Short communication
NeuriteTracer: A novel ImageJ plugin for automated quantification of neurite outgrowth

https://doi.org/10.1016/j.jneumeth.2007.08.029Get rights and content

Abstract

In vitro assays to measure neuronal growth are a fundamental tool used by many neurobiologists studying neuronal development and regeneration. The quantification of these assays requires accurate measurements of neurite length and neuronal cell numbers in neuronal cultures. Generally, these measurements are obtained through labor-intensive manual or semi-manual tracing of images. To automate these measurements, we have written NeuriteTracer, a neurite tracing plugin for the freely available image-processing program ImageJ. The plugin analyzes fluorescence microscopy images of neurites and nuclei of dissociated cultured neurons. Given user-defined thresholds, the plugin counts neuronal nuclei, and traces and measures neurite length. We find that NeuriteTracer accurately measures neurite outgrowth from cerebellar, DRG and hippocampal neurons. Values obtained by NeuriteTracer correlate strongly with those obtained by semi-manual tracing with NeuronJ and by using a sophisticated analysis package, MetaXpress. We reveal the utility of NeuriteTracer by demonstrating its ability to detect the neurite outgrowth promoting capacity of the rho kinase inhibitor Y-27632. Our plugin is an attractive alternative to existing tracing tools because it is fully automated and ready for use within a freely accessible imaging program.

Introduction

Proper formation of neuronal circuitry depends on correct migration of neurons, regulated outgrowth of axons and dendrites, and accurate target selection (Jessell, 1991). Understanding the regulatory mechanisms governing neuronal outgrowth during development and regeneration after injury is therefore of great importance for the development of treatments for both neuropathological disorders and spinal cord injury. Investigation of these mechanisms relies heavily on the use of in vitro neurite outgrowth assays to identify factors that cause inhibition or promotion of neuronal extension. In these assays, the most commonly used measure to assess the ability of a specific agent to affect neurite outgrowth is the length of outgrowth per cell (Ahmed et al., 2006, De Jaco et al., 2002, Fournier et al., 2002, Kleene et al., 2001, Koprivica et al., 2005, Sakurai et al., 1997, Smirnova et al., 2001, W. Zhang et al., 2007).

Both commercially available and public domain methods for automated measurement of neurite outgrowth are available. Commercially available automated analysis systems such as the IN Cell Analyzer (GE Healthcare) or ImageXpress (Molecular Devices) are prohibitively expensive for all but very well funded laboratories. Sophisticated tracing algorithms have been described in the literature (Al-Kofahi et al., 2006, Xiong et al., 2006, Y. Zhang et al., 2007) however the implementation of these algorithms is not readily accessible to biologists with limited programming experience. Neurite outgrowth experiments are therefore often analyzed by labor-intensive manual or semi-manual tracing (Meijering et al., 2004).

To develop an accessible tool for automated tracing, we have written NeuriteTracer, a plugin for the multi-platform free image-processing program, ImageJ (Abramoff et al., 2004). NeuriteTracer processes pairs of neuronal and nuclear marker images to obtain skeletons of neuronal extensions and masks of neuronal nuclei. We have validated the measurements obtained by our plugin by comparison to semi-manual tracing as well as analysis using the “Neuron outgrowth” module of MetaXpress (Molecular Devices Corp., Downingtown, PA) in cerebellar, hippocampal and dissociated dorsal root ganglion (DRG) cultures. We find that values obtained with NeuriteTracer or MetaXpress correlate equally with values obtained using semi-manual tracing. Further, NeuriteTracer is able to detect the increase in outgrowth promoted by the rho kinase inhibitor Y-27632 (Darenfed et al., 2007, Fournier et al., 2003) demonstrating the ability of the program to detect biologically relevant outgrowth changes.

Section snippets

Primary neuronal cultures

All culture materials were from Invitrogen (Burlington, ON) unless otherwise indicated. Total cerebellar neurons from post-natal day 8 (P8) Sprague-Dawley rat pups (Charles River Canada, Saint Constant, Que.) were prepared as previously described (Hsieh et al., 2006). Briefly, chopped cerebella were dissociated with 0.125% trypsin in Hank's balanced salt solution, mechanically triturated and plated on 0.01% poly-l-lysine- (150,000–300,000 MW; Sigma–Aldrich Canada, Oakville, Ont.) coated 96-well

NeuriteTracer: an automated neurite tracing ImageJ plugin

A schematic representation of the steps performed by the plugin is shown in Fig. 1. Given a directory containing pairs of images corresponding to nuclear (Hoechst 33342) and neurite marker (βIII tubulin) images, the plugin opens the images in a nuclear and a neuronal stack. Images are first pre-processed to optimize uniformity of illumination and contrast in the input images. This step consists of subtraction of a background image to reduce artifacts generated by the acquisition system as well

Discussion

We have developed NeuriteTracer, an ImageJ plugin for automated tracing of images from neuronal outgrowth assays. We have demonstrated that NeuriteTracer accurately labels and measures neurites in both complex and simple neuronal cultures and, in relatively pure neuronal cultures, identifies and counts neuronal nuclei. NeuriteTracer is able to detect changes in neurite outgrowth that are physiologically relevant.

Conclusion

NeuriteTracer is a fast simple-to-use ImageJ plugin for the analysis of outgrowth in two-dimensional fluorescence microscopy images of neuronal cultures. The plugin performed well on images from three different types of neurons with distinct morphologies. Furthermore, the sensitivity is sufficient to detect the modest increase in outgrowth induced by treatment with a known outgrowth enhancer, Y-27632. Thus, NeuriteTracer provides an accessible tool for high-throughput screening of neuronal

Acknowledgements

We thank Wiam Belkaid and Dalinda Liazoghli for providing hippocampal cultures for testing NeuriteTracer. This work was supported by grants from the MS Society of Canada, CIHR, and by the Program in Neuroengineering at McGill University. A.E.F. is a Tier 2 Canada Research Chair. A.B.-O. is a recipient of the MSSC Donald Paty Award. M.P. is supported by an MS Society of Canada Post-Doctoral Fellowship.

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