Labeling Neuronal Morphology Using Custom Diolistic Techniques

Summary

Here, we present a protocol for custom diolistic labeling. The customization of this fluorescent neuronal labeling method provides a modifiable technique that can be adapted to a wide variety of research goals and applications in the analysis of neuronal morphology.

Abstract

Diolistic labeling is increasingly utilized in neuroscience as a highly efficient and reproducible method for the visualization and analysis of neuronal morphology. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery, allows for the non-toxic fluorescent labeling of multiple neurons, including their dendritic arbors and spines, in both living and fixed tissue. Since first described, this novel labeling method has been modified and adapted to fit a variety of research goals and laboratory settings. Diolistic labeling has traditionally relied on the use of a commercially available, hand-held gene gun for the propulsion of coated micro-particles into tissue sections. Recently, laboratory-built biolistic devices have been developed and allow for the increased availability and customization of this method. Here, we discuss one such custom biolistic device and provide a detailed protocol for its use in diolistic labeling. In addition to decreasing the associated costs, the laboratory-built device also overcomes many of the obstacles normally experienced with traditional diolistics, allowing for reliable and reproducible neuronal labeling. The versatility of this method allows for its adaptation to a variety of laboratory settings and neuroscience-related research goals.

Introduction

Three-dimensional morphological reconstructions of individual neurons and their dendritic arbors have served as the bases for analyzing the structure-function relationships within the nervous system^1,2,3. For over a century, the main method for these studies consisted of various modifications of the famed Golgi staining procedure⁴, which has proved invaluable in developing our modern understanding of the nervous system^1,2. However, this method and the various modifications of the silver impregn....

Protocol

All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals. This protocol has been approved by the IACUC at Missouri State University.

NOTE: The following protocol will detail the methods used in the diolistic labeling of fixed brain tissue obtained from rats prepared using transcardial perfusion. Previous studies¹⁶ have utilized similar methods of labeling but without the transcardial perfusion and from various animal species.

1. DiI/T....

Results

Using the procedure presented here, custom diolistic labeling has been used to characterize alterations in neuronal morphology of the lateral/dentate nucleus of the cerebellum. Here, we show representative labeling results from developing rat pups exposed to abnormally high levels of the serotonergic agonist 5-methyloxytryptamine (5-MT), both pre-and postnatally¹⁸. With the aid of quantitative software, dendritic branching morphology (Figure 2), architecture (Figur.......

Discussion

Here, we demonstrate a method of custom diolistic labeling to quantitatively analyze neuronal morphology and synaptic connectivity. The versatility of this method allows for its adaptation to a variety of laboratory settings and research goals. While the results presented here exhibit its use in rat neural tissue, other studies have used diolistic labeling to investigate diverse species through various neuroscience-related applications¹⁶. The method is relatively fast, as it takes 1-2 days from ti.......

Materials

Name	Company	Catalog Number	Comments
Granular Paraformaldehyde	Sigma-Aldrich	P6148-5KG
Sodium Phosphate Monobasic Anhydrous	Sigma-Aldrich	S2554-500G
Sodium Phosphate Dibasic Anhydrous	Sigma-Aldrich	71640-1KG
Type 1-A, Low EEO Gel Agarose,	Sigma Aldrich	A0169-250G
Polyvinylpyrrolidone	Sigma Aldrich	77627-100G
Dichloromethane (Methylene Chloride)	Sigma Aldrich	D65100-1L
Yellow Pipet tips 1000pk	Fisher Scientific	2681151
Corning Transwell Multiple Well Plate with Permeable Polyester Membrane Inserts	Fisher Scientific	07-200-155
Carbocyanine fluorescent DiI 100mg	Invitrogen	D-282
ProLong Gold Antifade	Invitrogen	P36930
1.3-µm Tungsten particles 6G	BioRad	165-2269
Millipore Swinnex Filter Holder	EDMmillipore	SX0001300
Neurolucida Neuron Tracing Software	MicroBrightField	n/a