This work was supported by the King Abdullah University of Science and Technology (KAUST) Competitive Research Grants (CRG) grant &#34;KAUST-BBP Alliance for Integrative Modelling of Brain Energy Metabolism&#34; to P.J.M.

1. Image Processing Using Fiji
<ol>
	<li>Open the image stack by dragging and dropping the native file from the microscope containing the stack, or by dragging and dropping the folder containing the whole image stack into the software window. 
	NOTE: Fiji is able to automatically recognize all the standard image formats, such as .jpg, .tif, and .png, as well as proprietary file formats from microscope providers. While the following protocol has been optimized for image stacks from 3DEM, these steps might be used for light microscopy datasets as well.</li>
	<li>Once the stack is opened, go to Image &#62; Properties to make sure the voxel size has been read from the metadata. If not, it can be manually entered.</li>
	<li>Make sure to transform the image into 8-bit. Click on Image &#62; Type and select 8-bit.</li>
	<li>If the original stack was in different sequential files/folders, use Concatenate to merge them in a single stack by selecting Image &#62; Stacks &#62; Tools &#62; Concatenate.</li>
	<li>Save the stack by selecting File &#62; Save as. It will be saved as a single .tif file and can be used as a backup and for further processing.</li>
	<li>If the original stack is acquired as different tiles, apply stitching within TrakEM2.
	<ol>
		<li>Create a new TrakEM2 project using New &#62; TrakEM2 (blank).</li>
		<li>Within the viewport graphical user interface (GUI), import the stacks by clicking the right mouse button &#62; Import &#62; Import stack, and import all the tiles opened in the Fiji main GUI. Make sure to resize the canvas size to fit the entire stack by right-clicking &#62; Display &#62; Resize Canvas/Layer Set, and choose the y/x pixel size depending on the final size of the montage. 
		NOTE: For instance, if the montage should be finalized using four tiles of 4,096 x 4,096 pixels, the canvas size should be at least 8,192 x 8,192 pixels. Consider make it slightly bigger, and crop it later.</li>
		<li>Superimpose the common portions of individual tiles by dragging and dropping them on the layer set. Use the top left slider to modify the transparency to help with the superimposition.</li>
		<li>Montage and realign the stacks by clicking the right mouse button &#62; Align, and then select one of the options (see note below). 
		NOTE: SBEM or FIB-SEM are usually already well aligned, but minor misalignments might occur. Align layers is the automated pipeline for z alignment; Montage multiple layers is the automated pipeline for aligning tiles on each z stack, for the entire volume; Align multilayer mosaic combines the two previous options. This operation is time-consuming and subject to the random-access memory (RAM) of the machine. Consider using a high-end computer and let it run for hours/days, depending on the size of the stack.</li>
		<li>Finally, export the image stacks and save them by using mouse right click &#62; Make flat image, and then make sure to select the first to the last image in the drop-down menus Start and End.</li>
		<li>Using TrakEM2 embedded functions, segment structures of interest if needed.
		<ol>
			<li>In the TrakEM2&#39;s GUI, right-click on Anything under the Template window and select Add new child &#62; Area_list.</li>
			<li>Drag and drop Anything on top of the folder under Project Objects, and one Anything will appear there.</li>
			<li>Drag and drop the Area list from the template to the Anything located under Project Objects.</li>
			<li>On the image stack viewport, select the Z space with the cursor. The area list will appear, with a unique ID number. Select the brush tool on top (bottom right) and use the mouse to segment a structure by filling its cytosol, over the whole z stack.</li>
			<li>Export the segmented mask to be used in ilastik as seed for carving by right-clicking either on the Area list object on the Z space list, or on the mask in the viewport, and select Export &#62; Area lists as labels (tif).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Depending on the resolution needed for further reconstructions, if possible, reduce the pixel size of the image stack by downsampling, considering the memory requirements of the software that will be used for segmentation and reconstruction. Use Image &#62; Adjust &#62; Size. 
	NOTE: ilastik handles stacks of up to 500 pixels on xy well. Also, downsampling will reduce the resolution of the stack; therefore, take into account the minimum size at which the object still appears recognizable and, thus, can be segmented.</li>
	<li>To enhance contrast and help the segmentation, the Unsharp mask filter can be applied to make membranes crisper. Use Process &#62; Filter &#62; Unsharp mask.</li>
	<li>Export the image stack as single images for further processing in segmentation software, (e.g., ilastik), using File &#62; Save as... &#62; Image sequence and choose .tif format.</li>
</ol>
2. Segmentation (Semi-automated) and Reconstruction Using ilastik 1.3.2
<ol>
	<li>In the main ilastik GUI, select the Carving module.</li>
	<li>Load the image stack using Add New &#62; Add a single 3D/4D volume from sequence.</li>
	<li>Select Whole directory and choose the folder containing the image stack saved as single files. At the bottom of the new window, where options for loading the images are present, make sure to keep Z selected.</li>
	<li>For the following steps, all operations and buttons can be found on the left side of the main software GUI. Under the Preprocessing tab, use the standard options already checked. Use bright lines (ridge filters) and keep the filter scale at 1.600. This parameter can be modified afterward.</li>
	<li>Once the preprocessing is finished, select the next page in the drop-down menu of the Labeling module. One Object and one Background are present by default.</li>
	<li>Select the object seed by clicking on it, and draw a line on top of the structure of interest; then, select the background seed and draw one or multiple lines outside the object to be reconstructed. Then, click on Segment, and wait. 
	NOTE: Depending on the power of the computer and the size of the stack, segmentation could take from a few seconds to hours. Once it&#39;s done, a semitransparent mask highlighting the segmentation should appear on top of the segmented structure.
	<ol>
		<li>Scroll through the stack to check on the segmentation. The segmentation might not be accurate and not follow the structure of interest accurately or spill out from it. Correct any spillover by placing a background seed on the spilled segmentation, and add an object seed over the nonreconstructed segment of the object of interest.</li>
		<li>If the segmentation is still not correct, try to modify with the Bias parameter, which will increase or decrease the amount of uncertain classified pixels as accepted. Its value is 0.95 by default; decrease it to limit any spillover (usually to not less than 0.9) or increase if the segmentation is too conservative (up to 1).</li>
		<li>Another possibility is to go back to step 2.4 (by clicking on Preprocessing) and to modify the size of the filter; increasing the value (e.g., to 2) will minimize the salt-and-pepper noise-like effects but will also make membranes more blurred and smaller details harder to detect. This might limit spillover.</li>
	</ol>
	</li>
	<li>Reiterate as much as needed, as long as all desired objects have been segmented. Once an object is finished, click on Save current object, below Segment. Two new seeds will appear, to start the segmentation of a new object.</li>
	<li>Extract surface meshes right away as .obj files by clicking on Export all meshes.</li>
	<li>If further proofreading is needed, segmentation can be extracted as binary masks. Select the mask to export it by clicking Browse objects; then, right-click on Segmentation &#62; Export and, then, under Output file info, select tif sequence as format.</li>
</ol>
3. Proofreading/Segmentation (Manual) in TrakEM2 (Fiji)
<ol>
	<li>Load the image stack and create a new TrekEM2 project as per step 1.6.1.</li>
	<li>Import the masks that need proofreading using the option Import labels as arealists, available in the same import menu used to import the image stack.</li>
	<li>Select the arealist imported in the Z space and use the Brush tool to review the segmentation.</li>
	<li>Visualize the model in 3D by right-clicking on Area list &#62; Show in 3D. In the following window asking for a resample number, a higher value will generate a lower resolution mesh. 
	NOTE: Depending on the size of the object, consider using a value between 4 and 6, which usually gives the best compromise between resolution and morphological detail.</li>
	<li>Export the 3D mesh as wavefront .obj by choosing from the menu File &#62; Export surfaces &#62; Wavefront.</li>
</ol>
4. 3D Analysis
<ol>
	<li>Open Blender. For the following steps, make sure to install the NeuroMorph toolkit (available at&#160;available at https://neuromorph.epfl.ch/index.html) and the glycogen analysis toolkit (available at https://github.com/daniJb/glyco-analysis).
	<ol>
		<li>Import the objects using the NeuroMorph batch import by clicking Import Objects under the Scene menu, to import multiple objects at once. Make sure to activate Use remesh and Smooth shading. 
		NOTE: It is not recommended to click on Finalize remesh if there is uncertainty about the initial size of the object. Import tree depth at value 7 (default) is usually good to maintain an acceptable resolution and morphology of the object.</li>
		<li>Select the object of interest from the outliner and modify the octree depth of the remesh function under the Modifiers menu iteratively to minimize the number of vertices and to avoid losing details in resolution and correct morphology. When changing the octree depth, the mesh on the main GUI will change accordingly. Once finished, click on Apply to finalize the process. 
		NOTE: Values around 4 are usually good for small objects (such as postsynaptic densities), values around 7 for larger ones (such as long axons or dendrites), and values around 8 or 9 for complete cellular morphologies where details of different resolutions should be maintained.</li>
		<li>Use the image superimposition tool Image stack interactions on the left panel, under the NeuroMorph menu, to load the image stack. Make sure to enter the physical size of the image stack for x, y, and x (in microns or nanometers, depending on the units of the mesh) and select the path of the stack by clicking on Source_Z. X and Y are orthogonal planes; they are optional and will be loaded only if the user inserts a valid path.</li>
		<li>Then, select a mesh on the viewport by right-clicking on it, enter the edit mode by pressing Tab, select one (or more) vertices using mouse right click, and finally, click on Show image at vertex. One (or more) cut plane(s) with the micrograph will appear superimposed on top of the mesh.</li>
		<li>Select the cut plane by right-clicking on it, press Ctrl + Y, and scroll over the 3D model using the mouse scroll. This can also be used as proofreading method.</li>
		<li>Use Measurement tools for quantifications of surface areas, volumes, and lengths. These operations are documented in more details by Jorstad et al.19 and on the NeuroMorph website24.</li>
		<li>Use the Glycogen analysis tool for quantifying glycogen proximity toward spines and boutons. Operations are documented in more details in a previous publication10 and in the glycogen analysis repository25.</li>
	</ol>
	</li>
	<li>Run GLAM23. The code, executable, and some test files are available in the GLAM repository26.
	<ol>
		<li>Prepare two geometry files in .ply format: one source file containing glycogen granules and one target file containing the morphology surfaces.</li>
		<li>Prepare three .ascii colormap files (each line containing t_norm(0..1) R(0..255) G(0..255) B(0..255)) for representing local absorption values, peak values, and average absorption values.</li>
		<li>Execute the C++ script GLAM, with the geometry files (step 4.2.1) and colormap files (step 4.2.2), by setting the influence radius (in microns), the maximum expected absorption value, and a normalized threshold for clustering the absorption peaks. For information about other parameters, execute the script with the -help option.</li>
		<li>Export the results as .ply or .obj files: target objects color-mapped with GLAM values, absorption peak markers represented as color-coded spheres, and target objects color-coded with respect to the mean absorption value.</li>
	</ol>
	</li>
	<li>Open VR Data Interact. VR Data Interact-executable code is available in a public repository27.
	<ol>
		<li>Import meshes to be visualized, following the instructions in the VR menu. Make sure to import the .obj files computed in step 4.2.1, in case a GLAM analysis is needed.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Leighton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEM+images+of+block+faces,+cut+by+a+miniature+microtome+within+the+SEM+-+a+technical+note.">SEM images of block faces, cut by a miniature microtome within the SEM - a technical note.</a> Scanning Electron Microscopy. , 73-76 (1981).</li><li>Knott, G., Marchman, H., Wall, D., Lich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+section+scanning+electron+microscopy+of+adult+brain+tissue+using+focused+ion+beam+milling.">Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling.</a> Journal of Neuroscience. 28, 2959-2964 (2008).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> PLOS Biology. 2, e329 (2004).</li><li>Coggan, J. S., 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+Process+for+Digitizing+and+Simulating+Biologically+Realistic+Oligocellular+Networks+Demonstrated+for+the+Neuro-Glio-Vascular+Ensemble.">A Process for Digitizing and Simulating Biologically Realistic Oligocellular Networks Demonstrated for the Neuro-Glio-Vascular Ensemble.</a> Frontiers in Neuroscience. 12, (2018).</li><li>Tomassy, G. S., 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=Distinct+Profiles+of+Myelin+Distribution+Along+Single+Axons+of+Pyramidal+Neurons+in+the+Neocortex.">Distinct Profiles of Myelin Distribution Along Single Axons of Pyramidal Neurons in the Neocortex.</a> Science. 344, 319-324 (2014).</li><li>Wanner, A. A., Genoud, C., Masudi, T., Siksou, L., Friedrich, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense+EM-based+reconstruction+of+the+interglomerular+projectome+in+the+zebrafish+olfactory+bulb.">Dense EM-based reconstruction of the interglomerular projectome in the zebrafish olfactory bulb.</a> Nature Neuroscience. 19, 816-825 (2016).</li><li>Kasthuri, N., 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=Saturated+Reconstruction+of+a+Volume+of+Neocortex.">Saturated Reconstruction of a Volume of Neocortex.</a> Cell. 162, 648-661 (2015).</li><li>Cal&#236;, C., 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=The+effects+of+aging+on+neuropil+structure+in+mouse+somatosensory+cortex&#8212;A+3D+electron+microscopy+analysis+of+layer+1.">The effects of aging on neuropil structure in mouse somatosensory cortex&#8212;A 3D electron microscopy analysis of layer 1.</a> PLOS ONE. 13, e0198131 (2018).</li><li>Cal&#236;, C., Agus, M., Gagnon, N., Hadwiger, M., Magistretti, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Analysis+of+Glycogen+Derived+Lactate+Absorption+in+Dense+and+Sparse+Surface+Reconstructions+of+Rodent+Brain+Structures.">Visual Analysis of Glycogen Derived Lactate Absorption in Dense and Sparse Surface Reconstructions of Rodent Brain Structures.</a> , (2017).</li><li>Cal&#236;, C., 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=Three-dimensional+immersive+virtual+reality+for+studying+cellular+compartments+in+3D+models+from+EM+preparations+of+neural+tissues.">Three-dimensional immersive virtual reality for studying cellular compartments in 3D models from EM preparations of neural tissues.</a> Journal of Comparative Neurology. 524, 23-38 (2016).</li><li>Fiala, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruct:+a+free+editor+for+serial+section+microscopy.">Reconstruct: a free editor for serial section microscopy.</a> Journal of Microscopy. 218, 52-61 (2005).</li><li>Cardona, 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=TrakEM2+software+for+neural+circuit+reconstruction.">TrakEM2 software for neural circuit reconstruction.</a> PLOS ONE. 7, e38011 (2012).</li><li>Januszewski, M., 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=High-precision+automated+reconstruction+of+neurons+with+flood-filling+networks.">High-precision automated reconstruction of neurons with flood-filling networks.</a> Nature Methods. 1, (2018).</li><li>Shahbazi, 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=Flexible+Learning-Free+Segmentation+and+Reconstruction+of+Neural+Volumes.">Flexible Learning-Free Segmentation and Reconstruction of Neural Volumes.</a> Scientific Reports. 8, 14247 (2018).</li><li>Sommer, C., Straehle, C., K&#246;the, U., Hamprecht, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ilastik:+Interactive+learning+and+segmentation+toolkit.">Ilastik: Interactive learning and segmentation toolkit.</a> , (2011).</li><li>Holst, G., Berg, S., Kare, K., Magistretti, P., Cal&#236;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+large+EM+stack+support.">Adding large EM stack support.</a> , (2016).</li><li>Beyer, 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=ConnectomeExplorer:+query-guided+visual+analysis+of+large+volumetric+neuroscience+data.">ConnectomeExplorer: query-guided visual analysis of large volumetric neuroscience data.</a> IEEE Transactions on Visualization and Computer Graphics. 19, 2868-2877 (2013).</li><li>Beyer, 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=Culling+for+Extreme-Scale+Segmentation+Volumes:+A+Hybrid+Deterministic+and+Probabilistic+Approach.">Culling for Extreme-Scale Segmentation Volumes: A Hybrid Deterministic and Probabilistic Approach.</a> IEEE Transactions on Visualization and Computer. , (2018).</li><li>Jorstad, 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=NeuroMorph:+A+Toolset+for+the+Morphometric+Analysis+and+Visualization+of+3D+Models+Derived+from+Electron+Microscopy+Image+Stacks.">NeuroMorph: A Toolset for the Morphometric Analysis and Visualization of 3D Models Derived from Electron Microscopy Image Stacks.</a> Neuroinformatics. 13, 83-92 (2015).</li><li>Jorstad, A., Blanc, J., Knott, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuroMorph:+A+Software+Toolset+for+3D+Analysis+of+Neurite+Morphology+and+Connectivity.">NeuroMorph: A Software Toolset for 3D Analysis of Neurite Morphology and Connectivity.</a> Frontiers in Neuroanatomy. 12, 59 (2018).</li><li>Cal&#236;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglial+anatomy+in+the+times+of+connectomics.">Astroglial anatomy in the times of connectomics.</a> Journal of Translational Neuroscience. 2, 31-40 (2017).</li><li>Mohammed, H., 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=Abstractocyte:+A+Visual+Tool+for+Exploring+Nanoscale+Astroglial+Cells.">Abstractocyte: A Visual Tool for Exploring Nanoscale Astroglial Cells.</a> IEEE Transactions on Visualization and Computer Graphics. , (2017).</li><li>Agus, M., 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=GLAM:+Glycogen-derived+Lactate+Absorption+Map+for+visual+analysis+of+dense+and+sparse+surface+reconstructions+of+rodent+brain+structures+on+desktop+systems+and+virtual+environments.">GLAM: Glycogen-derived Lactate Absorption Map for visual analysis of dense and sparse surface reconstructions of rodent brain structures on desktop systems and virtual environments.</a> Computers &amp; Graphics. 74, 85-98 (2018).</li><li>&#201;cole Polytechnique F&#233;d&#233;rale de Lausanne. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NeuroMorph Analysis and Visualization Toolset. , (2018).</li><li>. GitHub - daniJb/glycol-analysis: Glycogen_analysis.py is a python-blender API based script that performs analysis on a reconstructed module of glycogen data Available from: <a target="_blank" href="https://github.com/daniJb/glyco-analysis">https://github.com/daniJb/glyco-analysis</a> (2018)</li><li>. GitHub &#8211; magus74/GLAM: Glycogen Lactate Absorption Model Available from: <a target="_blank" href="https://github.com/magus74/GLAM">https://github.com/magus74/GLAM</a> (2019)</li><li>Agus, M., 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=GLAM:+Glycogen-derived+Lactate+Absorption+Map+for+visual+analysis+of+dense+and+sparse+surface+reconstructions+of+rodent+brain+structures+on+desktop+systems+and+virtual+environments.">GLAM: Glycogen-derived Lactate Absorption Map for visual analysis of dense and sparse surface reconstructions of rodent brain structures on desktop systems and virtual environments.</a> Dryad Digital Repository. , (2018).</li><li>Cal&#236;, C., 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=Data+from:+The+effects+of+aging+on+neuropil+structure+in+mouse+somatosensory+cortex&#8212;A+3D+electron+microscopy+analysis+of+layer+1.">Data from: The effects of aging on neuropil structure in mouse somatosensory cortex&#8212;A 3D electron microscopy analysis of layer 1.</a> Dryad Digital Repository. , (2018).</li></ol>

The authors have nothing to disclose.

The method presented here is a useful step-by-step guide for the segmentation and 3D reconstruction of a multiscale EM dataset, whether they come from high-resolution imaging techniques, like FIB-SEM, or other automated serial sectioning and imaging techniques. FIB-SEM has the advantage of potentially reaching perfect isotropy in voxel size by cutting sections as thin as 5 nm using a focused ion beam, its FOV might be limited to 15-20 &#181;m because of side artifacts, which are possibly due to the deposition of the cut tissue if the FOV exceeds this value. Such artifacts can be avoided by using other techniques, such as SBEM, which uses a diamond knife to cut serial sections inside the microscope chamber. In this latter case, the z resolution can be around 20 nm at best (usually, 50 nm), but the FOV might be larger, although the pixel resolution should be compromised for a vast region of interest. One solution to overcome such limitations (magnification vs. FOV) is to divide the region of interest in tiles and acquire each of them at a higher resolution. We have shown here results from both an SBEM stack-dataset (i) in the representative results-and a FIB-SEM stack-dataset (ii) in the representative results.
As the generation of larger and larger datasets is becoming increasingly more common, efforts in creating tools for pixel classification and automated image segmentation are multiplying; nevertheless, to date, no software has proven reliability comparable to that of human proofreading, which is therefore still necessary, no matter how time-consuming it is. In general, smaller datasets that can be downsampled, like in the case of dataset (ii), can be densely reconstructed by a single, expert user in a week, including proofreading time.
The protocol presented here involves the use of three software programs in particular: Fiji (version 2.0.0-rc-65/1.65b), ilastik (version 1.3.2 rc2), and Blender (2.79), which are all open-source and multi-platform programs and downloadable for free. Fiji is a release of ImageJ, powered by plugins for biological image analysis. It has a robust software architecture and is suggested as it is a common platform for life scientists and includes TrakEM2, one of the first and most widely used plugins for image segmentation. One issue experienced by many users lately is the transition from Java 6 to Java 8, which is creating compatibility issues; therefore, we suggest refraining from updating to Java 8, if possible, to allow Fiji to work properly. ilastik is a powerful software providing a number of frameworks for pixel classification, each one documented and explained on their website. The carving module used for the semi-automated segmentation of EM stacks is convenient as it saves much time, allowing scientists to reduce the time spent on manual work from months to days for an experienced user, as with a single click an entire neurite can be segmented in seconds. The preprocessing step is very intense from a hardware point of view, and very large datasets, like the SBEM stack presented here, which was 26 GB, require peculiar strategies to fit into memory, considering that one would acquire large dataset because cannot compromise field of view and resolution. Therefore, downsampling might not be an appropriate solution in this case. The latest release of the software can do the preprocessing in a few hours with a powerful Linux workstation, but the segmentation would take minutes, and scrolling through the stack would still be relatively slow. We still use this method for a first, rough segmentation, and proofread it using TrakEM2. Finally, Blender is a 3D modeling software, with a powerful 3D rendering engine, which can be customized with python scripts that can be embedded in the main GUI as add-ons, such as NeuroMorph and glycogen analysis. The flexibility of this software comes with the drawback that, in contrast to Fiji, for instance, it is not designed for the online visualization of large datasets; therefore, visualizing and navigating through large meshes (exceeding 1 GB) might be slow and not efficient. Because of this, it is always advisable to choose techniques that reduce mesh complexity but are careful not to disrupt the original morphology of the structure of interest. The remesh function comes in handy and is an embedded feature of the NeuroMorph batch import tool. An issue with this function is that, depending on the number of vertices of the original mesh, the octree depth value, which is related to the final resolution, should be modified accordingly. Small objects can be remeshed with a small octree depth (e.g. 4), but the same value might disrupt the morphology of larger objects, which needs bigger values (6 at best, to 8 or even 9 for a very big mesh, such as a full cell). It is advisable to make this process iterative and test the different octree depths if the size of the object is not clear.
As mentioned previously, one aspect that should be taken into account is the computational power to be dedicated to reconstruction and analysis, related to the software that is being used. All the operations shown in the representative results of this manuscript were obtained using a MacPro, equipped with an AMD FirePro D500 Graphics card, 64 GB of RAM, and an Intel Xeon E5 CPU with 8 cores. Fiji has a good software architecture for handling large datasets; therefore, it is recommended to use a laptop with a good hardware performance, such as a MacBook Pro with a 2.5 GHz Intel i7 CPU and 16 GB of RAM. ilastik software is more demanding in terms of hardware resources, in particular during the preprocessing step. Although downsampling the image stack is a good trick to limit the hardware requests from the software and allows the user to process a stack with a laptop (typically if it is below 500 pixels in x,y,z), we suggest the use of a high-end computer to run this software smoothly. We use a workstation equipped with an Intel Xeon Gold 6150 CPU with 16 cores and 500 GB of RAM.
When provided with an accurate 3D reconstruction, scientists can discard the original micrographs and work directly on the 3D models to extract useful morphometric data to compare cells of the same type, as well as different types of cells, and take advantage of VR for qualitative and quantitative assessments of the morphologies. In particular, the use of the latter has proven to be beneficial in the case of analyses of dense or complex morphologies that present visual occlusion (i.e., the blockage of view of an object of interest in the 3D space by a second one placed between the observer and the first object), making it difficult to represent and analyze them in 3D. In the example presented, an experienced user took about 4 nonconsecutive hours to observe the datasets and count the objects. The time spent on VR analysis might vary as aspects like VR sickness (which can, to some extent, be related to car sickness) can have a negative impact on the user experience; in this case, the user might prefer other analysis tools and limit their time dedicated to VR.
Finally, all these steps can be applied to other microscopy and non-EM techniques that generate image stacks. EM generates images that are, in general, challenging to handle and segment, compared with, for instance, fluorescence microscopy, where something comparable to a binary mask (signal versus a black background), that in principle can be readily rendered in 3D for further processing, often needs to be dealt with.

The first proposed model for an electron microscopy setup allowing automated serial section and imaging dates back to 19811; the diffusion of such automated, improved setups to image large samples using EM increased in the last ten years2,3, and works showcasing impressive dense reconstructions or full morphologies immediately followed4,5,6,7,8,9,10.
The production of large datasets came with the need for improved pipelines for image segmentation. Software tools for the manual segmentation of serial sections, such as RECONSTRUCT and TrakEM211,12, were designed for transmission electron microscopy (TEM). As the whole process can be extremely time-consuming, these tools are not appropriate when dealing with the thousands of serial micrographs that can be automatically generated with state-of-the-art, automated EM serial section techniques (3DEM), such as block-face scanning electron microscopy (SBEM)3 or focused ion beam-scanning electron microscopy (FIB-SEM)2. For this reason, scientists put efforts into developing semi-automated tools, as well as fully automated tools, to improve segmentation efficiency. Fully automated tools, based on machine learning13 or state-of-the-art, untrained pixel classification algorithms14, are being improved to be used by a larger community; nevertheless, segmentation is still far from being fully reliable, and many works are still based on manual labor, which is inefficient in terms of segmentation time but still provides complete reliability. Semi-automated tools, such as ilastik15, represent a better compromise, as they provide an immediate readout for the segmentation that can be corrected to some extent, although it does not provide a real proofreading framework, and can be integrated using TrakEM2 in parallel16.
Large-scale segmentation is, to date, mostly limited to connectomics; therefore, computer scientists are most interested in providing frameworks for integrated visualizations of large, annotated datasets and analyze connectivity patterns inferred by the presence of synaptic contacts17,18. Nevertheless, accurate 3D reconstructions can be used for quantitative morphometric analyses, rather than qualitative assessments of the 3D structures. Tools like NeuroMorph19,20 and glycogen analysis10 have been developed to take measurements on the 3D reconstructions for lengths, surface areas, and volumes, and on the distribution of cloud points, completely discarding the original EM stack8,10. Astrocytes represent an interesting case study, because the lack of visual clues or repetitive structural patterns give investigators a hint about the function of individual structural units and consequent lack of an adequate ontology of astrocytic processes21, make it challenging to design analytical tools. One recent attempt was Abstractocyte22, which allows a visual exploration of astrocytic processes and the inference of qualitative relationships between astrocytic processes and neurites.
Nevertheless, the convenience of imaging sectioned tissue under EM comes from the fact that the amount of information hidden in intact brain samples is enormous and interpreting single section images can overcome this issue. The density of structures in the brain is so high that 3D reconstructions of even a few objects visible at once would make it impossible to distinguish them visually. For this reason, we recently proposed the use of virtual reality (VR) as an improved method to observe complex structures. We focus on astrocytes23 to overcome occlusion (which is the blocking of the visibility of an object of interest with a second one, in a 3D space) and ease qualitative assessments of the reconstructions, including proofreading, as well as quantifications of features using count of points in space. We recently combined VR visual exploration with the use of GLAM (glycogen-derived lactate absorption model), a technique to visualize a map of lactate shuttle probability of neurites, by considering glycogen granules as light-emitting bodies23; in particular, we used VR to quantify the light peaks produced by GLAM.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fiji</td>
			<td>Open Source</td>
			<td>2.0.0-rc-65/1.65b</td>
			<td>Open Source image processing editor 
			www.fiji.sc</td>
		</tr>
		<tr>
			<td>iLastik</td>
			<td>Open Source&#160;</td>
			<td>1.3.2 rc2</td>
			<td>Image Segmentation tool 
			www.ilastik.org</td>
		</tr>
		<tr>
			<td>Blender</td>
			<td>Blender Foundation</td>
			<td>2.79</td>
			<td>Open Source 3D Modeling software 
			www.blender.org</td>
		</tr>
		<tr>
			<td>HTC Vive Headset</td>
			<td>HTC</td>
			<td>Vive / Vive Pro</td>
			<td>Virtual Reality (VR) Head monted headset 
			www.vive.com</td>
		</tr>
		<tr>
			<td>Neuromorph</td>
			<td>Open Source</td>
			<td>---</td>
			<td>Collection of Blender Addons for 3D Analysis 
			neuromorph.epfl.ch</td>
		</tr>
		<tr>
			<td>Glycogen Analysis</td>
			<td>Open Source</td>
			<td>---</td>
			<td>Blender addon for analysis of Glycogen 
			https://github.com/daniJb/glyco-analysis</td>
		</tr>
		<tr>
			<td>GLAM</td>
			<td>Open Source</td>
			<td>---</td>
			<td>C++ Code For generating GLAM Maps 
			https://github.com/magus74/GLAM</td>
		</tr>
	</tbody>
</table>

a method for 3d reconstruction and virtual reality analysis of glial and neuronal cells

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction of high-resolution imaged stacks to reveal ultrastructural patterns that could not be resolved using 2D images. Indeed, the latter might lead to a misinterpretation of morphologies, like in the case of mitochondria; the use of 3D models is, therefore, more and more common and applied to the formulation of morphology-based functional hypotheses. To date, the use of 3D models generated from light or electron image stacks makes qualitative, visual assessments, as well as quantification, more convenient to be performed directly in 3D. As these models are often extremely complex, a virtual reality environment is also important to be set up to overcome occlusion and to take full advantage of the 3D structure. Here, a step-by-step guide from image segmentation to reconstruction and analysis is described in detail.

By using the procedure presented above, we show results on two image stacks of different sizes, to demonstrate how the flexibility of the tools makes it possible to scale up the procedure to larger datasets. In this instance, the two 3DEM datasets are (i) P14 rat, somatosensory cortex, layer VI, 100 &#181;m x 100 &#181;m x 76.4 &#181;m4 and (ii) P60 rat, hippocampus CA1, 7.07 &#181;m x 6.75 &#181;m x 4.73 &#181;m10.
The preprocessing steps (Figure 1) can be performed the same way for both datasets, simply taking into account that a larger dataset such as the first stack, which is 25 GB, requires more performing hardware to handle large data visualization and processing. The second stack is only 1 GB, with a perfectly isotropic voxel size.
The size of the data might not be directly related to the field of view (FOV), rather than to the resolution of the stack itself, which depends on the maximum pixel size of the sensor of the microscope, and the magnification of the stack. In any case, logically, larger FOVs are likely to occupy more physical space compared to smaller FOVs, if acquired at the same resolution.
Once the image stack is imported, as indicated in section 1 of the protocol, in Fiji software (Figure 1A), a scientific release of ImageJ12, one important point is to make sure that the image format is 8-bit (Figure 1B). This because many acquisition software different microscopy companies producers generate their proprietary file format in 16-bit to store metadata relevant to information about the acquiring process (i.e., pixel size, thickness, current/voltage of the electron beam, chamber pressure) together with the image stack. Such adjustments allow scientists to save memory, as the extra 8-bit containing metadata does not affect the images. The second important parameter to check is the voxel size, which then allows the reconstruction following the segmentation to be performed on the correct scale (micrometers or nanometers; Figure 1B).
Stacks might need realignment and/or stitching if they have been acquired using tiling; these operations can be performed within TrakEM2 (Figure 2A), although regarding realignment, automated 3DEM techniques like FIB-SEM or 3View are usually well realigned.
One last step requires filtering and, possibly, downsampling of the stack, depending on which objects need to be reconstructed and whether downsampling affects the recognition of the reconstructable features. For instance, for the larger stack (of the somatosensory cortex of P14 rats), it was not possible to compromise the resolution for the benefit of the reconstruction efficiency, while for the smaller stack (of the hippocampus CA1 of P60 rats), it was possible to do so because the resolution was far above what was needed for the smallest objects to be reconstructed. Finally, the use of the unsharp mask enhances the difference between the membranes and the background, making it favorable for the reconstructions of software like ilastik which uses gradients to pre-evaluate borders.
Following the image processing, reconstruction can be performed either manually using TrakEM2, or semi-automatically using ilastik (Figure 2C). A dataset like the smaller one listed here (ii), that can be downsampled to fit into memory, can be fully segmented using ilastik (Figure 2B) to produce a dense reconstruction. In the case of the first dataset listed here (i), we have managed to load and preprocess the entire dataset with a Linux workstation with 500 GB of RAM. The sparse segmentation of 16 full morphologies was obtained with a hybrid pipeline, by extracting rough segmentation that was manually proofread using TrakEM2.
The 3D analysis of features like surface areas, volumes, or the distribution of intracellular glycogen can be performed within a Blender environment (Figure 3) using custom codes, such as NeuroMorph19 or glycogen analysis10.
In case of datasets containing glycogen granules as well, analysis on their distribution can be inferred using GLAM, a C++ code that would generate colormaps with influence area directly on the mesh.
Finally, such complex datasets can be visualized and analyzed using VR, which has been proven to be useful for the analysis of datasets with a particular occluded view (Figure 4). For instance, peaks inferred from GLAM maps were readily inferred visually from dendrites in the second dataset discussed here.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59444/59444fig1.jpg" /> 
Figure 1: Image processing and preparation for image segmentation. (a) Main Fiji GUI. (b) Example of stacked images from dataset (i) discussed in the representative results. The panel on the right shows properties allowing the user to set the voxel size. (c) Example of a filer and resize operation applied to a single image. The panels on the right show magnifications from the center of the image. <a href="https://www.jove.com/files/ftp_upload/59444/59444fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59444/59444fig2.jpg" /> 
Figure 2: Segmentation and reconstruction using TrakEM2 and ilastik. (a) TrakEM2 GUI with objects manually segmented (in red). (b) The exported mask from panel a can be used as input (seed) for (c) semi-automated segmentation (carving). From ilastik, masks (red) can be further exported to TrakEM2 for manual proofreading. (d) Masks can be then exported as 3D triangular meshes to reveal reconstructed structures. In this example, four neurons, astrocytes, microglia, and pericytes from dataset (i) (discussed in the representative results) were reconstructed using this process. <a href="https://www.jove.com/files/ftp_upload/59444/59444fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59444/59444fig3.jpg" /> 
Figure 3: 3D analysis of reconstructed morphologies using customized tools. (a) Isotropic imaged volume from FIB-SEM dataset (ii) (as discussed in the representative results). (b) Dense reconstruction from panel a. Grey = axons; green = astrocytic process; blue = dendrites. (c) Micrograph showing examples of targets for quantifications such as synapses (dataset (i)) and astrocytic glycogen granules (dataset (ii)) in the right magnifications. (d) Mask from panel c showing the distribution of glycogen granules around synapses. (e) Quantification of glycogen distribution from the dataset from panel c, using the glycogen analysis toolbox from Blender. The error bars indicate standard errors. N = 4,145 glycogen granules. (f) A graphic illustration of the input and output visualization of GLAM. <a href="https://www.jove.com/files/ftp_upload/59444/59444fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59444/59444fig4.jpg" /> 
Figure 4: Analysis in VR. (a) A user wearing a VR headset while working on (b) the dense reconstruction from FIB-SEM dataset (ii) (as discussed in the representative results). (c) Immersive VR scene from a subset of neurites from panel b. The green laser is pointing to a GLAM peak. (d) Example of an analysis from GLAM peak counts in VR. N = 3 mice per each bar. Analysis of FIB-SEM from a previous publication28. The error bars indicate the standard errors; *p &#60; 0.1, one-way ANOVA.&#160;<a href="https://www.jove.com/files/ftp_upload/59444/59444fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Serial sectioning and subsequent high-resolution imaging of biological tissue using electron microscopy (EM) allow for the segmentation and reconstruction ...

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12.6K Views.  King Abdullah University of Science and Technology. The described pipeline is designed for the segmentation of electron microscopy datasets larger than gigabytes, to extract whole-cell morphologies. Once the cells are reconstructed in 3D, customized software designed around individual needs can be used to perform a qualitative and quantitative analysis directly in 3D, also using virtual reality to overcome view occlusion.

Video: A Method for 3D Reconstruction and Virtual Reality Analysis of Glial and Neuronal Cells

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major challenge in conducting conditioning studies in humans is the ability to strongly manipulate or simulate the environmental contexts that are associated with conditioned emotional behaviors. In this regard, virtual reality (VR) technology is a promising tool. Yet, adapting this technology to meet experimental constraints requires special accommodations. Here we address the methodological issues involved when conducting fear conditioning in a fully immersive 6-sided VR environment and present fear conditioning data.
In the real world, traumatic events occur in complex environments that are made up of many cues, engaging all of our sensory modalities. For example, cues that form the environmental configuration include not only visual elements, but aural, olfactory, and even tactile. In rodent studies of fear conditioning animals are fully immersed in a context that is rich with novel visual, tactile and olfactory cues. However, standard laboratory tests of fear conditioning in humans are typically conducted in a nondescript room in front of a flat or 2D computer screen and do not replicate the complexity of real world experiences. On the other hand, a major limitation of clinical studies aimed at reducing (extinguishing) fear and preventing relapse in anxiety disorders is that treatment occurs after participants have acquired a fear in an uncontrolled and largely unknown context. Thus the experimenters are left without information about the duration of exposure, the true nature of the stimulus, and associated background cues in the environment1. In the absence of this information it can be difficult to truly extinguish a fear that is both cue and context-dependent. Virtual reality environments address these issues by providing the complexity of the real world, and at the same time allowing experimenters to constrain fear conditioning and extinction parameters to yield empirical data that can suggest better treatment options and/or analyze mechanistic hypotheses.
In order to test the hypothesis that fear conditioning may be richly encoded and context specific when conducted in a fully immersive environment, we developed distinct virtual reality 3-D contexts in which participants experienced fear conditioning to virtual snakes or spiders. Auditory cues co-occurred with the CS in order to further evoke orienting responses and a feeling of "presence" in subjects 2 . Skin conductance response served as the dependent measure of fear acquisition, memory retention and extinction.

Classical fear conditioning paradigm was adapted for human participants in a fully immersive virtual reality setting. Using a discrimination paradigm, conditioned fear, cue and context memory retention, and extinction was measured with skin conductance response to dynamic virtual snakes and spiders (the conditioned stimuli) in two distinct virtual contexts.

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major ...

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through modulation of neural resting membranes. Compared to other techniques, tDCS is relatively safe, cost-effective, and can be administered while individuals are engaged in controlled, specific cognitive processes. This latter point is important as tDCS may predominantly affect intrinsically active neural regions. In an effort to test tDCS as a potential treatment for psychiatric illness, the protocol described here outlines a novel procedure that allows the simultaneous application of tDCS during exposure to trauma-related cues using virtual reality (tDCS+VR) for veterans with posttraumatic stress disorder (NCT03372460). In this double-blind protocol, participants are assigned to either receive 2 mA tDCS, or sham stimulation, for 25 minutes while passively watching three 8-minute standardized virtual reality drives through Iraq or Afghanistan, with virtual reality events increasing in intensity during each drive. Participants undergo six sessions of tDCS+VR over the course of 2-3 weeks, and psychophysiology (skin conductance reactivity) is measured throughout each session. This allows testing for within and between session changes in hyperarousal to virtual reality events and adjunctive effects of tDCS. Stimulation is delivered through a built-in rechargeable battery-driven tDCS device using a 1 (anode) x 1 (cathode) unilateral electrode set-up. Each electrode is placed in a 3 x 3 cm (current density 2.22 A/m2) reusable sponge pocket saturated with 0.9% normal saline. Sponges with electrodes are attached to the participant&#8217;s skull using a rubber headband with the electrodes placed such that they target regions within the ventromedial prefrontal cortex. The virtual reality headset is placed over the tDCS montage in such a way as to avoid electrode interference.

This manuscript outlines a novel protocol to allow the simultaneous application of transcranial direct current stimulation during exposure to warzone trauma-related cues using virtual reality for veterans with posttraumatic stress disorder.

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

An Open-Source Virtual Reality System for the Measurement of Spatial Learning in Head-Restrained Mice

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and optical imaging tools while delivering precise sensory stimuli to a behaving animal. Recently, human and rodent studies using virtual reality (VR) environments have shown VR to be an important tool for uncovering the neural mechanisms underlying spatial learning in the hippocampus and cortex, due to the extremely precise control over parameters such as spatial and contextual cues. Setting up virtual environments for rodent spatial behaviors can, however, be costly and require an extensive background in engineering and computer programming. Here, we present a simple yet powerful system based upon inexpensive, modular, open-source hardware and software that enables researchers to study spatial learning in head-restrained mice using a VR environment. This system uses coupled microcontrollers to measure locomotion and deliver behavioral stimuli while head-restrained mice run on a wheel in concert with a virtual linear track environment rendered by a graphical software package running on a single-board computer. The emphasis on distributed processing allows researchers to design flexible, modular systems to elicit and measure complex spatial behaviors in mice in order to determine the connection between neural circuit activity and spatial learning in the mammalian brain.

Here, we present a simplified open-source hardware and software setup for investigating mouse spatial learning using virtual reality (VR). This system displays a virtual linear track to a head-restrained mouse running on a wheel by utilizing a network of microcontrollers and a single-board computer running an easy-to-use Python graphical software package.

Head-restrained behavioral experiments in mice allow neuroscientists to observe neural circuit activity with high-resolution electrophysiological and ...

Embryonic Mouse Brain Mixed Cell Cultures to Study Infection and Innate Immunity

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Author Spotlight: Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...