Name: preparation of newborn rat brain tissue for ultrastructural morphometric analysis of synaptic vesicle distribution at nerve terminals
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This manuscript was supported by NIH/NIGMS K08 123321 (to N.L.) and by funds from the Department of Anesthesiology at the University of Virginia. The Authors wish to thank Alev Erisir (Department of Biology, University of Virginia, Charlottesville, VA) for excellent training and technical assistance with TEM, and for her invaluable manuscript criticism. The Authors also thank the Advanced Electron Microscopy facility at the University of Virginia for technical assistance with specimen sectioning and post-staining.

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...

<ol>
	<li>Lunardi, N., Oklopcic, A., Prillaman, M., Erisir, A., Jevtovic-Todorovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+exposure+to+general+anesthesia+disrupts+spatial+organization+of+presynaptic+vesicles+in+nerve+terminals+of+the+developing+subiculum.">Early exposure to general anesthesia disrupts spatial organization of presynaptic vesicles in nerve terminals of the developing subiculum.</a> Molecular Neurobiology. 52 (2), 942-951 (2015).</li><li>Lunardi, N., Ori, C., Erisir, A., Jevtovic-Todorovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anesthesia+causes+long-lasting+disturbances+in+the+ultrastructural+properties+of+developing+synapses+in+young+rats.">General anesthesia causes long-lasting disturbances in the ultrastructural properties of developing synapses in young rats.</a> Neurotoxicity Research. 17 (2), 179-188 (2010).</li><li>Sanchez, V., 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=General+anesthesia+causes+long-term+impairment+of+mitochondrial+morphogenesis+and+synaptic+transmission+in+developing+rat+brain.">General anesthesia causes long-term impairment of mitochondrial morphogenesis and synaptic transmission in developing rat brain.</a> Anesthesiology. 115 (5), 992-1002 (2011).</li><li>Boscolo, A., 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=The+abolishment+of+anesthesia-induced+cognitive+impairment+by+timely+protection+of+mitochondria+in+the+developing+rat+brain:+the+importance+of+free+oxygen+radicals+and+mitochondria+integrity.">The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: the importance of free oxygen radicals and mitochondria integrity.</a> Neurobiology of Disease. 45 (3), 1031-1041 (2012).</li><li>Aghajanian, G. 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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...

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 terminals1,2. 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 relations3,4.
The transmission electron microscope (TEM) is a powerful tool to study the morphology of organelles and other cellular structures quantitatively. As of this decade, there are no other methods of investigation that can provide the same degree of resolution of lipid membranes and organelles without immuno-tagging, with the exception of cryofixation by high pressure freezing. However, freeze substitution techniques are not widely used, and normally require expensive equipment and long preparation times.
In order to take advantage of TEM&#39;s high resolving power, optimal specimen preparation is of paramount importance. The main goals of specimen preparation are to preserve tissue structure with minimum alteration from the living state, enhance specimen contrast, and stabilize the tissue against extraction of cellular components during processing and exposure to the electron beam. Numerous protocols for TEM tissue preparation have been introduced and perfected by several laboratories over the years. Many of them have focused on methods for optimal visualization of synaptic vesicles5,6,7,8,9,10,11. Among a number of well-established, gold standard methods currently in use, we chose procedures for chemical fixation, post fixation, en bloc staining, sequential dehydration, resin embedding and post staining that aim to preserve optimal tissue structure and achieve excellent image contrast. Of note, preservation of fine ultrastructure can be particularly challenging when working with newborn rat brain tissue. In fact, the central nervous system of very young animals is characterized by a higher water content than the adult brain, more prominent enlargement of extracellular spaces, and looser connections between cells12. This makes newborn rat brain tissue profoundly sensitive to changes in osmolarity, and exquisitely prone to artifactual shrinkage and/or swelling when processed through sequential solutions of different tonicity12. Therefore, our methods employed solutions for specimen processing that are of osmolarity as close as possible to that of rat newborn brain. Our goal was to obtain high-quality, high-resolution electron microscopy images for quantitative assessment of synaptic vesicle spatial distribution at nerve terminals. Specifically, we sought to measure the number of vesicles within the nerve terminal, the distance of synaptic vesicles from the pre-synaptic plasma membrane, the number of vesicles docked at the pre-synaptic membrane, the size of synaptic vesicles and the inter-vesicle distances1.
Satisfactory chemical fixation is a prerequisite for obtaining high-quality electron micrographs that can provide the morphological detail necessary to study synaptic vesicle morphology and spatial organization. Although several modes of fixation exist, fixation of brain tissue by vascular perfusion is decidedly superior to other methods. Since fixation via vascular perfusion begins immediately after the arrest of systemic circulation, it shortens the interval between deoxygenation of the brain tissue and cross-linking of proteins with fixatives, resulting in minimum alterations in cell structure. Furthermore, it accomplishes fast and uniform penetration, because of the rapid flow of the fixative from the vascular bed to the extracellular and cellular compartments12,13,14. Primary fixation with glutaraldehyde, followed by secondary fixation (post-fixation) with osmium tetroxide, yields excellent preservation of the fine structure15,16,17. A mixture of glutaraldehyde and paraformaldehyde has the additional advantage of more rapid penetration into the tissue12.
Since biological tissues are not sufficiently rigid to be cut into thin sections without the support of a resin matrix, they need to be embedded in a medium before thin sectioning. Water-immiscible epoxy resins are commonly used as an embedding medium in TEM. When this type of matrix is used, all specimen&#39;s free water must be replaced with an organic solvent before resin infiltration. Water is removed by passing the specimen through a series of solutions of ascending concentrations of ethanol and/or acetone12. In this protocol, specimens are first flat embedded between flexible aclar sheets, then embedded in a capsule. The final result is tissue situated at the tip of a cylindrical resin block, which has the ideal geometry to be least affected by vibrations arising during microtome sectioning.
Staining with heavy metals to enhance endogenous tissue contrast is another important aspect of specimen preparation. Image contrast in TEM is due to electron scattering by the atoms in the tissue. However, biological materials consist largely of low atomic weight molecules (i.e., carbon, hydrogen, oxygen and nitrogen). Therefore, the generation of sufficient scattering contrast requires the incorporation of high atomic weight atoms into the cellular components of the tissue. This is achieved through staining of the specimen with heavy metals12,18,19. Osmium tetroxide, uranium and lead, which bind strongly to lipids, are the most common heavy metals used as electron stains.
Osmium (atomic number 76) is one of the densest metals in existence. It is both a fixative and a stain, although its primary role in TEM is as a reliable fixative12. Among various fixation protocols in use, the method of double fixation with glutaraldehyde followed by osmium is the most effective in reducing the extraction of cell constituents during specimen preparation. These two fixatives are used to stabilize the maximum number of different types of molecules, especially proteins and lipids, and result in superior preservation of tissue ultrastructure12,14,15,16,17.
Uranium (atomic number 92) is the heaviest metal used as electron stain, most typically in the form of uranyl acetate. Similarly to osmium, it acts as a stain and fixative, although its primary role in TEM is as a stain20,21. Nucleic acid-containing and membranous structures are strongly and preferentially stained with uranyl salts in aldehyde-fixed tissues22,23. Treatment of tissues with uranyl acetate after osmication and before dehydration results in stabilization of membranous and nucleic acid-containing structures, as well as enhanced contrast, and permits identification of some structural details that would not be easily detected in specimens stained with osmium alone12,24,25. It is thought that uranyl acetate may stabilize the fine structure by combining with reduced osmium that has been deposited on lipid membranes during osmication24. Maximum contrast is achieved when uranyl acetate is applied before embedding and as a post-stain in thin sections12.
Lead (atomic number 82) is the most common stain used for TEM and is mainly employed for post-staining of thin sections. Lead salts have high electron opacity and show affinity for a wide range of cellular structures, including membranes, nuclear and cytoplasmic proteins, nucleic acids and glycogen26,27. When the double staining method is employed (i.e., staining with uranyl acetate is followed by treatment with lead), the latter acts as a developer of uranyl acetate staining. For instance, lead post-staining of chromatin fixed with glutaraldehyde increases uranyl acetate uptake by a factor of three28,29,30,31,32. Lead also enhances the staining imparted by other metals such as osmium. It is thought that lead salts stain the membranes of osmium-fixed tissues by attaching to the polar group of phosphatides in the presence of reduced osmium33. A potential disadvantage of staining with both uranyl acetate and lead, especially for prolonged durations, is that many different structural elements are stained equally and non-specifically, and thus may not be easily distinguished from one another12.
The recent introduction of alternative light sources, such as in optical super-resolution photo-activated localization microscopy, has significantly improved light microscopy resolution34. However, because light microscopy relies on histochemical and immune-cytochemical methods to visualize individually-labelled proteins or enzymes, the power of TEM to display all structural elements at once remains unsurpassed for in-depth study of the morphology and dimensional relationships of tissue structures. In particular, no other technique can provide the morphological detail necessary to perform morphometric analysis of synaptic vesicle distribution at pre-synaptic nerve boutons. Nevertheless, it is important to note that electron micrographs capture the structure of the tissue after the organism dies, and therefore they cannot provide information regarding the dynamics of pre-synaptic vesicle trafficking and exocytosis. Hence, other tools, such as FM dye-live imaging and patch-clamp electrophysiology, should be considered when the main objective is to study dynamic and/or functional aspects of synaptic vesicle trafficking and exocytosis.

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			<td>4% Osmium tetroxide</td>
			<td>Electron Microscopy Sciences</td>
			<td>19170</td>
			<td>acqueous</td>
		</tr>
		<tr>
			<td>50% Glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16310</td>
			<td>EM grade, acqueous</td>
		</tr>
		<tr>
			<td>Aclar 33 C embedding film</td>
			<td>Electron Microscopy Sciences</td>
			<td>50425-25</td>
			<td>7.8 mil thickness, size 8&#34;x10&#34;</td>
		</tr>
		<tr>
			<td>BEEM capsule holder</td>
			<td>Electron Microscopy Sciences</td>
			<td>69916</td>
			<td>holds size &#34;00&#34; capsules</td>
		</tr>
		<tr>
			<td>BEEM embedding capsules</td>
			<td>Electron Microscopy Sciences</td>
			<td>70021</td>
			<td>size &#34;00&#34;, flat</td>
		</tr>
		<tr>
			<td>Butler block trimmer</td>
			<td>Electron Microscopy Sciences</td>
			<td>69945-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Camel hair paint brush</td>
			<td>Electron Microscopy Sciences</td>
			<td>65576-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Disc punch</td>
			<td>Electron Microscopy Sciences</td>
			<td>77850-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Embed 812 kit</td>
			<td>Electron Microscopy Sciences</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>17600</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Electron Microscopy Sciences</td>
			<td>17800</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead nitrate</td>
			<td>Electron microscopy Sciences</td>
			<td>17900</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica UC7 ultracut microtome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scale</td>
			<td>Electron Microscopy Sciences</td>
			<td>62091-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>19208</td>
			<td>EM grade, granular</td>
		</tr>
		<tr>
			<td>Precision Thelco laboratory oven</td>
			<td>Thelco</td>
			<td>51221159</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldricht</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>StatMark pen</td>
			<td>Electron Microscopy Sciences</td>
			<td>72109-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>11760-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td>powder</td>
		</tr>
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</table>

preparation of newborn rat brain tissue for ultrastructural morphometric analysis of synaptic vesicle distribution at nerve terminals

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.

General criteria that are mostly accepted as indicative of satisfactory or defective preservation of specimen for TEM have been established. These criteria are exemplified in four selected electron micrographs (two examples of optimal preparation, two examples of defective preparation) that were obtained by treating young rat brain tissue following the methods described in this protocol.
In general, a good-quality electron micrograph appears as an orderly, distinct and overall grayish image. I...

Watch this Scientific Journal Video about Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals at JoVE.com

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

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.

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Research

JoVE Journal

Neuroscience

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