Neuron Tracing

Neurons are highly specialized cells responsible for the transmission of electrical and chemical messages across the nervous system. The key morphological parts of an individual neuron are the cell body, the dendrites, and the axon. The dendrites, often referred to as a dendritic tree due to their arborization pattern, are responsible for receiving information from surrounding neurons or sensory cells, integrating the signals, and then transmitting them to the cell body. The degree of arborization of the dendrite and its location is critical as neurons need to be extremely close to transmit signals to each other. The axon is then responsible for the electrical signal initiation and conduction away from the cell body and towards other neurons or targets, such as muscles or glands. The axonal terminal ends at the synapse, the junction between the neuron and its target. The number of axonal terminals and synapses that a neuron creates depends on the neuronal subtype and its connectivity with the environment. Understanding neuronal structures are critical to understanding specialized cell functions or when comparing healthy versus diseased states.

In this case study, the Automatic Neuron Tracer in ZEISS arivis Pro software is used for neuron tracing to create three-dimensional digital reconstructions of neuronal structures, in a large group of neurons, as well as to automatically quantify several neuronal morphological parameters.

Cell Body Segmentation

The unprocessed image data was imported into ZEISS arivis Pro, and the dataset was visualized in 3D. While the dataset can be analyzed at native data resolution (100%), for this application the resolution was scaled down to 25%. Although this reduces the image details, it increases analysis speed and does not affect the quality of the segmentation trace.

The first part of the processing applies a Denoising function to reduce the image noise. Next, two morphology filters are run in sequence. Morphological filters are non-linear operations used to change the morphology of features in an image, such as enlarging or shrinking structures in the image. The erosion operation erodes away the boundaries of regions of foreground pixels, resulting in areas of foreground pixels shrinking in size. The dilation operation gradually enlarges the boundaries of regions of foreground pixels, resulting in areas of foreground pixels enlarging. The combined application of these two filters, results in the erosion of the neurite structures to highlight the cell body, which is returned to its original size by dilation. The result of the processed image is saved for future use and analysis. The Intensity Threshold Segmenter is then applied to the processed image to create segments based on a specific range of intensities. A Splitting operation follows to divide cell bodies touching each other. The cell body segments will be used as starting points for the automatic Neuron Tracer task, which is the core of the application.

Trace Editing

The neurite trace can be edited in 2D or 3D using the Trace Tool. Trace editing allows the user to join or split traces that are incorrectly created. Skeleton view is used to visualize the trace while editing, allowing you to draw, cut, or merge traces with interactive visualization of the changes in the viewer. Additional traces can be added and connected to pre-existing ones. Once the changes are completed, they can be confirmed by selecting the Apply Changes button.

In this video tutorial for neuron tracing, learn how to segment cell bodies and trace neurites with Automatic Neuron Tracer in ZEISS arivis Pro. The operations discussed include Denoising, Morphology filters, Intensity Thresholder, Splitting, and the Threshold-based reconstruction functions of the Automatic Neuron Tracer. Explore the different tools available for setting up the filters.

Neuronal morphology and visualization

Once the analysis is completed, the reconstruction can be visualized in 3D (Figure 2). The digital reconstruction of neurons can be overlaid with the imaging dataset, allowing correlation of the morphological features of neurons with their functional properties.

Branch Length

Different measurements can be used to quantitatively evaluate the morphological characteristics of the neuronal population. Here you can see a radar chart that highlights the variation in the number of neurites in the neurons traced. On average each neuron has 11 neurites, with most of them having under 20 neurites (Figure 7).

Tortuosity

Another neurite feature critical for neuronal function is tortuosity, which is a measure of the straightness of the neurite. Tortuosity is defined as the ratio of the displacement from root to terminal of the path to the path length. A perfectly straight path has a value of 1, and a tortuous path has a higher value. Neuronal tortuosity is altered in neurodegenerative diseases, which results in reduced connectivity and subsequent loss of function. In this neuron dataset there is variation in the path tortuosity, with the average value being 1.44 (Figure 9).