Automated analysis of NeuronJ tracing data

Abstract

Studies of neuronal differentiation in vitro often involve tracing and analysis of neurites. NeuronJ (Meijering et al., Cytometry Part A 2004;58A:167-176; http://www. imagescience.org/meijering/software/neuronj/) is a program that can be used for semiautomated tracing of individual neurons; when tracing is completed, a text file containing neurite length measurements is generated. Using cultured hippocampal neurons, we have found that to reach statistical significance it is generally necessary to trace about 100 neurons in each treatment group. Posttracing data analysis requires importing each text file into a statistics program. Analysis of distinct parameters, such as effects of a treatment on axonal versus dendritic branching, requires a great deal of time consuming posttracing data manipulation. We have developed XL_Calculations, a Java-based program that performs batch analysis on NeuronJ measurement files and automatically makes multiple calculations, including the number, length, and total output (sum length) of primary, secondary, and tertiary neurites on axons and dendrites, and writes the calculations into an Excel worksheet. Batch processing of NeuronJ measurement files dramatically reduces the time required to analyze neuronal morphology. In addition, our program performs more than 45 distinct calculations, enabling detailed determination of treatment effects on neuronal differentiation. Using this program to analyze NeuronJ tracing data, we demonstrate that continuous exposure of differentiating hippocampal neurons to Netrin 1 increases the number of secondary branches on both axons and dendrites, without significantly altering the length of the axon, dendrites, or branches. Similar results were obtained when neurons were grown on poly-D-lysine or laminin. '

Stages of neuronal morphological development in vitro—An automated assay

“…We took advantage of a tool to quantify morphological development of motor neurons in low density culture with software already at hand. Popko et al (2009) also demonstrate automated staging of hippocampal neurons, though using data obtained from NeuronJ and post-processed through a JAVA-based algorithm. We encourage other investigators to adapt these methods to their own image processing software of choice, with the hope of allowing more standardization and quantitative result reporting in the field of neuronal morphology.…”

“…MetaMorph ® has been used before to identify neurons and quantify neuronal morphology (Klimaschewski et al, 2002;Narayan et al, 2007), as have other software packages such as Neuromantic ® (Myatt and Nasuto, 2009), NeuronJ (Popko et al, 2009) and HCA-Vision (Vallotton et al, 2007). Our selection of MetaMorph ® (as opposed to one of the many other image processing tools available) was motivated simply by the fact that we already had access to the software.…”

Headless Myo10 Is a Negative Regulator of Full-length Myo10 and Inhibits Axon Outgrowth in Cortical Neurons

“…The neurons were imaged on a Nikon TE-2000 microscope with a 20ϫ lens. All processes of the neuron were traced using the ImageJ plugin NeuronJ, and analyzed with XLNJ_Cal-culations, a Java program that performs batch analysis of NeuronJ tracing data (35). For quantification of filopodia number, COS-7 cells were replated 2 h after transfection onto poly-Llysine-coated coverslips.…”

Orbitofrontal cortex 5-HT2A receptor mediates chronic stress-induced depressive-like behaviors and alterations of spine density and Kalirin7

“…For dendritic analysis, 5~8 pyramidal neurons in layer II/III of each rat were selected randomly from the left OFC as described (Gutierrez-Rojas et al, 2013) and imaged with a 100× oil objective. The total dendritic length and the number of dendritic branches per neuron were measured and analyzed as described previously using NeuronJ, a plugin for ImageJ (NIH) (Popko et al, 2009). For analysis of dendritic spine density and area, spines along two secondary and tertiary dendritic segments (20 µm long) from each neuron were selected as described (Jiang-Xie et al, 2014), and they were counted and measured using MetaMorph (Molecular Devices, USA) (Ma et al, 2008b).…”