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...

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P., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+dendritic+spine+neck+changes+are+correlated+with+synaptic+strength.">Activity-dependent dendritic spine neck changes are correlated with synaptic strength.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (28), (2014).</li><li>Bosch, M., Hayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+plasticity+of+dendritic+spines.">Structural plasticity of dendritic spines.</a> Current Opinion in Neurobiology. 22 (3), 383-388 (2012).</li><li>Mart&#237;nez-Cerde&#241;o, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrite+and+spine+modifications+in+autism+and+related+neurodevelopmental+disorders+in+patients+and+animal+models.">Dendrite and spine modifications in autism and related neurodevelopmental disorders in patients and animal models.</a> Developmental Neurobiology. 77 (4), 393-404 (2017).</li><li>Arenkiel, B. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brainbow:+New+resources+and+emerging+biological+applications+for+multicolor+genetic+labeling+and+analysis.">Brainbow: New resources and emerging biological applications for multicolor genetic labeling and analysis.</a> Genetics. 199 (2), 293-306 (2014).</li><li>Haggerty, D. L., Grecco, G. G., Reeves, K. 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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 ...

This protocol will allow researchers to answer important questions about neuronal patterns of connectivity and spatial organization that underlies function within a circuit. The main advantage of this technique is that it enables visualization of neuronal morphologies located deep within intact three-dimensional tissue. This method is straightforward and thus easily reproducible.First-time experimenters should be comfortable performing this technique. It is imperative to have a clear tissue sample in order to obtain high-quality data. The visual demonstration of appropriate tissue clarity and clearing techniques will enable easily reproducible results.To begin, place the hydrogel-submerged brain tissue in a vacuum incubator for three hours at 37 degrees Celsius with minus 90 kilopascal vacuum. Leave the tube top unscrewed to allow the vacuum to form properly. Wash the tissue with PBS for 10 minutes at room temperature with gentle shaking.Place the polymerized tissue sample in the electrophoresis chamber, keeping track of the orientation of the tissue within the chamber. Fill the chamber and reservoir with the supplied electrophoresis SDS buffer. Run the sample at 70 volts, one ampere, and 35 degrees Celsius with constant current for about two hours per milliliter of tissue.Check the sample periodically for sufficient clearing of brain tissue by removing it from the chamber and replacing it in the same orientation. After the sample has finished clearing, wash it in PBS overnight at room temperature, replacing the PBS with fresh PBS as often as possible. Following the final wash in PBS, wash the tissue for five minutes in deionized water at room temperature three times.The tissue will become opaque and may expand. Incubate the tissue in the refractive index matching solution for at least four hours at room temperature. During the incubation, construct a suitable housing chamber to image the sample.Start by using a glass slide as a base for mounting, then lay down either rubber or plastic spacers and secure them with super glue, making sure that there aren't any holes. Place the clear tissue into the mounting chamber pre-filled with refractive index matching solution. Securely mount the tissue by placing a glass coverslip on top and sealing it with nail polish.Image this tissue by adding a drop of refractive index matching mounting solution directly on top of the glass. To construct a large tissue imaging chamber for tissues with more than a five millimeter thickness, use a 10 centimeter glass dish with a high wall. Place a 50 milliliter conical tube in the center, making sure that the diameter of the tube is large enough to accept the barrel of the objective lens used.Make 3%agarose in water and pour it in the space between the glass dish and the conical tube, then allow it to cool for one hour. This will form a ring of solid agarose. Securely adhere the tissue to the bottom of the chamber using super glue and fill the chamber with refractive index matching solution, which may start to polymerize unless preserved from air and stored at four degrees Celsius.Acquire the image using a confocal microscope. Turn on all the relevant imaging equipment. Place the sample on the stage.Carefully approach the immersion media with the objective and form a continuous column of media. Using epifluorescence, find an appropriate imaging field. Start by setting the resolution and scan speed settings.If imaging using a confocal microscope, fully close the pinhole to obtain the smallest optical section and thus the best Z resolution. Gradually increase the laser power or sensor gain until a suitable image is obtained with a high signal-to-noise ratio. If utilizing standard EGFP or tdTomato two-color imaging, set the light collection settings.Set the Z-stack parameters based on the observed start and end points of the tissue. Set the step size based on the desired Z resolution. Smaller step sizes will yield a greater Z resolution, but may lead to sample bleaching.When satisfied with the image acquisition settings, acquire the image. Ensure that the image has a high signal-to-noise ratio and shows distinct boundaries of structures. After image acquisition, the representative cell morphology was analyzed using embedded statistics and classifying scripts within the analysis software.The collected data reflects that Neuron 2 has larger than dendritic structure with a higher density of spines compared to Neuron 1. To substantiate this result, standard Scholl analysis was performed, which affirms that Neuron 2 is more dendritically complex than Neuron 1 as denoted by the increased number of Scholl intersections at 50 to 100 micrometers from the soma. Neuron 1 contains a larger proportion of spines with more filopodia-like shapes and is an immature spine subtype.Spines with defined heads, called mushroom spines, likely contain more developed and mature synapses. Thus, Neuron 2 is more mature with a higher density of mature spines. After clearing, the tissue chunks can be stained using traditional immunohistochemistry.

imaging-quantification-intact-neuronal-dendrites-via-clarity-tissue

Research

JoVE Journal

Neuroscience

3.3K visualizações.  Baylor College of Medicine. Este protocolo permitirá que os pesquisadores respondam a perguntas importantes sobre padrões neuronais de conectividade e organização espacial que fundamentam a função dentro de um circuito. A principal vantagem dessa técnica é que ela permite a visualização de morfologias neuronais localizadas profundas dentro do tecido tridimensional intacto. Este método é simples e, portanto, facilmente reproduzível.Os experimentadores iniciantes devem estar confortáveis em executar e...