Name: large-scale reconstructions and independent, unbiased clustering based on morphological metrics to classify neurons in selective populations
Uploaded: 2017-02-15T12:30:00
Description: Watch this Scientific Journal Video about Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations at JoVE.com

We would like to thank Drs. Ed Callaway and Marty Usrey for allowing us to use the tissue prepared as a part of a prior study and Libby Fairless and Shiyuan Liu for help with neuronal reconstructions. This work was funded by the NIH (NEI: EY018683) and the Whitehall Foundation.

Note: The tissue examined in this study was prepared as a part of a separate study5. Therefore, all of the experimental methods involving the use of animals have been described in detail in the Experimental Methods section of Briggs et al. (2016). All procedures involving animals conducted as a part of the prior study were approved by the Institutional Animal Care and Use Committees. The steps for injection of virus into the dLGN and histological processing of brain tissue are described b...

<ol>
	<li>Cajal, S. R. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histologie du systeme nerveaux de l'homme et des vertebres. , (1911).</li><li>Seung, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+functional+connectomics.">Toward functional connectomics.</a> Nature. 471, 170-172 (2011).</li><li>Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transneuronal+circuit+tracing+with+neurotropic+viruses.">Transneuronal circuit tracing with neurotropic viruses.</a> Current Opinion in Neurobiology. 18, 1-7 (2009).</li><li>Wickersham, I. R., Finke, S., Conzelmann, K. K., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+neuronal+tracing+with+a+deletion-mutant+rabies+virus.">Retrograde neuronal tracing with a deletion-mutant rabies virus.</a> Nature Methods. 4, 47-49 (2007).</li><li>Briggs, F., Kiley, C. W., Callaway, E. M., Usrey, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+substrates+for+parallel+streams+of+corticogeniculate+feedback+originating+in+both+V1+and+V2+of+the+macaque+monkey.">Morphological substrates for parallel streams of corticogeniculate feedback originating in both V1 and V2 of the macaque monkey.</a> Neuron. 90, 388-399 (2016).</li><li>Cauli, 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=Classification+of+fusiform+neocortical+interneurons+based+on+unsupervised+clustering.">Classification of fusiform neocortical interneurons based on unsupervised clustering.</a> PNAS. 97, 6144-6149 (2000).</li><li>Bragg, E. M., Fairless, E. A., Liu, S., Briggs, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+visual+sector+thalamic+reticular+neurons+in+the+macaque+monkey+sugests+retinotopically-specialized,+parallel+stream-mixed+input+to+the+lateral+geniculate+nucleus.">Morphology of visual sector thalamic reticular neurons in the macaque monkey sugests retinotopically-specialized, parallel stream-mixed input to the lateral geniculate nucleus.</a> J. Comparative Neurology. 525 (5), 1273-1290 (2017).</li><li>Osakada, F., 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=New+rabies+virus+varients+for+monitoring+and+manipulating+activity+and+gene+expression+in+defined+neural+circuits.">New rabies virus varients for monitoring and manipulating activity and gene expression in defined neural circuits.</a> Neuron. 71, 617-631 (2011).</li><li>Callaway, E. M., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+circuit+tracing+with+glycoprotein-deleted+rabies+viruses.">Monosynaptic circuit tracing with glycoprotein-deleted rabies viruses.</a> Journal of Neuroscience. 35, 8979-8985 (2015).</li><li>Nhan, H. L., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+superior+colliculus-+and+middle+temporal+area-projecting+neurons+in+primate+primary+visual+cortex.">Morphology of superior colliculus- and middle temporal area-projecting neurons in primate primary visual cortex.</a> J. Comparative Neurology. 520, 52-80 (2012).</li><li>Pinault, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thalamic+reticular+nucleus:+structure,+function+and+concept.">The thalamic reticular nucleus: structure, function and concept.</a> Brain Research Reviews. 46, 1-31 (2004).</li><li>Gage, G. J., 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, e3564 (2012).</li><li>Wong-Riley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+visual+system+of+monocularly+sutured+or+enucleated+cats+demonstrable+with+cytochrome+oxidase+histochemistry.">Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry.</a> Brain Research. 171, 11-28 (1979).</li><li>Gerfen, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+neuroanatomical+methods.">Basic neuroanatomical methods.</a> Current Protocols in Neuroscience. Chapter 1, Unit 1.1 (2003).</li><li>Thorndike, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Who+belongs+in+the+family?.">Who belongs in the family?.</a> Psychometrika. 18, 267-276 (1953).</li><li>Talebi, V., Baker, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Categorically+distinct+types+of+receptive+fields+in+early+visual+cortex.">Categorically distinct types of receptive fields in early visual cortex.</a> Journal of Neurophysiology. 115, 2556-2576 (2016).</li><li>Helmstaedter, 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=Connectomic+reconstruction+of+the+inner+plexiform+layer+in+the+mouse+retina.">Connectomic reconstruction of the inner plexiform layer in the mouse retina.</a> Nature. 500, 168-174 (2013).</li></ol>

Neuroanatomical studies have remained a pillar of neuroscience and recent interest in connectomics and structure-function relationships has renewed enthusiasm for detailed morphological characterization of selective neuronal populations. Traditionally, neuroanatomical studies have relied on qualitative classifications of neurons into morphologically distinct classes of neurons defined by expert neuroanatomists. With advances in the techniques for reconstructing neurons and extracting morphological data, it is now possibl...

Neuroanatomy is one of the foundations of neuroscience1 and recent interest in &#34;connectomics&#34; has renewed enthusiasm for understanding the morphological diversity of neuronal populations and the connections between specific neurons2. Methods for labeling and reconstructing neurons have greatly improved with recent innovations, including genetic and virus-mediated circuit tracing approaches3,4, enabling more comprehensive morphological surveys of neuronal populations5. In addition to improvements in labeling individual neurons, quantitative data analysis techniques have also emerged that enable independent and unbiased classification of neurons into distinct subpopulations based on morphological data5,6. These unbiased techniques are an improvement upon more traditional qualitative classification methods that have been the standard in the field for over a century. The goal of this study is to outline, step-by-step, the combination of virus-mediated labeling of neurons within a selective population, large-scale reconstructions of a comprehensive sample of these neurons, and quantitative data analysis based on independent clustering with statistical evaluation. By combining these methods, we outline a novel approach toward the collection and analysis of neuroanatomical data to facilitate comprehensive sampling and unbiased classification of morphologically unique neuronal types within a selective neuronal population.
As an example of these methods, we describe our analysis of a large population of neurons within a single sector of the thalamic reticular nucleus (TRN) of the macaque monkey. These data are from a prior study7. Methods for selectively labeling TRN neurons projecting to the dorsal lateral geniculate nucleus of the thalamus (dLGN) using surgical injection of modified rabies virus encoding EGFP4,8 (see Table of Specific Materials/Equipment, row 2) are outlined. This modified rabies virus lacks the gene encoding an essential coat protein, eliminating trans-synaptic movement of the virus. Once the virus enters axon terminals at the injection site, it acts like a traditional retrograde tracer with the important benefit of driving EGFP expression throughout the full dendritic arborization of infected neurons5,9,10. Accordingly, this G-deleted rabies virus can be utilized to selectively infect and label any neuronal population following injection and retrograde transport.
In order to perform a comprehensive analysis of a specific neuronal population, it is important to sample from a broad distribution of neurons within the population. Because the virus-mediated labeling technique produces complete intracellular, &#34;Golgi-like&#34; fills of many neurons with axons at the virus injection site, it is possible to reconstruct a very large sample of neurons within the full extent of a brain structure. Additionally, because the modified rabies virus is so effective at infecting and labeling large numbers of neurons, it is possible to reconstruct hundreds of neurons per animal. Procedures for sampling 160 neurons throughout the visual sector of the TRN11 in order to generate a comprehensive sample of dLGN-projecting TRN neurons are outlined. The process of reconstructing individual neurons using a neuron reconstruction system including a microscope, camera, and reconstruction software is described. Also described are methods to determine positions of individual neurons within a brain structure (in this case within the TRN) and to verify virus injection site volume and location within a structure (in this case within the dLGN) using volumetric contour reconstructions. Steps to export morphological data and perform independent cluster analyses based on morphological metrics measured for each neuron are outlined. There are limitations to clustering methods and there are also a variety of different clustering algorithms available. Accordingly, these options and the benefits of some of the more commonly used algorithms are described. The cluster analysis does not provide statistical verification of the uniqueness of clusters. Therefore, additional steps are outlined to verify optimal clustering as well as the relationships between morphological data within and across clusters. Statistical methods for evaluating clusters for the TRN dataset to confirm that TRN neurons are grouped into three unique clusters based on 10 independent morphological metrics are described.
Thus, by outlining steps for selectively labeling, reconstructing, and analyzing morphological data from a specific neuronal population, we describe methods for quantifying morphological differences among neurons within a population. Prior findings of distinct neuronal types within the visual sector of the macaque monkey TRN are confirmed with separate statistical evaluation methods. Together, we hope these techniques will be broadly applicable to neuroanatomical datasets and help establish quantitative classification of the diversity of neuronal populations through the brain.

<table border="0" cellpadding="0" cellspacing="0" width="818">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">SAD&#916;G-EGFP</td>
			<td style="width:171px;">E.M. Callaway Laboratory, Salk Institute</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Prepared by Dr. F. Osakada. G-deleted rabies virus available through the Salk Institute Viral Core</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Recording electrode: platinum/iridium or tungsten</td>
			<td style="width:171px;">FHC</td>
			<td style="width:133px;">UEPSGGSE1N2M</td>
			<td style="width:281px;">Visit website (www.fh-co.com) for alternative order specifications</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:233px;">Nanoject II</td>
			<td style="width:171px;">Drummod Scientific</td>
			<td style="width:133px;">3-000-204, 110V</td>
			<td style="width:281px;">Alternatives: picospritzer, Hamilton syringe</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:233px;">Freezing microtome</td>
			<td style="width:171px;">Thermo Scientific</td>
			<td style="width:133px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">DAB</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:133px;">D5905-50TAB</td>
			<td style="width:281px;">3,3&#39;-Diaminobenzidine tetrahydrochloride, tablet, 10 mg substrate per tablet. Caution: carcinogen - must be bleached before discarding</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:233px;">Cytochrome C</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:133px;">C2037-100MG</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:233px;">Catalase</td>
			<td style="width:171px;">Sigma-Aldich</td>
			<td style="width:133px;">C9322-5G</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-GFP</td>
			<td style="width:171px;">Life Technologies/Thermo Fisher</td>
			<td style="width:133px;">#A-11122&#160;</td>
			<td style="width:281px;">Primary antibody</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:233px;">Biotinylated goat anti-rabbit</td>
			<td style="width:171px;">Vector Laboratories</td>
			<td style="width:133px;">#BA-1000</td>
			<td style="width:281px;">Secondary antibody</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Neurolucida System&#160;</td>
			<td style="width:171px;">MicroBrightField</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Software for neuron tracing and analysis. http://www.mbfbioscience.com/neurolucida</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:233px;">Neurolucida Explorer</td>
			<td style="width:171px;">MicroBrightField</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Data export software</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Microfire Camera&#160;</td>
			<td style="width:171px;">Optronics</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">2-Megapixel true color microscope camera. http://www.simicroscopes.com/pdfs/microfire.pdf</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Nikon E800 Microscope</td>
			<td style="width:171px;">Nikon Instruments Inc.</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Biological research microscope. http://www.microscopyu.com/museum/eclipseE800.html</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Matlab</td>
			<td style="width:171px;">The MathWorks Inc.&#160;</td>
			<td style="width:133px;"></td>
			<td style="width:281px;">Matrix-based computational mathematics software. http://www.mathworks.com</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Microsoft Office Excel</td>
			<td style="width:171px;">Microsoft</td>
			<td style="width:133px;"></td>
			<td>Spreadsheet program</td>
		</tr>
	</tbody>
</table>

large-scale reconstructions and independent, unbiased clustering based on morphological metrics to classify neurons in selective populations

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population.
The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing &#34;Golgi-like&#34; complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills.
Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

We have shown previously that large-scale reconstructions of neurons within a selective population is feasible following injection of modified rabies virus into the dLGN5. Recently, the same tissue was utilized to reconstruct 160 neurons in the visual sector of the TRN (Bragg et al., in review; Figure 2A-B) following the detailed methodological steps described above. In the TRN study, three unique clusters of TRN neurons were identified based on indepe...

Watch this Scientific Journal Video about Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations at JoVE.com

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a ...

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