Scott W. Emmons emmons@aecom.yu David Hall hall@aecom.yu Meng Xu mxu@aecom.yu

1. NERVOUS SYSTEM RECONSTRUCTION USING ELEGANCE May, 2007 Scott W. Emmons emmons@aecom.yu.edu David Hall hall@aecom.yu.edu Meng Xu mxu@aecom.yu.edu

2. Overview Elegance is a JAVA-based program that facilitates the tracing and reconstruction of neurons across serial section images. The location of a neuron profile in an image is represented by a single point and neuron reconstructions are non-volumetric stick figures. Neuron diagrams created by Elegance show the pathway of the neuron through the tissue or organism and neuron architecture along with the locations of synapses along each branch. Multiple neurons can be traced simultan-eously. To reconstruct a neuron, the locations of structures in images, both neurite profiles and synapses, are entered from the computer screen with the mouse. Coordinates and associated attributes are stored in a MYSQL database. Relationships across images are stored in a Connectivity Table. From the information in the database tables, Elegance draws 2D or 3D neuron diagrams.

3. 2D diagram of a C. elegans sensory neuron dorsal anterior posterior ventral ~1 µm Entrance point into the ganglion section number

4. Diagrams show locations of synapses and synaptic partners

5. Overview of steps in reconstruction: Entering and loading images, defining objects • To begin a reconstruction project using Elegance, the user first enters information about a stack of digitized images (TIFF format). Key information includes section number and directory location. Section number is used to calculate the z coordinate. • To enter information from images into the database, multiple images are loaded into RAM. Elegance displays a specified number of images side-by-side on the computer screen in section number order. The user can scroll through the stack in RAM with left and right arrows. Data can be entered from one image at a time by selecting it. • Double clicking with the mouse anywhere on a selected image creates an object with a unique identification number. The object identifier and its associated X, Y, Z coordinate are entered into an Object Table in the database. (0,0) is at the lower left-hand corner of the image. • There are two object types: neuron and synapse. Double click creates an object of neuron type. Neuron type objects are shown on the screen as a blue square. They are used to identify and mark the centers of neuron profiles. • ALT double click creates an object of synapse type. After entering a synapse object, the program prompts for the kind of synapse, chemical or gap junction, and waits for the identities of the pre and post-synaptic neurons to be entered. To enter this information, while continuing to hold the ALT key, first click on the pre-synaptic neuron object and then on post-synaptic neuron objects. Post-synaptic neurons can be of any number, allowing for polyadic synapses. When the ALT key is released, a red circle appears indicating the location of the synapse object and pre- and post-synaptic objects are listed beside it.

6. Overview of steps in reconstruction: Connecting neurons across sections, creating “contins” and neuron diagrams • Neurons are traced out by connecting neuron type objects in adjacent sections. To connect two objects, in a selected image CTRL click on the first object (it turns green). Select an adjacent image (by clicking anywhere). CTRL click the second object. Elegance draws a line across the images connecting the two objects. On user command, Elegance enters a record in the Connectivity Table consisting of the two object names. • To reconstruct a neuron, the user asks Elegance to calculate a “contin.” A contin is a string of connected neuron objects representing a partially or fully reconstructed neuron. Starting from a user selected neuron object, Elegance queries the Connectivity Table and collects all the objects joined directly to the chosen object or indirectly to it via other objects in a continuous chain, moving through the stack in both directions and along all branches until all ends are found. Elegance assigns a unique contin number to this chain (or reassigns the previous number to a recalculated contin). The contin number and a user assignable alternate name (such as a neuron name) are displayed over the lines connecting objects in the contin. • To obtain a printable diagram of a neuron, the user asks Elegance to first find all the synapses belonging to a particular contin. Then the user asks Elegance to create a diagram of the neuron.

7. The following are a series of screen shots illustrating the stages of reconstruction

8. 1. Appearance of the Elegance screen upon opening the program. Elegance allows for entering a username at login. Multiple users can log in simultaneously and enter data into the database (but they should avoid simultaneously opening the same images). When data is entered or modified in the Object Table, username and date/time are recorded in appropriate fields for every record, so that the person who created or modified the record can be later determined.

10. 2. To enter information about image files, click Image, Enter. Image Number is the key field for the Image Table. Directory tells Elegance where to find the image, Section Number is used to calculate the Z coordinate for neuron diagrams.

12. 3. To select images to be loaded into RAM, click Image, Find and enter criteria for a range of images. In the following example, to see all the images in the PAG series, “P” was entered in the “Image Number” and “Approximately Matches” box. When the list of images appears, select those desired and click Load.

14. 4. The 10 images selected are loaded into RAM and 3 are displayed on the screen. Initially they may be out of order. Click left arrow or right arrow to automatically order the stack and to scroll through it. The selected image is shown by the purple bar at the top. Use Zoom and the other tools at the bottom of the screen to adjust magnification and other aspects of the selected image. These changes are applied to overlays and do not affect the original image. To see different areas, use the arrow as a “hand” tool by holding down the left button while moving the mouse (there must be no selected object in the image, see below). The X and Y coordinates of the mouse arrow are given at the lower left-hand corner. Translations, as well as zoom and other operations, may be applied to all the images in RAM simultaneously by clicking Image and checking “Lock Images.” The images shown here are scans of paper prints from a C. elegans reconstruction project. The prints were previously marked with pen and colored ink.

16. 5. To create a neuron object, double click on a selected image. The object number appears next to the object, and object number and coordinates are entered into the Object Table. Initially, neuron objects are shown as blue squares. After the user has marked all the synapses of a particular profile, this may be indicated by converting the square to a circle (hold m and click the object to “mark” it; hold “u” and click an object to “unmark” it and convert it back to a square). Thus the progress of the reconstruction can be recorded and visualized on the screen. Create a synapse object by holding ALT and double clicking at the location of the synapse. When prompted, select the type of synapse, chemical or gap junction, then, while continuing to hold ALT, click in order the presynaptic object and one or more post-synaptic objects. When ALT is released, this information appears next to the synapse object. Objects, both neuron and synapse types, can be selected and moved. To select an object, click it. It turns amber. To unselect an object, click anywhere away from the object. Delete key deletes a selected object along with its record in the data table. Delete connections to other objects before deleting an object (see below). To move a selected object, hold the mouse button and drag. The new coordinates are updated to the database. All operations on objects (except connections, see below) are updated in the database and refreshed on the screen automatically.

18. Here we see that object 4930 (red 45) is presynaptic (the presynaptic density is in red 45, which contains synaptic vesicles) at two dyadic chemical synapses, one with 1628 (red 68) and 4931 (red 67), and one with 4366 (violet 29) and 4931 (red 67). 4366 (violet 29) is presynaptic at a dyadic chemical synapse with 4929 (red 84) and 4931 red (67).

20. 6. To connect corresponding profiles in adjacent sections, select the first object by CTRLclick (it turns green), then select an adjacent image and CTRL click the second object. Elegance connects the objects with a line. This information is in the overlay only, it is not yet entered into the Connectivity Table. To save the connection, click Save, Connectivities. To delete a connection, follow the same procedure with two connected objects. Elegance responds that these objects are already connected and asks for instructions. Click delete.

22. 7. Calculating a “contin.” Before Elegance can make a neuron diagram, it must collect all connected objects that are part of the neuron from the connectivity table. A chain of connected objects is called a contin. There are two ways to calculate a contin: within Elegance, click an object while holding down “c”, a calculation will be initiated from this object; or, in save menu, choose “calculate” and enter an object number where the calculation should start. If adding newly connected objects to a preexisting contin, start the calculation from a previously included object to preserve the original contin number. As Elegance calculates a contin, it identifies objects that are either branchpoints or ends and assigns a segment number to all the objects in the segment lying between them. Pairs of branchpoint and endpoint objects and the assigned segment number they define are entered in a Segment Endpoint Table, which is used in drawing neuron diagrams. After a calculation, select View, Refresh. Elegance displays contin number, alternate contin name, and segment number over the lines connecting objects on the screen. Here we see that red 45 is contin 211, the neuron HOB, and lies on segment 1; red 67 is contin 201, the neuron EF1, segment 3; and violet 27 is contin 760, the neuron R3BL, segment 8.

24. 8. To calculate a neuron diagram, first calculate the synapse list for that neuron by choosing graphical display menu -> calculate synapses. Elegance places these in a temporary table. Then select the type of diagram desired. 2D-Y displays the Z coordinate (section number) along the X axis and the Y value along the Y axis. 2D-X displays the Z coordinate along the X axis and the X value along the Y axis. For a series in which sectioning was perpendicular to the anteroposterior axis with numbers increasing moving from anterior to posterior, Y represents the dorsoventral axis (numbers increasing ventral to dorsal), and X represents the left/right axis (numbers increasing left to right), which is the case in the examples shown here, 2D-Y is a view of the neuron from the left side, with left/right deviations projected onto the plane of the image, while 2D-X is a view from the top, with dorsoventral deviations projected onto the plane of the image. Versions of these two programs that display the stick figure diagram without synapses are also available. Elegance makes a neuron diagram by connecting endpoints and branchpoints with straight lines. The object number and section number of these key points are displayed. Both axes are scaled to fit the JAVA screen image created. To print the image, select Print Screen and paste the image into another program (like Powerpoint or Microsoft Office Picture Manager). The following example is contin 760, R3BL, 2D-Y diagram.

25. 2D diagram of a C. elegans sensory neuron dorsal anterior posterior ventral ~1 µm Entrance point into the ganglion section number

26. Vertical arrows indicate chemical synapses and their polarity, vertical lines indicate gap junctions. For each synapse, a line running parallel to the neuron branch indicates the sections over which that synapse runs continuously. In the region shown here, R3BL is pre-synaptic to EF1 in several places, and postsynaptic to HOA, HOB, R6BR, and several other neurons.

27. A region of R3BL showing the synapses.

28. List of programs • Elegance • Calculate Contin • Calculate Synapses • 2D-X • 2D-Y • 2D-X without synapses • 2D-Y without synapses

29. Nervous system wiring using Elegance Scott Emmons David Hall Programming: Meng Xu Initial version of software: Metahelix Life Sciences Ltd. Electron micrographs: N. Thomson, D. Albertson Previous Support: NIH, Albert Einstein College of Medicine Current support: Mathers Foundation, Mt. Kisco, NY WE INVITE SOFTWARE USERS!