Name: a micro-ct-based method for characterizing lesions and locating electrodes in small animal brains
Uploaded: 2018-11-08T14:00:00
Description: Watch this Scientific Journal Video about A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains at JoVE.com

The authors thank Greg Lin and Arthur McClelland for their expertise with the micro-CT machine, David Richmond and Hunter Elliott at the Image and Data Analysis Core (IDAC) at Harvard Medical School for their image processing advice, and William Liberti at Boston University for graciously providing a zebra finch brain. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF award no. 1541959. CNS is a part of Harvard University. This work was supported by the Richard and Susan Smith Family Foundation and IARPA (contract #D16PC00002). S.B.E.W. was supported by fellowships from the Human Frontier Science Program (HFSP; LT000514/2014) and the European Molecular Biology Organization (EMBO; ALTF1561-2013). G.G. was supported by the National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP).

All care and experimental manipulation of animals were reviewed and approved by the Harvard Institutional Animal Care and Use Committee. The perfusion described here is specific for rats, but the procedure is applicable to any animals with smaller or similarly sized brains.
1. Perfusion
<ol>
	<li>Prepare 1x phosphate-buffered saline (PBS). For a rat (age: 0.5&#8211;1.5 years old, weight: 250&#8211;600 g), 800&#8211;1,000 mL should be sufficient. Use 400 mL to perfuse the animal and an additi...

<ol>
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The following are critical steps to the protocol: first, the use of a combination of PFA and GA to perfuse the animal and subsequently post-fix the brain was paramount to achieving consistent full osmium penetration of the tissue. Although we did not test this explicitly, a plausible explanation is that PFA fixation is reversible15, whereas GA fixation is not reversible16,17. Because a two-week incubation in osmium tetroxide is required fo...

Neuroscientists have relied on lesions for a long time in order to understand the relationship between function and location in the brain. For example, our understanding of the hippocampus as being indispensable for learning and memory and of the prefrontal cortex as being key for impulse control were both products of serendipitous lesions in humans1,2. The use of animal models, however, has allowed neuroscientists to harness the power of lesions by going beyond serendipity, and the function of countless brain areas has been elucidated through systematic studies of structure-function relationships through lesions3,4.
To correctly assign function to a structure, however, lesion studies require precise quantification procedures, which is an area that has been lacking. The current gold standard for quantifying lesions is to section, mount, and image brains with a light microscope. The imaged slices are then matched to the closest sections on an atlas, and the approximate coordinates of the lesions across subjects are indirectly reported, often through the use of camera lucida images or example histological slices3,4,5,6,7,8,9,10.
Beyond the imprecision of current lesion quantification procedures, these techniques are time-consuming and prone to failure. Small changes in brain stiffness, blade sharpness, and temperature can lead to botched, warped, or torn sections. Sections can also stain unevenly and be improperly imaged because of bubbles in the mounting medium. Importantly, upon sectioning, the three-dimensional context of the lesion&#39;s location in the brain is lost, making precise 3D reconstruction of the lesion in the brain challenging.
Another common application for lesions has been to determine the location of single and multiple electrode recordings in the brain. At the end of the final recording session, researchers induce small electrolytic lesions at the electrode tip and process the brain histologically as done in a conventional lesion experiment11. This technique suffers from the same drawbacks described above, with additional problems being that the electrolytic lesions are usually larger than the electrodes used to make them but are usually small enough that they are challenging to find histologically. When multiple electrodes are inserted, as in the case of a tetrode array, verification through electrolytic lesions is even more challenging. An alternative to electrolytic lesions is the use of a dye on the electrode to later verify histologically12, but this technique suffers from the same drawbacks that come with conventional histology.
Here, we describe in-depth a recently described method13 based on staining techniques in electron microscopy (EM) and X-ray computed tomography (micro-CT) that quantifies lesions and locates electrodes in small animal brains better than current methods. Micro-CT is an imaging technique in which X-rays are shot at a sample that is rotated 360&#176; while a scintillator collects the X-rays not deflected by the sample. The result is a high-resolution digital 3D reconstruction of the sample that can be visualized in any orientation and quantified precisely. Many academic institutions have micro-CT scanners, which are also available commercially.

<table border="0" cellpadding="0" cellspacing="0" style="width:1016px;" width="1016">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:288px;">Paraformaldehyde (PFA)</td>
			<td style="width:217px;">Electron Microscopy Sciences (EMS)</td>
			<td align="right" style="width:344px;">15710</td>
			<td style="width:167px;">2% (w/v/) in 1X PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glutaraldehyde (GA)</td>
			<td>EMS</td>
			<td align="right">16220</td>
			<td>2.5% (w/v) GA in 1X PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OsO4</td>
			<td>EMS</td>
			<td align="right">19190</td>
			<td>Work in fume hood</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ethanol</td>
			<td>Decon Labs</td>
			<td>Koptec</td>
			<td>140, 190, 200 proof</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acetone</td>
			<td>EMS</td>
			<td align="right">10015</td>
			<td>Glass-distilled</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Durcupan ACM resin</td>
			<td>Sigma-Aldrich</td>
			<td align="right">44610</td>
			<td>A, B, C and D components, resin for embedding</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable molds</td>
			<td>Ted Pella</td>
			<td align="right">27114</td>
			<td>Suggested</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">milliQ water (ultrapure water)</td>
			<td>Millipore Sigma</td>
			<td>QGARD00R1 (or related purifier)</td>
			<td>Suggested</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Parafilm (paraffin film)</td>
			<td>Millipore Sigma</td>
			<td>P7793</td>
			<td>Suggested paraffin film</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micro-CT scanner</td>
			<td>Nikon Metrology Ltd., Tring, UK</td>
			<td>X-Tek HMS ST 225</td>
			<td>Used by authors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Software for visualizing and analyzing micro-CT scans:</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td>Volume Graphics</td>
			<td>VG Studio Max</td>
			<td>Used by authors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td>FEI / Thermo Scientific</td>
			<td>Avizo</td>
			<td>Used by authors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td>FEI / Thermo Scientific</td>
			<td>Amira</td>
			<td>Similar to Avizo</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td>Mark Sutton &#38; Russell Garwood</td>
			<td>Spiers</td>
			<td>Free, http://spiers-software.org/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td>Pixmeo Sarl</td>
			<td>Osirix Lite</td>
			<td>Free, https://www.osirix-viewer.com/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td>Open Source</td>
			<td>FIJI</td>
			<td>Free, https://fiji.sc/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;"></td>
			<td>Adobe</td>
			<td>Photoshop</td>
			<td>Good for analyzing one slice at a time</td>
		</tr>
	</tbody>
</table>

a micro-ct-based method for characterizing lesions and locating electrodes in small animal brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

Traditionally, brains are sectioned and stained in order to quantify lesions and locate electrodes, but this method is error-prone, labor-intensive, and typically requires estimation of the results. By preparing whole brains for micro-CT imaging, the probability of damaging the samples is greatly reduced, features of interest may be analyzed in the context of the entire brain, and the method lends itself to parallel processing of many samples, considerably speeding up sample preparation.<...

Watch this Scientific Journal Video about A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains at JoVE.com

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

This method can help answer key questions in the field of neuroscience by providing a simpler, less error prone, and more quantitative method for verifying lesions and locating electrode locations. The main advantage of this technique is that it allows for the verification of lesions and electrode sites in the whole brain without the need for sectioning. The end product is a digital 3D volume of the brain and multiple brains can be processed in parallel.Though this method will be demonstrated in the rat, it can also be applied to other organisms, such as mice and zebra finches. Generally, individuals new to this method will struggle because some steps require particular care to ensure best results. To begin, place an extracted brain in profusion solution in a 50 milliliter conical tube.Store the sample for two to three days with gentle shaking at four degrees Celsius. Make sure that the osmium is diluted in water and not a buffer. This is because the water will act as a mild detergent, allowing the osmium to penetrate deep into the tissue.Prepare 50 milliliters of 2%osmium tetroxide in double-distilled water. Then place the brain in a new 50 milliliter conical tube and add the osmium tetroxide solution. Close the tube and seal it with paraffin film to prevent leaks.Store the sealed tube at four degrees Celsius with gentle shaking for two weeks. Make sure that the tube is placed horizontally. The brain is very thick and a long ways to travel by diffusion.Placing the tube horizontally will allow the osmium to travel deeper into the tissue. After the sample incubates for two weeks, wash it with double-distilled water five times to remove all the unbound osmium tetroxide from the sample. Next, wash the sample with double-distilled water for 30 minutes at four degrees Celsius.Replace the double-distilled water with four dilutions of ethanol to dehydrate the sample. To begin the resin infiltration process, move the sample through three acetone and three acetone resin dilutions, then immerse the sample in 100%resin at room temperature to continue the infiltration process. After this, transfer the sample to a disposable mold.Infuse the sample with fresh 100%resin for four hours at room temperature. Degas the sample in a vacuum oven for 15 minutes at 45 degrees Celsius. Then cure the sample in an oven for 48 hours at 60 degrees Celsius.Finally, when the sample has finished curing, peel off the disposable mold and scan it with the Micro-CT machine. In traditional sectioning, the orientation of the brain must be predetermined. Using this Micro-CT method, the digital volume of the sample brain can be manipulated in three dimensions and virtually sliced in any direction.Scanning electron microscopy of the prepared rat brain confirm that the tissue was not significantly damaged for the purposes of Micro-CT imaging. A zebra finch brain was also prepared and scanned according to this protocol to test the utility of this method on other small animal brains. This technique can be used to find surface lesions in the brain, as well as lesions deep within the brain.This technique can also be applied to the location of single tetrodes in situ, electrolytic lesions, electrode tracks, tetrode arrays in situ, and silicon probes in situ. While attempting this procedure, it's important to remember to allow the brain to incubate for long enough and with enough agitation. Following this procedure, other methods like optogenetics and two-photon imaging can be performed in order to answer additional questions about the role different brain areas play in different behaviors.This technique could allow researchers in the field of neuroscience to better explore structure-function relationships between the brain and behavior in rats, mice, songbirds, and other small animals. Don't forget that working with osmium can be extremely hazardous and precautions, such as wearing gloves and working in a vacuum hood should always be taken while performing this procedure.

Research

JoVE Journal

Neuroscience

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