Name: imaging and quantification of intact neuronal dendrites via clarity tissue clearing
Uploaded: 2021-04-20T15:00:00
Description: Watch this Scientific Journal Video about Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing at JoVE.com

We would like to thank the viral core NRDDC at the Jan and Dan Duncan Neurological Institute for producing the AAVs and lentiviruses used in these experiments. Additionally, we would like to thank the Baylor College of Medicine Center for Comparative Medicine for mouse husbandry and general maintenance of the mice used. We would like to thank the American Heart Association for their support under award number 20PRE35040011, and BRASS: Baylor Research Advocates for Student Scientists for their support (PJH). Finally, we would like to thank Logos for providing our lab with the Logos X-Clarity electrophoretic tissue clearing system.

The following protocol follows all animal care guidelines for Baylor College of Medicine.
1. Dissection and tissue preparation
<ol>
	<li>Euthanize the mouse with an overdose of isoflurane by placing the mouse in a closed container with a towel soaked in isoflurane (or by other IUCAC approved means).</li>
	<li>Perfuse the animal transcardially using a 25 G needle with 10 mL of ice cold PBS, followed by 10 mL of 4% PFA.</li>
	<li>Dissect the brain region (or tissue) of interest.</li>
	<li>Plac...

<ol>
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R., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+and+imaging+technologies+for+circuit-based+neuroanatomy.">Molecular genetics and imaging technologies for circuit-based neuroanatomy.</a> Nature. 461 (7266), 900-907 (2009).</li><li>Kim, E. H., Chin, G., Rong, G., Poskanzer, K. E., Clark, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+probes+for+neurobiological+sensing+and+imaging.">Optical probes for neurobiological sensing and imaging.</a> Accounts of Chemical Research. 51 (5), 1023-1032 (2018).</li><li>Weissman, T. A., Pan, Y. 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Before the advent of contemporary tissue clearing techniques, studying neuronal morphology consisted of time-intensive sectioning, imaging, and reconstruction of adjacent very thin sections. Using electrophoretic tissue clearing in combination with confocal imaging provides an unobstructed view of complete neuronal morphology. From intact dendritic trees, down to the smallest synaptic bouton, imaging and quantifying neuronal morphology has never been more feasible.
The preparation of cleared b...

Understanding the spatial organization, patterns of connectivity, and morphology of complex three-dimensional biological structures is essential for delineating the functions of specific cells and tissues. This is especially true in neuroscience, in which tremendous effort has been dedicated to building high-resolution neuroanatomical maps of the central nervous system1,2. Close examination of the neurons that comprise these maps yields varied morphologies, with connections and locations that reflect the function of these diverse sets of neurons3,4. Moreover, investigation of subcellular structures, especially dendritic spines, can inform the maturity of synapses, thereby reflecting developmental processes and neurological disease states5,6,7. Thus, approaches that improve imaging resolution and throughput are essential toward better understanding brain function at all scales.
Recent advances have expanded the molecular and genetic toolkit for marking and manipulating populations of neurons. The development of new fluorescent markers, combined with new methods of introducing these markers into neurons, allows for differential labeling of populations of interacting neurons within the same animal or brain sample8,9,10,11. Because light is scattered by opaque lipids, and given the high lipid content of brain tissue, imaging neuronal populations has primarily been limited to thin sections or has relied on advanced microscropy techniques (e.g., confocal, multi-photon, and light-sheet microscopy) to image deep structures. However, these efforts have been greatly bolstered by advances in tissue clearing techniques. Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/immunostaining/in situ hybridization-compatible Tissue-hYdrogel (CLARITY) is one such technique, in which tissues of interest are infused with hydrogel monomers (acrylamide and bis-acrylamide) and then washed with detergents12. The hydrogel monomers hybridize to create a stable 3D hydrogel scaffold that is optically transparent and permeable to macromolecule labels. Nucleic acids and proteins are maintained within the hybridized matrix, whereas lipids are removed by the detergent washes (Figure 1). This results in a stable tissue that is rigid enough to maintain the original shape and orientation of cells and non-lipid molecules, while optically transparent enough to easily image deep structures at high resolution. This maintenance of tissue structure and orientation allows for the imaging of thick slices, thereby preserving cell-to-cell connections and spatial relationships. Moreover, because the location and availability of proteins and nucleic acids is maintained during the clearing process, cleared tissues are able to hold expression-based markers, as well as exogenous labels. Thus, CLARITY lends itself as a potent method for imaging large amounts of deep brain structures and the connections between these structures at high resolution.
The use of CLARITY greatly improves approaches to imaging neuronal populations. This technique is especially adept at generating large amounts of imaging data. CLARITY works well with multiple forms of protein-based fluorescence. This protocol utilizes a lentiviral-based approach to sparsely label cells with EGFP and tdTomato; however, transgenic reporter alleles expressing tdTomato or EGFP to label cells for reconstruction have been routinely used. It is important to choose a fluorophore that is both photo-stable and bright (e.g., EGFP or tdTomato). Additionally, using a strong promoter to express the fluorophore yields superior contrast and image quality. The drawbacks of this technique arise as properly analyzing this large amount of data can be both labor- and time-intensive. Specialized microscopes can help improve throughput and decrease workload. However, building, owning, and/or operating advanced microscopes are often cost-prohibitive for many laboratories. This work presents a high throughput, relatively rapid, and simple method of visualizing large amounts of neural tissue at high-resolution using CLARITY tissue clearing of large sections, combined with standard confocal microscopy. This protocol describes this approach through the following steps: 1) dissecting and preparing the neural tissue, 2) clearing the tissue, 3) mounting the tissue, 4) imaging the prepared slices, and 5) processing full slice images using microscopy visualization software reconstruction and analysis (Figure 2). These efforts result in high-resolution images that can be used to analyze populations of neurons, neuronal connection patterns, 3D dendritic morphology, dendritic spine abundance and morphology, and molecular expression patterns within intact brain tissue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Conical Tube</td>
			<td>Thermo Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G x 1&#34; Needle</td>
			<td>BD</td>
			<td>305127</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide (No-Bis)</td>
			<td>National Diagnostics</td>
			<td>EC-810</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Tube</td>
			<td>Thermo Scientific</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoretic Tissue Clearing Solution</td>
			<td>Logos</td>
			<td>C13001</td>
			<td></td>
		</tr>
		<tr>
			<td>Histodenz</td>
			<td>Sigma</td>
			<td>D2158-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel Solution Kit</td>
			<td>Logos</td>
			<td>C1310X</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Oxford Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>EMS</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 1x, 500 mL, 6 bottles/case</td>
			<td>fisher</td>
			<td>MT21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>VA-044</td>
			<td>Wako</td>
			<td>925-41020</td>
			<td></td>
		</tr>
		<tr>
			<td>X-CLARITY Polymerization System</td>
			<td>Logos</td>
			<td>C20001</td>
			<td></td>
		</tr>
		<tr>
			<td>X-CLARITY Tissue Clearing System II</td>
			<td>Logos</td>
			<td>C30001</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging and quantification of intact neuronal dendrites via clarity tissue clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

After image acquisition, the representative cell morphology was analyzed using embedded statistics and classifying scripts within the analysis software. The collected data (Figure 6A) reflects that neuron 2 has a larger dendritic structure with a higher density of spines. As a whole, the data suggests that neuron 2 has a more complex dendritic structure compared to neuron 1. To substantiate this result, standard Sholl analysis was performed, which affirms that neuron 2 is more dendritically ...

Watch this Scientific Journal Video about Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing at JoVE.com

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

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