Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

Summary

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

Abstract

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population.

The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing "Golgi-like" complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills.

Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

Introduction

Neuroanatomy is one of the foundations of neuroscience¹ and recent interest in "connectomics" has renewed enthusiasm for understanding the morphological diversity of neuronal populations and the connections between specific neurons². Methods for labeling and reconstructing neurons have greatly improved with recent innovations, including genetic and virus-mediated circuit tracing approaches^3,4, enabling more comprehensive morphological surveys of neuronal populations⁵. In addition to improvements in labeling individual neurons, quan....

Protocol

Note: The tissue examined in this study was prepared as a part of a separate study⁵. Therefore, all of the experimental methods involving the use of animals have been described in detail in the Experimental Methods section of Briggs et al. (2016). All procedures involving animals conducted as a part of the prior study were approved by the Institutional Animal Care and Use Committees. The steps for injection of virus into the dLGN and histological processing of brain tissue are described b.......

Discussion

Neuroanatomical studies have remained a pillar of neuroscience and recent interest in connectomics and structure-function relationships has renewed enthusiasm for detailed morphological characterization of selective neuronal populations. Traditionally, neuroanatomical studies have relied on qualitative classifications of neurons into morphologically distinct classes of neurons defined by expert neuroanatomists. With advances in the techniques for reconstructing neurons and extracting morphological data, it is now possibl.......

Materials

Name	Company	Catalog Number	Comments
SADΔG-EGFP	E.M. Callaway Laboratory, Salk Institute		Prepared by Dr. F. Osakada. G-deleted rabies virus available through the Salk Institute Viral Core
Recording electrode: platinum/iridium or tungsten	FHC	UEPSGGSE1N2M	Visit website (www.fh-co.com) for alternative order specifications
Nanoject II	Drummod Scientific	3-000-204, 110V	Alternatives: picospritzer, Hamilton syringe
Freezing microtome	Thermo Scientific
DAB	Sigma Aldrich	D5905-50TAB	3,3'-Diaminobenzidine tetrahydrochloride, tablet, 10 mg substrate per tablet. Caution: carcinogen - must be bleached before discarding
Cytochrome C	Sigma Aldrich	C2037-100MG
Catalase	Sigma-Aldich	C9322-5G
Rabbit anti-GFP	Life Technologies/Thermo Fisher	#A-11122	Primary antibody
Biotinylated goat anti-rabbit	Vector Laboratories	#BA-1000	Secondary antibody
Neurolucida System	MicroBrightField		Software for neuron tracing and analysis. http://www.mbfbioscience.com/neurolucida
Neurolucida Explorer	MicroBrightField		Data export software
Microfire Camera	Optronics		2-Megapixel true color microscope camera. http://www.simicroscopes.com/pdfs/microfire.pdf
Nikon E800 Microscope	Nikon Instruments Inc.		Biological research microscope. http://www.microscopyu.com/museum/eclipseE800.html
Matlab	The MathWorks Inc.		Matrix-based computational mathematics software. http://www.mathworks.com
Microsoft Office Excel	Microsoft		Spreadsheet program