Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

The goals of this protocol are to describe steps to reconstruct the morphology of virus-labeled neurons and to perform independent, unbiased, cluster analyses on populations of labeled neurons to enable comprehensive characterization of morphological metrics along distinct neuronal subclasses. We outline a novel approach for collecting and analyzing neuroanatomical data to facilitate more comprehensive sampling and unbiased classification of morphologically unique neuronal types within a selective neuronal population. The main advantage of this technique is that it enables large scale analysis of neuronal morphology and utilizes unbiased methods to classify distinct neuronal subclasses.

The most challenging aspects of morphological reconstructions are selecting well isolated neurons to reconstruct, assigning the proper z-depth, and aligning arbors to continue reconstructions across adjacent tissue sections. Begin a reconstruction by selecting a slide bearing a stained 50 micron brain tissue section from a primate stereotaxically injected with modified rabies virus expressing EGFP. Place the slide in the microscope slide holder and focus on a single section of interest using a low magnification objective.

Make sure the camera image is visible in the live image by properly positioning the camera shutter. Choose a reasonably isolated labeled neuron within the brain structure of interest to enable unambiguous reconstruction of dendritic arborization. Switch to 10x magnification and bring the area into focus.

Preferentially choose neurons if the cell body is fully within a single section to accurately estimate its area and roundness. Once a well stained, well isolated neuron is chosen, click the new section button in the software to add the home section containing the cell body to the section manager. Enter the number of sections to include in the reconstruction.

Recommend inserting two to three sections to start. Assign a section thickness of 50 microns. Choose the two x objective then click the contour button in the top toolbar and choose the contour type from the dropdown menu.

Trace the contour of the target structure by using the mouse to click on points along the contour. When finished tracing the contour, right-click the mouse and select close contour from the menu to close the contour. Next, with the view still at two x, click on the desired marker symbol on the left toolbar.

Then click on the center of the target structure to place the marker. Once the contours are complete, select the 40x or 60x objective to trace the outline of the cell body. Then select the neuron tracing button on the top toolbar and select the neuron structure to trace, in this case, cell body, from the dropdown menu.

Trace the cell body by clicking on points around the largest extent of the cell body, adjusting the z-depth as needed to bring the cell body in focus. And right-clicking the mouse to finish the cell body contour. Next, place a different style marker at the center of the cell body contour, ensuring that the marker is roughly at the center in z-depth.

It's critical to establish the correct z-depth in the home section. as well as in adjacent sections, before beginning each tracing so that z-axis values are accurate. To begin tracing dendritic arbors, select dendrite from the top dropdown menu.

Then trace each dendrite starting at the cell body. When a dendrite branches, right-click the mouse and select bifurcating node or trifurcating node to place a node at this branch point. Be sure to adjust the z-depth throughout the tracing to accurately capture the angle and direction of the dendrite.

At the end of the dendrite in this section, right-click the mouse and select ending from the dropdown menu. When all dendritic arbors are traced in the home section, identify those dendritic endings that will likely continue into the adjacent section. And bring these into focus at the appropriate z-depth in the microscope image at 20x.

Include major landmarks nearby such as blood vessels or easily recognizable dendrite patterns or bundles in the microscope image. Next, take a picture of the live image using a digital camera handheld to the computer screen. Then switch the microscope objective to two x and move to the adjacent section on the slide.

Then line up the contours of the previously traced section in the software view with the boundaries of the LGN and TRN in the new section. Return to 20x and align using landmarks. Then, with the aid of the photo of the dendrite endings from the previously traced section, move the tracing to align the dendrites by first using the arrow tool to select the reconstruction.

Then right-click and select move from the dropdown menu and move the tracing accordingly. To rotate the tracing, right-click and select rotate from the dropdown menu and rotate the tracing accordingly. Alternatively, select the match tool from the top tools dropdown and select the number of points to match.

Then click on the ending in the reconstruction and the corresponding continuation point in the live image to match dendritic endings to beginnings. Once the tracing from the previous section is lined up with the dendrites in the new section, add a new section to the section manager as before. Alternatively, simply select the adjacent section that was created previously and adjust the z-depth in the same manner.

Make sure the corresponding new section is selected in the section manager by clicking on the current section. Then, after increasing the magnification to 40x or 60x, select the dendrite using the arrow. Right-click the mouse on the end of the dendrite from the previous section and select add to ending from the dropdown menu.

A prompt will ask whether the continuation is in a new section. Click yes. Then trace the continuation of the dendrite using the mouse as a draw tool.

Take images of the endings in preparation for aligning to the next section. Then add continuations to dendritic tracings until at least three sections are traced for the neuron or until the dendrites can no longer be followed or found. Using an extraction program associated with the neuron reconstruction system, open a completed reconstruction file.

From the edit dropdown menu, select select all objects. Select branch structure analysis from the top analysis toolbar and then click on each tab and select the desired analysis options in each tab. Extract all desired data by clicking the OK button in the analysis window and save in a spreadsheet format by right-clicking on the output windows and selecting export to Excel for further analysis.

This photograph shows a single coronal section through the dorsal LGN of one animal. Cytochrome oxidase staining is used to visualize LGN layers. And GFP staining marks the virus injection site.

The arrow indicates regions of dense, retrogradely labeled, thalamic reticular nucleus neurons. Section orientation is according to the dorsal ventral medial lateral compass illustrated. The scale bar is illustrated in the bottom left corner.

This image shows the contour outlines of the LGN in red and the TRN in maroon for all sections containing virus injection as indicated by the black contours. Yellow stars indicate centers of injection sites. This image shows the 3D rendering of the contours and injection site.

This is a map of the locations of reconstructed TRN neurons color-coded according to cluster assignment within a single aggregate TRN contour shown in maroon. The scale bar represent 500 microns. This image shows aggregate contours of TRN in black and the LGN in gray with five reconstructed TRN neurons colored warm to cool according to their medial lateral position within the TRN.

The scale bar represents 500 microns. These photographs show the detail of the same five TRN neurons with color matched scale bars representing 100 microns. This hierarchical dendrogram tree illustrates linkage distances between 160 reconstructed TRN neurons based on 10 independent morphological metrics and shows the overall results of the cluster analysis.

Three distinct clusters of TRN neurons are illustrated in blue, green, and red. Once these neuronal reconstruction techniques are mastered a single neuron can be completely reconstructed in one to two hours. It is important to constantly monitor and adjust z-depth while reconstructing neurons.

The protocol outlined here is an improvement upon traditional, more biased, neuroanatomical analysis methods and enables researchers to explore novel subclasses of neurons based on large-scale morphological data. After watching this video, you should have a good understanding of how to reconstruct neurons through adjacent tissue sections and extract morphological data for further analysis.