJ features and thus are likely to operate in common molecular pathways. Finally, it opens the possibility to investigate how different synaptic parameters correlate with one another in undisturbed conditions17 and which genes ensure such coordinated morphometric correlations.
Taken together, this protocol illustrates how to use the two macros &#34;Drosophila NMJ Morphometrics&#34; and &#34;Drosophila NMJ Bouton Morphometrics&#34;, which perform objective and sensitive quantification of ten morphological NMJ features in a high-throughput manner.

Cognitive disorders such as intellectual disability, autism spectrum disorder and schizophrenia are often characterized by abnormal synaptic function1,2,3. Synapse morphology and function are tightly intertwined; morphological defects can cause synaptic malfunction and, reversely, aberrant synaptic transmission will impact synaptic maturation and morphology4,5,6.
A number of model organisms have been employed in order to better understand synapse biology and shed light on how synaptic changes affect brain function in health and disease7,8,9. The Drosophila NMJ is an extensively studied and well-established in vivo model for glutamatergic synapse biology10,11. In the past decades, this model has been used for physiological and gene-focused studies, as well as for large scale genetic screens, with the aim to detect morphological differences between NMJs. In particular, forward genetic screens have identified many crucial regulators and mechanisms underlying synapse development and function12,13,14,15,16. However, most of these screens relied on visual assessment of NMJ terminal morphology and on qualitative detection of the synaptic abnormalities or semi-quantitative scoring of few morphological features. As a consequence, rather subtle synaptic morphological abnormalities that are non-obvious to the human eye are easily missed. In order to be able to comprehensively detect quantitative differences, the NMJ has to be accurately evaluated by systematic quantification of the morphological parameters of interest. Measuring NMJ features manually is laborious, especially when there are several NMJ features of interest and/or when performing large-scale genetic screenings. In order to support multiparametric, high-throughput morphological analysis and to achieve objective quantification, two macros &#34;Drosophila NMJ Morphometrics&#34; and &#34;Drosophila NMJ Bouton Morphometrics&#34; were developed17. Both macros run in the open source image analysis software Fiji18, and enable quantification of both confocal and nonconfocal images.
&#34;Drosophila NMJ Morphometrics&#34; measures NMJ terminals immunostained with the postsynaptic marker disc large-1 (Dlg-1) or the presynaptic horseradish peroxidase (Hrp), co-labeled with the active zone marker bruchpilot (Brp). It quantifies nine morphological parameters (further described below): NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the synaptic terminal (Figure 1). Although an algorithm for determining the number of boutons is present in this macro, it did not meet the criteria for accuracy17. To properly assess the number of boutons, it is necessary to use the &#34;Drosophila NMJ Bouton Morphometrics&#34; macro, which is specifically designed to quantify boutons using NMJ preparations immunostained by anti-Synaptotagmin (Syt) or anti-Cysteine string protein (Csp), and co-immunolabeled with Brp. The &#34;Drosophila NMJ Bouton Morphometrics&#34; macro quantifies the following parameters: number of boutons, NMJ bouton area, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones (Figure 2).
The macros consist of 3 sub-macros: (I) &#34;Convert to stack&#34; identifies all available image files and creates Z-hyperstacks and maximum intensity projections of both channels. As output, this macro will generate two new files per synapse called &#34;stack_image_name&#34; and &#34;flatstack_image_name&#34;. II) &#34;Define ROI&#34; will open all maximum projection images &#34;flatstack_image_name&#34; consecutively and present them with the request to manually define the region of interest (ROI) in which the specific synaptic terminal of interest is present. This was implemented to allow exclusion of synapses connecting to adjacent muscles and/or other types of synaptic terminals (such as 1s) that can be present in the images11. (III) &#34;Analyze&#34; applies fully automated analysis to the image area within the borders of the ROI. As a result of this step, the user will obtain two new files: &#34;results.txt&#34; where all the numerical measurement will be annotated and a &#34;res_image_name.tif&#34; where the underlying image segmentations produced by the macro will be illustrated. During image analysis three structures are derived from each synaptic terminal: the NMJ outline, the NMJ skeleton, and the number of Brp-positive active zones. The NMJ outline is used to determine the NMJ area and its perimeter and a subsequent watershed separation provides the number of boutons. From the skeleton, five NMJ features are deduced: the total NMJ length, the sum of the length of the longest continuous path connecting any two end points (longest branch length), the number of unconnected compartments per NMJ (referred to as &#34;islands&#34;), the number of branches, and the number of branching points (one branching point connects three or more branches). The number of active zones is determined in the Brp-channel by counting Brp-positive spots. The annotated NMJ outline (yellow line), the NMJ skeleton (blue line), and the number of Brp-positive active zones (indicated by white foci) are displayed in a results picture and the measurements of the parameters are processed to an (.txt) output file (Figure 3).
Drosophila NMJ Morphometrics&#34; and &#34;Drosophila NMJ Bouton Morphometrics&#34; were first described and extensively validated by Nijhof et al.17. This manuscript focuses on the methodology to analyze NMJ morphology using the macros &#34;Drosophila NMJ Morphometrics&#34; and &#34;Drosophila NMJ Bouton Morphometrics&#34;. Prior to macro-assisted analyses, NMJ dissections and immunostainings need to be performed. These are crucial steps, and the combination of markers used for immunohistochemistry needs to be suitable for macro analyses. These steps are briefly mentioned in section 1 of this protocol and direct the user to references describing in detail the protocols to execute these procedures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Immunostainings</td>
			<td></td>
			<td></td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Mouse anti-discs large 1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AFFN-DLG1-4D6</td>
			<td>1/25 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)</td>
		</tr>
		<tr>
			<td>Rabbit anti-horseradish peroxidase</td>
			<td>Jackson IR</td>
			<td>323-005-021</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Synaptotagmin</td>
			<td>Gift from Hugo Bellen</td>
			<td></td>
			<td>Jan-00</td>
		</tr>
		<tr>
			<td>Mouse anti-Cysteine string protein</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>DCSP-1(ab49)</td>
			<td>1/10 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit)&#160;</td>
		</tr>
		<tr>
			<td>Mouse anti-Bruchpilot</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82</td>
			<td>Jan-50</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 488</td>
			<td>Life technologies</td>
			<td>A11029</td>
			<td>1/200</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 568</td>
			<td>Life technologies</td>
			<td>A11011</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Zenon Alexa Fluor 568 Mouse IgG1 Labeling Kit</td>
			<td>ThermoFisher</td>
			<td>Z25006</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Confocal microscope or fluorescence microscope</td>
			<td>Leica SP5</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio imager</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Mac or Pc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two algorithms for high-throughput and multi-parametric quantification of drosophila neuromuscular junction morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Watch this Scientific Journal Video about Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology at JoVE.com

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

two-algorithms-for-high-throughput-multi-parametric-quantification

Research

JoVE Journal

Neuroscience

9.5K Views.  Radboud University Medical Center. Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ). 

Video: Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...