The morphology of dendrites and the axon determines how a neuron processes and transmits information. Neurite morphology is frequently analyzed by Sholl analysis or by counting the total number of neurites and branch tips. However, the time and resources required to perform such analysis by hand is prohibitive for the processing of large data sets and introduces problems with data auditing and reproducibility. Furthermore, analyses performed by hand or using course-grained morphometric data extraction tools can obscure subtle differences in data sets because they do not store the data in a form that facilitates the application of multiple analytical tools. To address these shortcomings, we have developed a program (titled “Bonfire”) to facilitate digitization of neurite morphology and subsequent Sholl analysis. Our program builds upon other available open-source morphological analysis tools by performing Sholl analysis on subregions of the neuritic arbor, enabling the detection of local level changes in dendrite and axon branching behavior. To validate this new tool, we applied Bonfire analysis to images of hippocampal neurons treated with 25 ng/ml Brain-Derived Neurotrophic Factor (BDNF) and untreated control neurons. Consistent with prior findings, conventional Sholl analysis revealed that global exposure to BDNF increases the number of neuritic intersections proximal to the soma. Bonfire analysis additionally uncovers that BDNF treatment affects both root processes and terminal processes with no effect on intermediate neurites. Taken together, our data suggest that global exposure of hippocampal neurons to BDNF results in a reorganization of neuritic segments within their arbors, but not necessarily a change in their number or length. These findings were only made possible by the neurite-specific Sholl data returned by Bonfire analysis.

Previous studies have shown that dendrites are influenced by substrate stiffness when neurons are plated in either pure or mixed cultures. However, because substrate rigidity can also affect other aspects of culture development known to impact dendrite branching, such as overall cell number, it is unclear whether substrate stiffness exerts a direct or indirect affect on dendrite morphology. In this study, we determine whether substrate stiffness plays a critical role in regulating dendrite branching independent of cell number. We plated primary mixed hippocampal cultures on soft and stiff gels, with Young’s moduli of 1 kPa and 7 kPa, respectively. We found that neurons plated on stiffer substrates showed increased branching relative to neurons grown on softer substrates at the same cell number. On the stiff gels, we also observed a cell number-dependent effect, in which increasing initial plating density decreased dendrite branching This change correlates with an increase in extracellular glutamate. We concluded that both cell number and substrate stiffness play roles in determining dendrite branching, and that the two effects are independent of one another.

Determining the shape of cell-specific dendritic arbors is a tightly regulated process that occurs during development. When this regulation is aberrant, which occurs during disease or injury, alterations in dendritic shape result in changes to neural circuitry. There has been significant progress on characterizing extracellular and intrinsic factors that regulate dendrite number by our laboratory and others. Generally, changes to the dendritic arbor are assessed by Sholl analysis or simple dendrite counting. However, we have found that this general method often overlooks local changes to the arbor. Previously, we developed a program (titled Bonfire) to facilitate digitization of neurite morphology and subsequent Sholl analysis and to assess changes to root, intermediate, and terminal neurites. Here, we apply these different Sholl analyses, and a novel Sholl analysis, to uncover previously unknown changes to the dendritic arbor when we overexpress an important regulator of dendrite branching, cytosolic PSD-95 interactor (cypin), at two developmental time points. Our results suggest that standard Sholl analysis and simple dendrite counting are not sufficient for uncovering local changes to the dendritic arbor.

Neuronal morphology plays a significant role in determining how neurons function and communicate [1][2][3] . Specifically, it affects the ability of neurons to receive inputs from other cells 2 and contributes to the propagation of action potentials 4,5 . 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 network 6,7 . 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 disease 8 , schizophrenia 9,10 , and mental retardation 11 . 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 variations 12 .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 Before You Begin: 1) E18 rat dissection:Standard dissection methods of E18 hippocampal neurons have previously been described 13 . 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 e...