We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.
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Method Article
Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales
* These authors contributed equally
In This Article
Summary
Abstract
Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.
Protocol
1. Before You Begin:
1) E18 rat dissection:
Standard dissection methods of E18 hippocampal neurons have previously been described13. In order to use the Bonfire program to analyze the morphological characteristics of the neurites, 8 bit .tif images of individual neurons must be obtained. This can be accomplished in a number of ways depending on the experimental protocol you are following. Neurons can be plated at a low enough density so that single neurons appear in the microscope field. Alternatively, to image individual neurons that are grown in a dense culture, neurons can be transfected using a var....
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Discussion
The Bonfire program is a semi-automated program for the analysis of dendrite and axon morphology. It greatly increases the efficiency and accuracy of Sholl analysis over performing the analysis manually. In addition, the Bonfire program saves the data at every step of the process, making it possible to audit the data and to verify the accuracy of the analysis. Therefore, the task of data analysis can be distributed to numerous individuals without compromising accuracy. Lastly, by performing the analysis on subregions.......
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Disclosures
The authors declare no competing interests. The funding agencies had no scientific role in the development of Bonfire.
Acknowledgements
This work was supported in part by a Busch Biomedical Grant, NSF grant IBN-0548543, NSF grant IBN-0919747, March of Dimes Foundation Grant 1-FY04-107, March of Dimes Foundation Grant 1-FY08-464 (to B.L.F). M.K.K. and C.G.L. were supported by NIH Biotechnology Training Grant T32 GM008339-20, and C.G.L. was also supported by a NJ Commission on Spinal Cord Research Predoctoral Fellowship 08-2941-SCR-E-0.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| NeuronJ plugin | http://www.imagescience.org/meijering/software/neuronj/ | ||
| ImageJ software | http://rsbweb.nih.gov/ij/ | ||
| Bonfire program | http://lifesci.rutgers.edu/~firestein | ||
| NeuronStudio | http://research.mssm.edu/cnic/tools-ns.html | ||
| MatLab Program | Mathworks |
References
- Elston, G. N. Pyramidal cells of the frontal lobe: all the more spinous to think with. J Neurosci. 20, (2000).
- Koch, C., Segev, I. The role of single neurons in information processing. Nat Neurosci. , Suppl 3. 1171-1177 (2000).
- Poirazi, P., Mel, B. W.
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