Name: analyzing dendritic morphology in columns and layers
Uploaded: 2017-03-23T15:00:00
Description: Watch this Scientific Journal Video about Analyzing Dendritic Morphology in Columns and Layers at JoVE.com

This work was supported by the Intramural Research Program of the National Institutes of Health, the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant HD008913 to C.-H.L.), and the Center for Information Technology (P.G.M., N.P., E.S.M., and M.M.).

Note: The protocol contains three sections: dual-view imaging (sections 1 - 3), dendritic tracing and registration (sections 4 - 6), and dendritic analysis (sections 7 - 9) (Figure 1). The codes and example files are provided in Table of Materials/Equipment.
1. Dual-image Acquisition
NOTE: This step is designed to acquire two image stacks of the neuron of interest in two orthogonal (horizontal and frontal) orientations.
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<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiling+of+R7+axons+in+the+Drosophila+visual+system+is+mediated+both+by+transduction+of+an+activin+signal+to+the+nucleus+and+by+mutual+repulsion.">Tiling of R7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion.</a> Neuron. 56 (5), 793-806 (2007).</li><li>Peng, H., Ruan, Z., Long, F., Simpson, J. H., Myers, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V3D+enables+real-time+3D+visualization+and+quantitative+analysis+of+large-scale+biological+image+data+sets.">V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets.</a> Nat Biotechnol. 28 (4), 348-353 (2010).</li><li>Takemura, S. Y., Lu, Z., Meinertzhagen, I. 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Here, we show how to image and analyze dendritic arbors of Drosophila medulla neurons. The first section, dual-view imaging, describes the deconvolution and combination of two image stacks into a high-resolution image stack. The second section, dendrite tracing and registration, describes the tracing and registration of dendrites of medulla neurons to the reference column array. The third section, dendritic analysis, describes the use of custom scripts to analyze dendritic patterns. Together these protocols prov...

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 information processing1,2. In such a column and layer organization, presynaptic neurons of a distinct modality project axons to terminate at a specific layer (so-called layer-specific targeting) and to form an orderly two-dimensional array (so-called topographic map), while postsynaptic neurons extend dendrites of appropriate sizes in specific layers to receive presynaptic inputs of the correct types and numbers. While axonal targeting to layers and columns has been well studied3,4, much less is known about how dendrites are routed to specific layers and expand appropriately sized receptive fields to form synaptic connections with the correct presynaptic partners5. The difficulty of imaging and quantifying dendritic targeting to layers and columns has hindered the study of dendritic development in columnar and laminated brain structures.
Drosophila medulla neurons are an ideal model for studying dendritic routing and circuit assembly in columns and layers. The fly medulla neuropil is organized as a 3D lattice of 10 layers and approximately 750 columns. Each column is innervated by a set of afferents, including photoreceptors R7/R8 and lamina neurons L1 - L5, whose axonal terminals form topographic maps in a layer-specific fashion6. About 38 types of medulla neurons are present in every medulla column and elaborate dendrites in specific layers and with appropriate field sizes to receive inputs from these afferents7. The synaptic circuits in the medulla have been reconstructed at the electron microscopic level; thus, the synaptic partnerships are well established7,8. Furthermore, genetic tools for labeling various types of medulla neurons are available9,10,11. By examining three types of transmedulla (Tm) neurons (Tm2, Tm9 and Tm20), we have previously identified two cell-type-specific dendritic attributes: (i) Tm neurons project dendrites in either the anterior or posterior direction (planar projection direction), depending on the cell types and (ii) dendrites of medulla neurons terminate in specific medulla layers in a cell-type-specific fashion (layer-specific termination)12. Planar projection direction and layer-specific termination are sufficient to differentiate these three types of Tm neurons, while mutations that disrupt Tm responses to layer and column cues affect distinct aspects of these attributes.
Here, we present a complete workflow for examining the dendritic patterning of Drosophila medulla neurons in columns and layers (Figure 1). First, we show a dual-view imaging method, which uses customized software to combine two confocal image stacks to generate high-quality isotropic images. This method requires only conventional confocal microscopy to generate high-quality images that allow for the reliable tracing of dendritic branches, without resorting to super-resolution microscopy, such as STED (Stimulated Emission Depletion) or structural illumination. Second, we present a method for tracing dendritic arbors and for registering the resulting neurite traces to a reference column array. Third, we show the computational methods for extracting information on the planar projection direction and layer-specific termination of dendrites, as well as for deriving estimates for dendritic branching and termination frequencies. Together, these methods allow for the characterization of dendritic patterns in 3D, the classification of cell types based on dendritic morphologies, and the identification of potential defects in mutants.

<table border="0" cellpadding="0" cellspacing="0" width="1632">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Software</td>
			<td style="width:301px;"></td>
			<td style="width:227px;"></td>
			<td style="width:640px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Huygens Professional&#160;</td>
			<td>Scientific Volume Imaging</td>
			<td>version 16.05</td>
			<td>for image deconvolution (https://svi.nl).&#160; commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV</td>
			<td></td>
			<td>version 7.3.0</td>
			<td>for image recombination and registration (http://mipav.cit.nih.gov/.).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV plugin: PlugInDrosophilaRetinalRegistration.class</td>
			<td></td>
			<td></td>
			<td>freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MIPAV plugin: PlugInDrosophilaStandardColumnRegistration.class</td>
			<td></td>
			<td></td>
			<td>freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td>for tracing neurites and assigning reference points for image registration (http://www.bitplane.com). commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaa3D</td>
			<td></td>
			<td></td>
			<td>for visualizing swc files (https://github.com/Vaa3D/release/releases/).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab</td>
			<td>Mathworks</td>
			<td>R2014b</td>
			<td>for morphometric analysis of dendrites (http://www.mathworks.com).&#160; commercial software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab toolbox: TREES1.14</td>
			<td></td>
			<td>v1.14</td>
			<td>for analyzing dendritic morphometric parameters (http://www.treestoolbox.org/download.html).&#160; freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab toolbox: Dendritic_Tree_Toolbox</td>
			<td></td>
			<td>v1.0</td>
			<td>for calculating morphometric parameters (https://science.nichd.nih.gov/confluence/display/snc/Data+collections+for+imagines+combination+and+standardize+column+registration). Freeware</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sample files</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SWC file definition</td>
			<td></td>
			<td></td>
			<td>http://www.neuronland.org/NLMorphologyConverter/MorphologyFormats/SWC/Spec.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The codes and sample files for image combination and registration</td>
			<td></td>
			<td></td>
			<td>https://science.nichd.nih.gov/confluence/display/snc/Data+collections+for+imagines+combination+and+standardize+column+registration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reference point example&#160;</td>
			<td></td>
			<td></td>
			<td>https://science.nichd.nih.gov/confluence/download/attachments/117216914/points.csv?version=1&#38;modificationDate=1471880596000&#38;api=v2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS Windows Windows 7 x64 or Macintosh OS X 10.7 or later</td>
			<td></td>
			<td></td>
			<td>3GHz 64-bit quad-core processor, 16G RAM (minimal)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: Quadro4000&#160; (or above) graphic card</td>
			<td>Nvidia</td>
			<td></td>
			<td>for stereographic visualization of dendrites.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: NVIDIA 3D vision2</td>
			<td>Nvidia</td>
			<td></td>
			<td>http://www.nvidia.com/object/3d-vision-main.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional: 120 Hz LCD display for NVIDIA 3D vision2</td>
			<td></td>
			<td></td>
			<td>http://www.nvidia.com/object/3d-vision-system-requirements.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents for imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24B10 antibody</td>
			<td>The Developmental Studies Hybridoma Bank</td>
			<td>24B10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP Tag Antibody</td>
			<td>Thermofisher Scientific</td>
			<td>G10362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-Rabbit (H+L), Alexa Fluor 488</td>
			<td>Thermofisher Scientific</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-Mouse (H+L), Alexa Fluor 568</td>
			<td>Thermofisher Scientific</td>
			<td>A21124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting&#160;Clay&#160;</td>
			<td>Fisher</td>
			<td>S04179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glasses, high performance, D=0.17mm</td>
			<td>Zeiss</td>
			<td>474030-9000-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing dendritic morphology in columns and layers

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.

Using the dual-view imaging procedure presented here, a fly brain containing sparsely labeled Tm20 neurons was imaged in two orthogonal directions. Prior to imaging, the brain was stained with appropriate primary and secondary antibodies for visualizing membrane-tethered GFP and photoreceptor axons. For imaging, the brain was first mounted in the horizontal orientation (Figure 2A,&#160;B). A GFP-labeled Tm20 neuron and the surrounding photoreceptor axons were imaged using...

Watch this Scientific Journal Video about Analyzing Dendritic Morphology in Columns and Layers at JoVE.com

Analyzing Dendritic Morphology in Columns and Layers

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.

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 ...

The overall goal of this protocol is to analyze dendric routing of adult drosophila medulla neurons in columns and layers to characterize dendric patterns, determine cell types based on morphology, and identify mutants. This method can help answer key questions in the field of neuroscience, including mapping the brain circuitry, and understanding the wiring mechanism of the dendrite. The main advantage of this technique is that it passes down the dendritic properties in three dimensional space, thus improving the understanding of dendritic wiring logic and functions.The goal of this procedure is to acquire two image stacks of the neurons of interest in two orthogonal, horizontal, and frontal orientations. Using the standard techniques, stain dissected fly brains with rabbit anti-GFP and an antibody that labels photoreceptor axons primary antibodies. Then label the primary antibodies with fluorescent secondary antibodies and clear the brain in 70%glycerol and 1X PBS.To mount the brain in the horizontal orientation, transfer a glycerol cleared brain into a 20 microliter drop of antifade mounting medium deposited centrally on the microscope slide. Then on a coverslip, attach small patches of clay at the four corners to prevent the coverslip from crushing the brain. Now under a dissecting microscope, position the brains in the ventral up position.Use the convex dorsal surface of the brain as a landmark to identify the orientation of the brain sample. This will provide a horizontal view of the neuron. Then attach the coverslip.Using a confocal microscope, ideally equipped with GaAsP detectors, obtain the horizontal view image stack with a high numerical aperture objective and digital zoom of 2.5. Acquire at least 180 optical sections, have 512 square pixels, and a step size of 2 microns. After taking the first image stack, remove the coverslip from the slide and reposition the brain in the anterior up position which provides a frontal view.Then using the same technique, make a new image stack of the frontal view of the same neurons imaged in the horizontal view. Be sure to also record the location of the neuron of interest with respect to the medulla neuropal. Identifying the same neurons is feasible under lower magnification.However, if signal is lost because the tissue is deep, just image the ventral half of your brain. Also check if the sample moved while acquiring images by examining the image stacks. Image de convolution and image combining are detailed in the text protocol.The goal of this procedure is to trace neurites and to assign reference points for registration. First open the recombined image file. Then go to edit.Then show display adjustment and turn off the photoreceptor channel, which is red. Next visualize the image in the surpass mode. If the computer is equipped with a stereograph system, turn on stereo and use the quad buffer mode to visualize 3D images.Add new filaments, go to surpass, then filaments, and click on the tab labeled skip automatic creation, edit manually. Then click on the draw tab and select AutoDepth. Next select settings.Toggle the line option and input the appropriate pixel number for better visualization. Then toggle show dendrites, beginning point, and branching point, and set the render quality to 100%Now select the draw tab, and start tracing neurites. Start with the axon, then move to the dendrites.The axon and dendrites of the trans medulla neurons should be easy to differentiate. After tracing the neurites, go back to settings, and de toggle both beginning point and branching point. Then go to surpass, then export.Select object, and save the filament as an inventor file. The next step is to assign reference points. To begin, select show display adjustment and turn on both imaging channels.Then go to surpass, then measurement. And under the edit tab, toggle specific channel, and select the photoreceptor channel in this case. Now assign reference points for the top layer.Select surpass, then measurement points, and mark the beginning of the m1 layer as a top layer. Use the clipping plane function to facilitate the point assignment. The order of the points is equatorial, anterior equatorial, anterior, anterior ventral, ventral, posterior ventral, posterior, posterior equatorial, and center.To find the center photoreceptor, it's the one associated with the most dendritic processes. Next assign the reference points for the R8 and R7 layers using the same technique. After defining each set of reference points, export the coordinates.Select statistics, then detailed, specific values, and position. Then select export statistics on tab display to file, and save the coordinates as a CSV file. Next open the three CSV files in a spreadsheet editor.Copy and paste the coordinates of the 27 reference points into a new file, following the order of top, R8, and then R7.Proceed by following text protocol for rigid body and TPS non-linear registration standardization to right ventral configuration in calculation of dendritic branching and terminating frequencies. Ultimately, the data is used to make various informative plots. Using the dual view imaging procedure presented here, a fly brain containing sparsely labeled tm 20 neurons was imaged in two orthogonal directions.Image recombination improves axial resolution. The recombined image has a better resolution than those of the horizontal view and frontal view images. After consulting 3D models, dendritic traces were collected from 15 tm 20 neurons and compared with dendric traces from 15 tm 2 neurons.The branching and terminating probabilities of these neurons were analyzed. Both types were similar for segments of less than 4 microns. Layer-specific analysis of dendritic termination showed the two neuron types distribute in distinct layer specific manners.Tm 2 dendrites receive inputs in m2 and in m5. While tm 20 dendrites terminate in the m1 to m3 layer. Planar analysis show that tm 2 dendrites project anteriorly, while tm 20 dendrites project posteriorly.After watching this video, you should have a good understanding of how to acquire two of your confocal images. Read your study marks on the imaging files and extract the dendritic properties from these images. While attempting this procedure, it's important to mark the sample properly before confocal imaging.As the imaging combination will fail if the sample moves during imaging.

Research

JoVE Journal

Neuroscience

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...