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>
	<li>Scoville, W., Milner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+recent+memory+after+bilateral+hippocampal+lesions.">Loss of recent memory after bilateral hippocampal lesions.</a> Journal of Neuropsychiatry and Clinical Neuroscience. 12, 103-113 (2000).</li><li>Damasio, H., Grabowski, T., Frank, R., Galaburda, A. M., Damasio, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+return+of+Phineas+Gage:+clues+about+the+brain+from+the+skull+of+a+famous+patient.">The return of Phineas Gage: clues about the brain from the skull of a famous patient.</a> Science. 264 (5162), 1102-1105 (1994).</li><li>Kawai, R., 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=Motor+cortex+is+required+for+learning+but+not+for+executing+a+motor+skill.">Motor cortex is required for learning but not for executing a motor skill.</a> Neuron. 86, 800-812 (2015).</li><li>Otchy, T., 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=Acute+off-target+effects+of+neural+circuit+manipulations.">Acute off-target effects of neural circuit manipulations.</a> Nature. 528, 358-363 (2015).</li><li>Wright, N., Vann, S., Aggleton, J., Nelson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+the+anterior+thalamus+in+directing+attention+to+task-relevant+stimuli.">A critical role for the anterior thalamus in directing attention to task-relevant stimuli.</a> Journal of Neuroscience. 35, 5480-5488 (2015).</li><li>Kapgal, V., Prem, N., Hegde, P., Laxmi, T., Kutty, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+exposure+to+combination+paradigm+of+environmental+enrichment,+physical+exercise+and+diet+reverses+the+spatial+memory+deficits+and+restores+hippocampal+neurogenesis+in+ventral+subicular+lesioned+rats.">Long term exposure to combination paradigm of environmental enrichment, physical exercise and diet reverses the spatial memory deficits and restores hippocampal neurogenesis in ventral subicular lesioned rats.</a> Neurobiology of Learning and Memory. 130, 61-70 (2016).</li><li>Hosseini, N., Alaei, H., Reisi, P., Radahmadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+NBM-+lesion+on+synaptic+plasticity+in+rats.">The effects of NBM- lesion on synaptic plasticity in rats.</a> Brain Research. 1655, 122-127 (2017).</li><li>Palagina, G., Meyer, J., Smirnakis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+visual+motion+representation+in+mouse+area+V1.">Complex visual motion representation in mouse area V1.</a> Journal of Neuroscience. 37, 164-183 (2017).</li><li>Ranjbar, H., Radahmadi, M., Reisi, P., Alaei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+electrical+lesion+of+basolateral+amygdala+nucleus+on+rat+anxiety-like+behavior+under+acute,+sub-chronic,+and+chronic+stresses.">Effects of electrical lesion of basolateral amygdala nucleus on rat anxiety-like behavior under acute, sub-chronic, and chronic stresses.</a> Clinical and Experimental Pharmacology and Physiology. , (2017).</li><li>Wood, R., 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+honeycomb+maze+provides+a+novel+test+to+study+hippocampal-dependent+spatial+navigation.">The honeycomb maze provides a novel test to study hippocampal-dependent spatial navigation.</a> Nature. , (2018).</li><li>Vermaercke, B., 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=Functional+specialization+in+rat+occipital+and+temporal+visual+cortex.">Functional specialization in rat occipital and temporal visual cortex.</a> Journal of Neurophysiology. 112, 1963-1983 (2014).</li><li>Jun, J. J., 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=Fully+integrated+silicon+probes+for+high-density+recording+of+neural+activity.">Fully integrated silicon probes for high-density recording of neural activity.</a> Nature. 551, 232-236 (2017).</li><li>Mas&#237;s, J., 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=micro-CT-based+method+for+quantitative+brain+lesion+characterization+and+electrode+localization.">micro-CT-based method for quantitative brain lesion characterization and electrode localization.</a> Scientific Reports. 8, 5184 (2018).</li><li>Gage, G., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> Journal of Visualized Experiments. 65, 3564 (2012).</li><li>Helander, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+studies+of+formaldehyde+binding+in+tissue.">Kinetic studies of formaldehyde binding in tissue.</a> Biotechnic &amp; Histochemistry. , (1994).</li><li>Palj&#228;rvi, L., Garcia, J., Kalimo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+aldehyde+fixation+for+electron+microscopy:+stabilization+of+rat+brain+tissue+to+withstand+osmotic+stress.">The efficiency of aldehyde fixation for electron microscopy: stabilization of rat brain tissue to withstand osmotic stress.</a> Histochemical Journal. , (1979).</li><li>Okuda, K., Urabe, I., Yamada, Y., Okada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+of+glutaraldehyde+with+amino+and+thiol+compounds.">Reaction of glutaraldehyde with amino and thiol compounds.</a> Journal of Fermentation and Bioengineering. 71, (1991).</li><li>Bahr, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmium+tetroxide+and+ruthenium+tetroxide+and+their+reactions+with+biologically+important+substances:+electron+stains+III.">Osmium tetroxide and ruthenium tetroxide and their reactions with biologically important substances: electron stains III.</a> Experimental Cell Research. , (1954).</li><li>Khan, A. A., Riemersma, J. C., Booij, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reactions+of+osmium+tetroxide+with+lipids+and+other+compounds.">The reactions of osmium tetroxide with lipids and other compounds.</a> Journal of Histochemistry &amp; Cytochemistry. 9, 560-563 (1961).</li><li>Riemersma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmium+tetroxide+fixation+of+lipids+for+electron+microscopy+a+possible+reaction+mechanism.">Osmium tetroxide fixation of lipids for electron microscopy a possible reaction mechanism.</a> Biochimica et Biophysica Acta. 152, (1968).</li><li>Mikula, S., Binding, J., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+and+embedding+the+whole+mouse+brain+for+electron+microscopy.">Staining and embedding the whole mouse brain for electron microscopy.</a> Nature Methods. 9, 1198-1201 (2012).</li><li>Mikula, S., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+whole-brain+staining+for+electron+microscopic+circuit+reconstruction.">High-resolution whole-brain staining for electron microscopic circuit reconstruction.</a> Nature Methods. 12, 541-546 (2015).</li><li>Crespigny, 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=3D+micro-CT+imaging+of+the+postmortem+brain.">3D micro-CT imaging of the postmortem brain.</a> Journal of Neuroscience Methods. 171, 207-213 (2008).</li><li>Anderson, R., Maga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+procedure+for+rapid+imaging+of+adult+mouse+brains+with+MicroCT+using+Iodine-Based+contrast.">A novel procedure for rapid imaging of adult mouse brains with MicroCT using Iodine-Based contrast.</a> PLoS One. 10, 0142974 (2015).</li><li>Zhou, Z., 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=Cerebral+cavernous+malformations+arise+from+endothelial+gain+of+MEKK3-KLF2/4+signalling.">Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.</a> Nature. 532, 122-126 (2016).</li><li>Choi, J., 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=Micro-CT+imaging+reveals+mekk3+heterozygosity+prevents+cerebral+cavernous+malformations+in+Ccm2-Deficient+mice.">Micro-CT imaging reveals mekk3 heterozygosity prevents cerebral cavernous malformations in Ccm2-Deficient mice.</a> PloS One. 11, 0160833 (2016).</li><li>Choi, J., Yang, X., Foley, M., Wang, X., Zheng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+Micro-CT+imaging+of+cerebral+cavernous+malformations+in+mouse+model.">Induction and Micro-CT imaging of cerebral cavernous malformations in mouse model.</a> Journal of Visualized Experiments. , (2017).</li><li>Benveniste, H., Kim, K., Zhang, L., Johnson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+microscopy+of+the+C57BL+mouse+brain.">Magnetic resonance microscopy of the C57BL mouse brain.</a> Neuroimage. 11, 601-611 (2000).</li><li>Weninger, W. J., 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=High-resolution+episcopic+microscopy:+a+rapid+technique+for+high+detailed+3D+analysis+of+gene+activity+in+the+context+of+tissue+architecture+and+morphology.">High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology.</a> Anat Embryol. 211, 213-221 (2006).</li><li>Schneider, J. E., 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=high-throughput+magnetic+paragraph+sign+resonance+imaging+of+mouse+embryonic+paragraph+sign+anatomy+using+a+fast+gradient-echo+sequence.">high-throughput magnetic paragraph sign resonance imaging of mouse embryonic paragraph sign anatomy using a fast gradient-echo sequence.</a> MAGMA. 16, 43-51 (2003).</li><li>Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography.">Optical projection tomography.</a> Annual Review of Biomedical Engineering. 6, 209-228 (2004).</li><li>Cox, D. D., Papanastassiou, A., Oreper, D., Andken, B., James, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Three-Dimensional+microelectrode+brain+mapping+using+stereo+microfocal+x-ray+imaging.">High-Resolution Three-Dimensional microelectrode brain mapping using stereo microfocal x-ray imaging.</a> Journal of Neurophysiology. 100, 2966-2976 (2008).</li><li>Borg, J. S., 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=Localization+of+metal+electrodes+in+the+intact+rat+brain+using+registration+of+3D+microcomputed+tomography+images+to+a+magnetic+resonance+histology+atlas.">Localization of metal electrodes in the intact rat brain using registration of 3D microcomputed tomography images to a magnetic resonance histology atlas.</a> eNeuro. 2, (2015).</li><li>Fu, T. -. M., 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=Stable+long-term+chronic+brain+mapping+at+the+single-neuron+level.">Stable long-term chronic brain mapping at the single-neuron level.</a> Nature Methods. 13, 875-882 (2016).</li></ol>

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.

a-micro-ct-based-method-for-characterizing-lesions-locating

Research

JoVE Journal

Neuroscience

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Rabies Necropsy Techniques in Large and Small Animals

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the entire state, with the exception of New York City. The Rabies laboratory necropsies a variety of animals ranging in size from bats to bovids. Most of these specimens are animals exhibiting neurological signs, however, less than 10% actually test positive for rabies; implying trauma, lesions or other infectious agents as the cause of these symptoms. Due to the risk of aerosolizing undiagnosed infectious agents, the Rabies Laboratory does not use power tools or saws. Three necropsy techniques will be presented for animals whose skulls are impenetrable with scissors. The laboratory has implemented these techniques to decrease potential exposure to infectious agents, eliminate unnecessary manipulation of the specimen and reduce processing time. The advantages of a preferred technique opposed to another is subject to the trained individual processing the specimen.

The goal of this protocol is to demonstrate safe necropsy techniques in small and large animals to obtain satisfactory tissue samples for rabies testing.

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the ...

Abbiategrasso Brain Bank Protocol for Collecting, Processing and Characterizing Aging Brains

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find preventive and curative measures. The most effective way to achieve this is through direct examination of diseased and healthy brain tissue. The authors present a protocol to obtain, process, characterize and store good quality brain tissue donated by individuals registered in an antemortem brain donation program. The donation program includes a face-to-face empathic approach to people, a collection of complementary clinical, biological, social and lifestyle information and serial multi-dimensional assessments over time to track individual trajectories of normal aging and cognitive decline. Since many neurological diseases are asymmetrical, our brain bank offers a unique protocol for slicing fresh specimens. Brain sections of both hemispheres are alternately frozen (at -80 &#176;C) or fixed in formalin; a fixed slice on one hemisphere corresponds to a frozen one on the other hemisphere. With this approach, a complete histological characterization of all frozen material can be obtained, and omics studies can be performed on histologically well-defined tissues from both hemispheres thus offering a more complete assessment of neurodegenerative disease mechanisms. Correct and definite diagnosis of these diseases can only be achieved by combining the clinical syndrome with the neuropathological evaluation, which often adds important etiological clues necessary to interpret the pathogenesis. This method can be time consuming, expensive and limited as it only covers a limited geographical area. Regardless of its limitations, the high degree of characterization it provides can be rewarding. Our ultimate goal is to establish the first Italian Brain Bank, all the while emphasizing the importance of neuropathologically verified epidemiological studies.

This protocol describes a method to track individual brain-aging trajectories through a brain donation program and proper characterization of brains. Brain donors are involved in a long-term longitudinal study including serial multi-dimensional assessments. The protocol contains a detailed description of brain processing and an accurate diagnostic methodology.

In a constantly aging population, the prevalence of neurodegenerative disorders is expected to rise. Understanding disease mechanisms is the key to find ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...