Analyzing Dendritic Morphology in Columns and Layers

Chun-Yuan Ting; Philip G. McQueen; Nishith Pandya; Evan S. McCreedy; Matthew McAuliffe; Chi-Hon Lee

doi:10.3791/55410

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Method Article

Analyzing Dendritic Morphology in Columns and Layers

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DOI:

10.3791/55410

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March 23rd, 2017

Chun-Yuan Ting¹ , Philip G. McQueen² , Nishith Pandya³ , Evan S. McCreedy³ , Matthew McAuliffe³ , Chi-Hon Lee¹

¹Section on Neuronal Connectivity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (NIH), ²Mathematical and Statistical Computing Laboratory, Center for Information Technology, National Institutes of Health (NIH), ³Biomedical Imaging Research Services Section, Center for Information Technology, National Institutes of Health (NIH)

Summary

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Abstract

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Introduction

During development, neurons elaborate dendrites in complex but stereotyped branched patterns to form synapses with their presynaptic partners. Dendritic branching patterns correlate with neuronal identity and functions. The locations of dendritic arbors determine the type of presynaptic inputs they receive, while the dendritic branching complexity and field sizes govern the input number. Thus, dendritic morphological properties are critical determinants for synaptic connectivity and neuronal computation. In many regions of complex brains, such as the fly optic lobes and the vertebrate retina, synaptic circuits are organized in columns and layers to facilitate informat....