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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The Authors have nothing to disclose.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
	</tbody>
</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...

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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 ...

Among several existing protocols for tissue preparation for transmission electron microscopy, we selected methods that yield optimal structural preservation and high resolution micrographs to perform morphometric analysis of synaptic vesicles distribution at presynaptic terminals. The high contrast images obtained with these methods allow to quantify parameters such as distance of synaptic vesicles from the presynaptic membrane number of vesicles docked at the presynaptic membrane and inter vesicle distances. All of these parameters are indicative of synaptic function.Since alterations in the spatial organization of synaptic vesicles may be associated with the disruption of synaptic function, these methods have been used to examine the effects of general anesthetics on synaptic development. These methods could also be used to examine the effects of any drug or treatment on the detailed morphology of synapses to provide insight into their function. Begin by preparing osmium tetroxide for post fixation of rat brain sections previously fixed with paraformaldehyde and glutaraldehyde via vascular perfusion.Sections become rigid after treatment with osmium tetroxide. Therefore tissue handling requires attention from then on. Also, before adding osmium, make sure that your sections are flattened as any fold will likely result in tissue fracture.Open one five milliliter glass ampule of 4%osmium tetroxide and water and drop it inside a brown glass bottle. Add five milliliters of 0.2 molar phosphate buffer and then add an additional 10 milliliters of 0.1 molar phosphate buffer to achieve a 1%final solution. Use a 20 milliliter syringe fitted with a long needle to draw up the 1%osmium tetroxide and then add the osmium tetroxide to a brown glass jar covered with aluminum foil.After making sure that the specimen has been unfolded and flattened, add 1%osmium tetroxide and 0.1 molar PB to the specimen vial. Incubate the specimen in osmium tetroxide for one hour at room temperature. Extract the osmium tetroxide with a micropipet after incubation is finished and rinse the sections as described in the protocol.To prepare uranyl acetate, place a 200 milliliter volumetric glass flask containing 100 milliliters of 70%ethanol on a stirring plate. Add four grams of uranyl acetate to the flask, wrap an aluminum foil to prevent precipitation by light and stir continuously. Dehydrate the sections in 50%ethanol.Stain with the prepared the 4%uranyl acetate for one hour and follow with serial dehydration and rinsing as described in the manuscript. Remove the syringe plunger from a 60 milliliter gavage syringe and cap it with a safety needle. Position the syringe with the open side up and add each ingredient of the resin mix one by one.Finish by adding 525 microliters of DMP30 with a pipet. Moving the plunger of the pipet very slowly as DMP is very viscous. Put the plunger back in the syringe.Invert the syringe a few times to mix the contents. The color will change from yellow to amber. Evacuate the air inside the syringe.Then place it on a rocker with continuous shaking for at least 30 minutes. Add one volume of epoxy resin and one volume of acetone to a scintillation vial and shake to mix. Apply this one to one resin to acetone mixture on the tissue section after the last acetone rinse keeping in covered to avoid acetone evaporation.Remove the one to one mixture of resin and acetone after two to four hours and replace it with full resin and incubate for four hours. Put all resin waste in a collection container under the hood to polymerize and to be disposed of later. Cut two rectangular pieces of clear polychlorotrifluoroethylene film and wipe them with 70%ethanol.Trim them so that there is at least 1.5 centimeter of tissue-free plastic on every side of the section, making sure that they have the same width but the height of the sheet on top is approximately 2/3 of the bottom sheet. Slowly tilt the vial and use a fine camel paintbrush and a flexible micro-spatula to very gently lift the specimen from the bottom. Then carefully move the section along the vial walls and transfer onto the bottom PCTFE sheet.Remove excess resin with a micro-spatula and gently cover the specimen with the top sheet. Gently push out any trapped air bubbles without applying direct pressure onto the tissue section and wipe out the excess resin. Use a solvent resistant pen to label the sheets and place in an oven at 60 degrees celsius for two to three days to polymerize.Gently pull apart the sandwiched PCTFE sheets to open them. Use a solvent resistant pen to mark the side of the sheet that contains the adhering section marking it near the tissue. Use a disc punch to obtain a circle sample of the section making sure that the pen mark is part of the punched out tissue.The pen mark will gleam when light is shined on the specimen. Place the cap of an embedding capsule side up on a capsule holder. Place a drop of resin on the cap and insert the punched disk inside the cap with the tissue section facing up.Insert a capsule into the cap using a gentle corkscrew movement. Use fine tweezers to roll and lower a printed two centimeter long piece of paper label into the capsule, making sure it fits to the curvature of the side walls of the capsule. Pour the embedding resin inside the capsule, fill up to edge of the capsule and use a pointed wooden stick to push the tissue specimen down to the bottom.Place the capsule in the oven at 60 degree celsius for two to three days for polymerization. And then continue with sectioning and staining as described in the protocol. Electron micrograph of satisfactory preservation of neuronal cell structure shows layered cell membranes without breaks.The cytoplasm is finally granular and without empty spaces. Mitochondria are neither swollen nor shrunken with a conserved outer double membrane and intact internal cristae. Optimal preservation of neuronal ultrastructure and fine morphological detail is essential in order to visualize synaptic vesicles and measure their distance from the presynaptic membrane.Synaptic vesicles should be distinct and lined by an unbroken single membrane. In order to facilitate morphometric analysis of synaptic vesicle distribution, presynaptic and postsynaptic membranes should be parallel in their continuity preserved. Electron micrograph of defective preservation of tissue structure shows the distortion and breakage of neuronal cell membranes and the presence of markedly enlarged extracellular spaces.Mitochondria appear distended and have swollen cristae. In another example of defective tissue structure preservation, there were large white empty spaces within the cytoplasm in place of finely granular cytoplasmic substance with enlarged extracellular spaces. An artificial membrane as forall likely resulted from mobilization of lipids during fixation with glutaraldehyde.When working young brain tissue use 1 molar phosphate buffer as a solvent for all your solution so that you keep tonicity as close as possible to that of newborn rat brain and minimize artefactual tissue shrinkage and or swelling. The micrographs obtained with these methods can be used to study the detailed morphology and dimensional structural relations of a number of cellular components other than synaptic vesicles. For example, mitochondria, golgi apparatus and nuclear chromatin.Several steps in this protocol require chemicals that can be toxic. Always work under a fume hood and wear personal protective equipment when handling aldehydes, osmium, uranium and lead compounds.

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JoVE Journal

Neuroscience

6.8K visualizações.  University of Virginia Health System. Entre vários protocolos existentes para preparação de tecidos para microscopia eletrônica de transmissão, selecionamos métodos que produzem preservação estrutural ideal e micrografos de alta resolução para realizar a análise morfósica da distribuição de vesículas sináptica em terminais pré-sinápticos. As imagens de alto contraste obtidas com esses métodos permitem quantificar parâmetros como distância de vesículas sinápticas do número de membrana pré-sináptica de ves...