Protocol for Tracing Neurons Using Vaa3D TeraFly

To download Vaa3D, and for the latest information & help visit the Vaa3D website at http://vaa3d.org

This protocol was written to familiarize new users with the Vaa3D TeraFly plug in, which allows for the visualization and analysis of large-scale multidimensional images. In this protocol we describe how to open an image series using TeraFly, offer tips on how to manually trace neurons, and explain how to identify and resolve common tracing errors. The goal of this protocol is to simultaneously accelerate the reconstruction process and reduce the number of errors made during neuron tracing.

Launching TeraFly and Uploading an Image Series:

1. Download Vaa3D from the website mentioned above, then double-click on the vaa3d_msvc.exe file in order to launch the program

2. From the Vaa3D menu bar, select Advanced > Big-Image-Data > TeraFly in order to launch the TeraFly plug-in

3. Once TeraFly is open, use File > Open TeraFly Image (3-5D) to select the desired image series

4. This will launch the 3D View window, from which the user can either start a new annotation file, or load an already existing one by using the “Load annotations” button shown below

Tracing Neurons Using TeraFly:

Three tracing methods:

• BBox trace (Alt+B)
  • This is the most common tracing option and can be used in most instances. It only takes into consideration local space, making it the fastest choice.
• Global trace (Alt+G)
  • This tracing method takes into consideration global space, making it slower than BBox. It’s useful when a dendrite/axon is in dense clusters, when there is low signal, or when a dendrite/axon contains tight curves.
• Polyline (Alt+Y)

  • The Polyline trace uses a series of user-defined markers to create a trace. When neither BBox nor Global trace methods work, Polyline can be a great option. It can be used to make a trace crossing a dense intersection, or when there is strong interfering signal from nearby objects (Figure 1).

    

    Figure 1: In this instance, BBox and Global traces are not effective at tracing Dendrite #1 due to interfering signal from the soma (red marker). Instead, Polyline can be used.

Where to begin:

• In order to ensure that every trace made is accurately marking a dendrite or axon that belongs to the desired neuron, it is advised to start tracing dendrites that have direct connections to the soma and then follow those dendrites outwards (Figure 2). This also facilitates keeping track of bifurcating dendrites, ensuring none of their branches have been overlooked. Once the terminal points of all dendritic branches with the same origin have been reached, the process can be repeated, once again starting from the soma and working outwards.

  

  Figure 2: Tracing should begin at dendrites with direct connections to the soma (yellow markers) and followed outwards.

Making high quality traces:

• Using a high resolution setting is advised to ensure that traces made are as accurate and precise as possible. This is especially important when tracing in high density or clustered areas, or in areas where there is interfering intensity from nearby objects.
• Higher resolution can be achieved in various ways:

1. By double-clicking on a desired area

2. By using the drop-down menu on the TeraFly window

   

3. By placing a marker in the desired area and then zooming in to the marker location

   

• Additionally, marker placement can be a great tool to keep track of bifurcations, dendrites and axons that have been completely traced, and traces that still need to be completed.

  

  Figure 3: On the left, red markers are used to signal unfinished traces. On the right, green markers indicate two terminal endpoints of a dendrite, while a red marker points to a dendritic branch whose trace still needs to be completed.

Identifying synapses, bifurcations, and terminal boutons:

• One of the most common goals for tracing neurons is structure analysis. The correct identification of synapses, bifurcations and terminal boutons is then critical to this goal because mistakes can result in erroneous addition of structure or loss of structure.

• A bifurcation occurs when a dendrite splits into two dendritic branches. These branches always occur in pairs, meaning that a dendrite can never split into more than two branches at a time. These are typically joined by an uninterrupted V-shaped connection (Figure 4).

  • If what seems like three branches are encountered, one of them must be either a synapse or a terminal bouton of another dendrite, careful observation is needed.

  • Overlooking one of the dendritic branches of a bifurcation can lead to significant structure loss that once overlooked is hard to recover because it is not very apparent that a mistake has been made.

    

    Figure 4: Example of a bifurcating dendrite with an uninterrupted V-shape

• Terminal boutons are usually characterized by a short bright intensity, followed by a sudden loss of signal (Figure 5). Being able to identify these neuronal endpoints ensures that no elements of the dendritic branch are missing and guarantees that a neuron’s structure is being captured properly.

  

  Figure 5: Examples of terminal boutons #1 and #2 characterized by a bright intensity followed by sudden loss of signal

• Synapses are perhaps the most difficult neuronal elements to identify. They can be easily confused as dendritic branches, and can occur anywhere along the dendrite or axon length. Synapses tend to be characterized by fuzzy gray areas between two dendrites, a sign that there might be some exchange going on between them (Figure 6). Although identifying synapses also helps accurately capture neuronal structure, synapse data can also be used later when trying to establish relationships within or across neurons

  

  Figure 6: Example of a synapse occurring between two dendrites

Where to find help:

• More help and information about the program can be found by clicking the information button on the main Vaa3D window, imaged below. Here the user can find a list of supported file types, keyboard, mouse and wheel operations, and neuron tracing and editing short keys.

  

Identifying and Resolving Common Tracing Errors:

Questionable intersections:

• It can be difficult to follow the path of dendrites and axons being traced in areas where these neuronal objects intersect. At any given intersection, it can be uncertain whether one dendrite is going over or under another, or if two dendrites are merely making contact due to close proximity, for example.

• In order to construct the most accurate trace, it is advised to use the highest resolution possible and take advantage of observing said intersection with all degrees of view. In addition, manipulating Z-thickness can clear up the path of neuronal objects at an intersection.

• It is important to take these precautions and to guarantee an intersection is traced correctly because misinterpretation could lead to mistakes that are time consuming to fix.

• Examples:

  Figure 7: Example of an intersection where one dendrite (red arrow) is going over another (green dendrite)

  Figure 8: Example of an instance where two dendrites are making contact (yellow and blue arrows) but not making a complete intersection

  Figure 9: Image on the left shows what looks like a bifurcating dendrite. The image on the right shows that after close inspection, this is actually two different dendrites. It is important to view from different perspectives.