Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Summary

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Abstract

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

Introduction

We describe methods for the preparation of newborn rat brain tissue to obtain high-quality electron micrographs for in-depth morphometric analysis of synaptic vesicle spatial distribution at nerve terminals^1,2. The high-contrast micrographs that can be obtained by processing specimens following these methods can also be used to study the detailed morphology of a number of cellular components and their dimensional structural relations^3,4.

The transmission electron microscope (TEM) is a powerful tool to study the morphology of....

Protocol

All studies were approved by the Institutional Animal Care and Use Committee at the University of Virginia (Charlottesville, VA) and conducted in accordance with the National Institutes of Health guidelines.

1. Fixation by Vascular Perfusion

NOTE: A general description of the method for rat brain vascular perfusion has been already detailed in this journal¹³ and is beyond the scope of this protocol. However, the following steps.......

Discussion

Handling of tissue sections during specimen preparation for TEM requires a considerable degree of finesse, concentration and patience. When using a micropipette to add and remove solutions, specimens can be sucked into the pipette tip by surface tension, so great care should be taken to avoid tissue damage by the pipette. Also, certain steps of the dehydration sequence can be as quick as 1 min, hence the operator needs to work swiftly to ensure that the next dehydration step is started on time and the specimen does not d.......

Materials

Name	Company	Catalog Number	Comments
4% Osmium tetroxide	Electron Microscopy Sciences	19170	acqueous
50% Glutaraldehyde	Electron Microscopy Sciences	16310	EM grade, acqueous
Aclar 33 C embedding film	Electron Microscopy Sciences	50425-25	7.8 mil thickness, size 8"x10"
BEEM capsule holder	Electron Microscopy Sciences	69916	holds size "00" capsules
BEEM embedding capsules	Electron Microscopy Sciences	70021	size "00", flat
Butler block trimmer	Electron Microscopy Sciences	69945-01
Camel hair paint brush	Electron Microscopy Sciences	65576-01
Disc punch	Electron Microscopy Sciences	77850-09
Embed 812 kit	Electron Microscopy Sciences	14120
Lead acetate	Electron Microscopy Sciences	17600
Lead citrate	Electron Microscopy Sciences	17800
Lead nitrate	Electron microscopy Sciences	17900
Leica UC7 ultracut microtome	Leica
Micro scale	Electron Microscopy Sciences	62091-23
Paraformaldehyde	Electron Microscopy Sciences	19208	EM grade, granular
Precision Thelco laboratory oven	Thelco	51221159
Sodium azide	Sigma-Aldricht	S2002
StatMark pen	Electron Microscopy Sciences	72109-01
Tyrode solution	Electron Microscopy Sciences	11760-05
Uranyl acetate	Electron Microscopy Sciences	22400	powder