The authors thank Thomas C. Mettenleiter and Verena te Kamp for critically reading the manuscript. This work was supported by the Federal Excellence Initiative of Mecklenburg Western Pomerania and the European Social Fund (ESF) Grant KoInfekt (ESF/14-BM-A55-0002/16) and an intramural collaborative research grant on Lyssaviruses at the Friedrich-Loeffler-Institute (Ri-0372).

RABV-infected, PFA-fixed archived brain material was used. The respective animal experimental studies were evaluated by the responsible animal care, use, and ethics committee of the State Office for Agriculture, Food Safety, and Fishery in Mecklenburg-Western Pomerania (LALFF M-V) and gained approval with permissions 7221.3-2.1-002/11 (mice) and 7221.3-1-068/16 (ferrets). General care and methods used in the animal experiments were carried out according to the approved guidelines.
CAUTION: This protocol uses various toxic and/or harmful substances, including PFA, methanol (MeOH), hydrogen peroxide (H2O2), sodium azide (NaN3), TBA, BA, BB, and DPE. MeOH and TBA are highly flammable. Avoid exposure by wearing appropriate personal protective equipment (a lab coat, gloves, and eye protection) and conducting experiments in a fume hood. Collect waste separately in appropriate containers and dispose of it according to local regulations. Rabies virus is classified as a biosafety level (BSL)-2 pathogen and can, therefore, generally be handled under BSL-2 conditions. Some activities, including procedures that may generate aerosols, work with high virus concentrations, or work with novel lyssaviruses, may require BSL-3 classification. Pre-exposure prophylaxis is recommended for high-risk personnel, including animal caretakers and laboratory workers18,19. Refer to local authority regulations.
1. Fixation of brain tissue and sectioning
<ol>
	<li>Fix brain samples in an appropriate volume of 4% PFA in phosphate-buffered saline (PBS [pH 7.4]) for at least 48 h at 4 &#176;C (with an approximate tissue-to-fixative ratio of 1:10 [v/v]).</li>
	<li>Wash the tissue samples 3x in PBS for at least 30 min each wash and store them in 0.02% NaN3/PBS at 4 &#176;C until use.</li>
	<li>Section the tissue into 1 mm-thick sections using a vibratome (blade feed rate: 0.3&#8211;0.5 mm/s, amplitude: 1 mm, slice thickness: 1,000 &#181;m).</li>
	<li>To retain the correct slice sequence, store each tissue section separately in a well of a multiwell cell culture plate. Add 0.02% NaN3/PBS and store the tissue sections at 4 &#176;C until use.</li>
</ol>
2. Sample pretreatment with methanol
NOTE: Perform all incubation steps with gentle oscillation and, if not indicated otherwise, at room temperature. Protect the samples from light. The sample pretreatment serves the overall purpose of improving antibody diffusion and reducing tissue autofluorescence by exposure to MeOH and H2O2, respectively14.
<ol>
	<li>Prepare 20% (v/v), 40%, 60%, and 80% MeOH solutions in distilled water. For instance, for 20% MeOH, add 10 mL of 100% MeOH to 40 mL of distilled water in an appropriate sealable vessel and mix by inverting it.</li>
	<li>Transfer the samples to reasonably sized vessels (e.g., 5 mL reaction tubes). Take care to use materials chemically resistant to the reagents used in this protocol. For instance, note that while polypropylene is suitable, polystyrene is not. 
	NOTE: The volume specifications in this protocol refer to 5 mL reaction tubes. If a different vessel is used, adjust the volumes accordingly.</li>
	<li>Incubate the samples in 4 mL of each concentration of the prepared series of MeOH solutions in ascending order for 1 h each.</li>
	<li>Incubate the samples 2x for 1 h each in pure (100%) MeOH.</li>
	<li>Cool the samples to 4 &#176;C (e.g., in a laboratory-safe refrigerator).</li>
	<li>Prepare bleaching solution (5% H2O2 in MeOH) by, for instance, diluting a 30% H2O2 stock solution at 1:6 in pure (100%) MeOH, and chill it at 4 &#176;C.</li>
	<li>Remove the 100% MeOH from the refrigerated samples and add 4 mL of prechilled bleaching solution (5% H2O2 in MeOH). Incubate overnight at 4 &#176;C.</li>
	<li>Exchange the bleaching solution for 4 mL of 80% MeOH and incubate for 1 h. Continue using the prepared series of MeOH solutions in descending order for 1 h each until the samples have been incubated for 1 h in 4 mL of 20% MeOH.</li>
	<li>Wash the samples 1x for 1 h with 4 mL of PBS.</li>
</ol>
3. Immunostaining
NOTE: Perform all incubation steps with gentle oscillation and, if not indicated otherwise, at room temperature. Protect the samples from light. To prevent microbial growth, add NaN3 to a final concentration of 0.02% to the solutions in this section. Tissue samples are further permeabilized by treatment with nonionic detergents Triton X-100 and Tween 20. Normal serum is used to block unspecific antibody binding. Glycine and heparin are added to reduce the immunolabeling background14.
<ol>
	<li>Wash the samples 2x for 1 h each in 4 mL of 0.2% Triton X-100/PBS.</li>
	<li>Permeabilize the samples for 2 days at 37 &#176;C with 4 mL of 0.2% Triton X-100/20% DMSO/0.3 M glycine/PBS.</li>
	<li>Block the unspecific binding of antibodies by incubating the samples for 2 days at 37 &#176;C in 4 mL of 0.2% Triton X-100/10% DMSO/6% normal serum/PBS. 
	NOTE: Use normal serum from the same species the secondary antibody was raised in to achieve ideal blocking results.</li>
	<li>Incubate the samples in 2 mL of primary antibody solution (3% normal serum/5% DMSO/PTwH [PBS-Tween 20 with heparin] + primary antibody/antibodies) for 5 days at 37 &#176;C. Refresh the primary antibody solution&#160;after 2.5 days.
	<ol>
		<li>For PTwH, reconstitute the heparin sodium salt in distilled water to make a 10 mg/mL stock solution (store this solution, aliquoted, at 4 &#176;C). Add the stock solution to 0.2% Tween 20/PBS to a final concentration of 10 &#181;g/mL. 
		NOTE: Choosing the correct antibody dilution may require optimization. Generally, standard immunohistochemistry concentrations are a good starting point.</li>
	</ol>
	</li>
	<li>Wash the samples for 1 day in 4 mL of PTwH, exchanging the wash buffer at least 4x&#8211;5x during the course of the day and leaving the final wash on overnight.</li>
	<li>Incubate the samples in 2 mL of secondary antibody solution (3% normal serum/PTwH + secondary antibody/antibodies) for 5 days at 37 &#176;C. Refresh the secondary antibody solution after 2.5 days.
	<ol>
		<li>Dilute the secondary antibody/antibodies at 1:500 in secondary antibody solution (i.e., 4 &#181;L in 2 mL).</li>
	</ol>
	</li>
	<li>Wash the samples for 1 day as described in step 3.5, leaving the final wash on overnight.</li>
</ol>
4. Nuclear staining
NOTE: Perform all incubation steps with gentle oscillation and, if not indicated otherwise, at room temperature. Protect the samples from light. If no nuclear staining is required or the excitation wavelength/emission spectrum of TO-PRO-3 is required for the excitation or detection of another fluorophore, skip this step.
<ol>
	<li>Dilute the nucleic acid stain TO-PRO-3 at 1:1,000 in PTwH and incubate the samples in 4 mL of nuclear staining solution for 5 h.</li>
	<li>Wash the samples for 1 day as described in step 3.5, leaving the final wash on overnight. 
	NOTE: Following the washes, the samples can be stored in PBS at 4 &#176;C until optical clearing.</li>
</ol>
5. Tissue clearing
NOTE: Perform all incubation steps with gentle oscillation and, if not indicated otherwise, at room temperature. Protect the samples from light. The tissue samples are dehydrated in a graded series of TBA solutions. As immunostaining requires aqueous solutions, all staining procedures have to be finished prior to tissue clearing. Optical clearance and refractive index matching are achieved by treatment with a mixture of BA, BB, and DPE. The clearing solution is supplemented with DL-&#945;-tocopherol as an antioxidant13.
<ol>
	<li>Prepare 30% (v/v), 50%, 70%, 80%, 90%, and 96% TBA solutions in distilled water. For instance, for 30% TBA, add 15 mL of 100% TBA to 35 mL of distilled water in an appropriate sealable vessel and mix by inverting. 
	NOTE: TBA has a melting point of 25&#8211;26 &#176;C; thus, it tends to be solid at room temperature. In order to prepare TBA solutions, heat the well-sealed bottle at 37 &#176;C in an incubator or water bath.</li>
	<li>Dehydrate the samples with 4 mL of each concentration of the prepared series of TBA solutions in ascending order for 2 h each. Leave the 96% TBA on overnight.</li>
	<li>Dehydrate the samples further in pure (100%) TBA for 2 h.</li>
	<li>Prepare clearing solution BABB-D15. 
	NOTE: BABB-D15 is a combination of BA and BB (BABB) which is mixed with DPE at a ratio of x:1, where x is specified in the solution&#8217;s name, in this case 15.
	<ol>
		<li>For BABB, mix one-part BA with two parts BB.</li>
		<li>Mix BABB and DPE at a ratio of 15:1.</li>
		<li>Add 0.4 vol% DL-&#945;-tocopherol (vitamin E). 
		NOTE: For example, for 20 mL of BABB-D15, mix 6.25 mL of BA with 12.5 mL of BB. Add 1.25 mL of DPE and supplement it with 0.08 mL of DL-&#945;-tocopherol.</li>
	</ol>
	</li>
	<li>Clear the samples in clearing solution until they are optically transparent (2&#8211;6 h).</li>
	<li>The samples can be stored at 4 &#176;C in BABB-D15, protected from light, until mounting and imaging.</li>
</ol>
6. Sample mounting
<ol>
	<li>Using a 3D printer, print the&#160;imaging chamber and the lid (material: copolyester [CPE], nozzle: 0.25 mm, layer height: 0.06 mm, wall thickness: 0.88 mm, wall count: 4, infill: 100%, no support structure; the corresponding .STL file can be found in the <a href="https://www.jove.com/files/ftp_upload/59402/3D_ImagingChamber.stl" target="_blank">Supplementary Materials</a> of this protocol).</li>
	<li>Assemble the imaging chamber (Figure 2).
	<ol>
		<li>Mount a round coverslip (diameter: 30 mm) on the imaging chamber with RTV-1 (one-component room-temperature-vulcanizing) silicone rubber. Remove the excess silicone rubber with a water-wetted cotton swab and cure overnight.</li>
		<li>Mount a round coverslip (diameter: 22 mm) on the lid with RTV-1 silicone rubber. Remove the&#160;excess silicone rubber&#160;with a water-wetted cotton swab and cure overnight.</li>
	</ol>
	</li>
	<li>Place the sample in an imaging chamber, add a small volume of BABB-D15, and insert the lid. Fill the chamber up with BABB-D15 through the inlet, using a hypodermic needle (27 G x 3/4 inch [0.40 mm x 20 mm]).</li>
	<li>Plug the inlet and seal the imaging chamber with RTV-1 silicone rubber. Cure overnight in the dark.</li>
</ol>
7. Imaging and image processing
<ol>
	<li>Set up the image acquisition by selecting the respective laser lines to match the fluorophores used. Adjust the detection ranges of each detector to prevent signal overlap between channels. 
	NOTE: Exemplary detection ranges for Alexa Fluor 488, Alexa Fluor 568, and TO-PRO-3 are 500&#8211;550 nm, 590&#8211;620 nm, and 645&#8211;700 nm, respectively.</li>
	<li>Choose the acquisition parameters, define the upper and lower border of the z-stack, and acquire the image stack. 
	NOTE: Exemplary acquisition parameters are a sequential scan with a pixel size of 60-90 nm, a z-step size of 0.5 &#181;m, a line average of 1, a scan speed of 400 Hz, and a pinhole size of 1 Airy unit.</li>
	<li>Process the image stack using appropriate image analysis software (e.g., Fiji) to generate 3D projections or perform in-depth analyses. 
	NOTE: Because of the large size of the acquired image files, the use of a workstation is usually necessary.
	<ol>
		<li>Open the acquisition or image file(s) in Fiji (File | Open | Select files).
		<ol>
			<li>If using, for instance, .LIF files, select or deselect the desired options in the Bio-Formats dialog window. View stack with Hyperstack. Other than that, no specific selections or ticks are necessary. Press OK.</li>
			<li>If the file contains multiple image stacks, select the ones to be analyzed and confirm by pressing OK.</li>
		</ol>
		</li>
		<li>Perform bleach correction by splitting the merged image into individual channels (Image | Color | Channels Tool, then select More | Split Channels). For each channel, select bleach correction (Image | Adjust | Bleach Correction) and choose Simple Ratio (background intensity: 0.0). 
		NOTE: In some cases, for instance, when there is no linear decay of the signal or the signal is too weak overall, Simple Ratio may fail. Alternatively, try Exponential Fit or skip the bleach correction.</li>
		<li>Adjust the brightness and contrast for each channel using the sliders (Image | Adjust | Brightness | Contrast).</li>
		<li>Merge the channels (Image | Color | Merge Channels), make a composite (Image | Color | Channels Tool, then select More | Make Composite), and convert it to RGB format (Image | Color | Channels Tool, then select More | Convert to RGB).</li>
		<li>If necessary, resize the image stack to reduce the computation time and file size (Image | Adjust | Size, both options ticked plus bilinear interpolation).</li>
		<li>Generate a 3D projection (Image | Stacks | 3D Project). Choose Brightest Point as projection method and set the slice spacing to match the z-step size of the acquired image stack. For maximum quality, set the rotation angle increment to 1 and enable interpolation. Modify the total rotation, transparency thresholds, and opacity as needed.</li>
		<li>If necessary, readjust the contrast and brightness by converting the 3D projection back to 8-bit format (Image | Type | 8-bit). Use the respective sliders (Image | Adjust | Brightness | Contrast) and reconvert the image stack to RGB format as described in step 7.3.4.</li>
		<li>Save the 3D projection as .TIF file (image file format) and .AVI file (video file format).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The resurgence and further development of tissue clearing techniques in recent years2,3,4,5,6,7,8,9,10,11,12,13,...

Conventional histology techniques mostly rely on thin sections of organ tissues, which can inherently provide only 2D insights into a complex 3D environment. Although feasible in principle, 3D reconstruction from serial thin sections requires demanding technical pipelines for both slicing and subsequent in silico alignment of the acquired images1. Moreover, seamless reconstruction of z-volumes after microtome slicing is critical as both mechanical and computational artifacts can remain because of suboptimal image registration caused by nonoverlapping image planes, staining variations, and physical destruction of tissue by, for instance, the microtome blade. In contrast, pure optical slicing of intact thick tissue samples allows the acquisition of overlapping image planes (oversampling) and, thereby, facilitates 3D reconstruction. This, in turn, is highly beneficial for the analysis of infection processes in complex cell populations (e.g., neuronal networks in the context of the surrounding glial and immune cells). However, inherent obstacles of thick tissue sections include light scattering and limited antibody penetration into the tissue. In recent years, a variety of techniques has been developed and optimized to overcome these issues2,3,4,5,6,7,8,9,10,11,12,13. Essentially, target tissues are turned optically transparent by treatment with either aqueous2,3,4,5,6,7,8,9 or organic solvent-based10,11,12,13 solutions. The introduction of 3DISCO (3D imaging of solvent-cleared organs)11,12 and its successor uDISCO (ultimate 3D imaging of solvent-cleared organs)13 provided a relatively fast, simple, and inexpensive tool with excellent clearing capabilities. The main constituents of the clearing protocol are the organic solvents tert-butanol (TBA), benzyl alcohol (BA), benzyl benzoate (BB), and diphenyl ether (DPE). The development and addition of iDISCO (immunolabeling-enabled 3D imaging of solvent-cleared organs)14, a compatible immunostaining protocol, constituted another advantage over existing methods and enabled the deep-tissue labeling of antigens of interest, as well as the long-term storage of immunostained samples. Thus, the combination of iDISCO14 and uDISCO13 allows for the high-resolution imaging of antibody-labeled proteins in large tissue sections (up to 1 mm) using conventional CLSM.
The preservation of an organ&#8217;s complex structure in all three dimensions is particularly important for brain tissue. Neurons comprise a very heterogeneous cellular subpopulation with highly diverse 3D morphologies based on their neurite projections (reviewed by Masland15). Furthermore, the brain consists of a number of compartments and subcompartments, each composed of different cellular subpopulations and ratios thereof, including glial cells and neurons (reviewed by von Bartheld et al.16). As a neurotropic virus, the rabies virus (RABV, reviewed by Fooks et al.17) primarily infects neurons, using their transport machinery to travel in retrograde direction along axons from the primary site of infection to the central nervous system (CNS). The protocol described here (Figure 1A) allows for the immunostaining-assisted detection and visualization of RABV and RABV-infected cells in large, coherent image stacks obtained from infected brain tissue. This enables an unbiased, 3D high-resolution assessment of the infection environment. It is applicable to brain tissue from a variety of species, can be performed immediately after fixation or after the long-term storage of samples in paraformaldehyde (PFA), and allows the storage and reimaging of stained and cleared samples for months.

<table border="0" cellpadding="0" cellspacing="0" style="width:827px;" width="827">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Reagents</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Benzyl alcohol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">41218</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Benzyl benzoate</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:160px;">BB6630-500ML</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dimethyl sulfoxide</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">4720.2</td>
			<td style="width:297px;">Various buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Diphenyl ether</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:160px;">240834-100G</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">DL-&#945;-Tocopherol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">A17039</td>
			<td style="width:297px;">Antioxidant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Donkey serum</td>
			<td style="width:119px;">Bio-Rad</td>
			<td style="width:160px;">C06SBZ</td>
			<td style="width:297px;">Blocking reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glycine</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">3908.2</td>
			<td style="width:297px;">Background reduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Goat serum</td>
			<td style="width:119px;">Merck</td>
			<td style="width:160px;">S26-100ML</td>
			<td style="width:297px;">Blocking reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Heparin sodium salt</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">7692.1</td>
			<td style="width:297px;">Background reduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Hydrogen peroxide solution (30 %)</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">8070.2</td>
			<td style="width:297px;">Sample bleaching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methanol</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">4627.4</td>
			<td style="width:297px;">Sample pretreatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Paraformaldehyde</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">0335.3</td>
			<td style="width:297px;">Crystalline powder to make fixative solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium azide</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">K305.1</td>
			<td style="width:297px;">Prevention of microbial growth in stock solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">tert-Butanol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">33278</td>
			<td style="width:297px;">Sample dehydration for tissue clearing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">TO-PRO-3</td>
			<td style="width:119px;">Thermo Fisher</td>
			<td style="width:160px;">T3605</td>
			<td style="width:297px;">Nucleic acid stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Triton X-100</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">3051.2</td>
			<td style="width:297px;">Detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tween 20</td>
			<td style="width:119px;">AppliChem</td>
			<td style="width:160px;">A4974,0500</td>
			<td style="width:297px;">Detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Miscellaneous</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">5 mL reaction tubes</td>
			<td style="width:119px;">Eppendorf</td>
			<td style="width:160px;">0030119401</td>
			<td style="width:297px;">Sample tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Coverslip, circular (diameter: 22 mm)</td>
			<td style="width:119px;">Marienfeld</td>
			<td style="width:160px;">0111620</td>
			<td style="width:297px;">Part of imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Coverslip, circular (diameter: 30 mm)</td>
			<td style="width:119px;">Marienfeld</td>
			<td style="width:160px;">0111700</td>
			<td style="width:297px;">Part of imaging chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Hypodermic needle (27 G x &#190;&#8221; [0.40 mm x 20 mm])</td>
			<td style="width:119px;">B. Braun</td>
			<td style="width:160px;">4657705</td>
			<td style="width:297px;">Filling of the imaging chamber with clearing solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">RTV-1 silicone rubber</td>
			<td style="width:119px;">Wacker</td>
			<td style="width:160px;">Elastosil E43</td>
			<td style="width:297px;">Adhesive for the assembly of the imaging chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Ultimaker CPE 2.85 mm transparent</td>
			<td style="width:119px;">Ultimaker</td>
			<td style="width:160px;">8718836374869</td>
			<td style="width:297px;">Copolyester filament for 3D printer to print parts of the imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Technical equipment and software</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">3D printer</td>
			<td style="width:119px;">Ultimaker</td>
			<td style="width:160px;">Ultimaker 2+</td>
			<td style="width:297px;">Printing of imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Automated water immersion system</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">15640019</td>
			<td style="width:297px;">Software-controlled water pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Benchtop orbital shaker</td>
			<td style="width:119px;">Elmi</td>
			<td style="width:160px;">DOS-20M</td>
			<td style="width:297px;">Sample incubation at room temperature (~ 150 rpm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Benchtop orbital shaker, heated</td>
			<td style="width:119px;">New Brunswick Scientific</td>
			<td style="width:160px;">G24 Environmental Shaker</td>
			<td style="width:297px;">Sample incubation at 37 &#176;C (~ 150 rpm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Confocal laser scanning microscope</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">DMI 6000 TCS SP5</td>
			<td style="width:297px;">Inverted confocal microscope for sample imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Fiji</td>
			<td style="width:119px;">NIH (ImageJ)</td>
			<td style="width:160px;">open source software (v1.52h)</td>
			<td style="width:297px;">Image processing package based on ImageJ</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Long working distance water immersion objective</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">15506360</td>
			<td style="width:297px;">HC PL APO 40x/1.10 W motCORR CS2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vibratome</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">VT1200S</td>
			<td style="width:297px;">Sample slicing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:251px;">Workstation</td>
			<td style="width:119px;">Dell</td>
			<td style="width:160px;">Precision 7920</td>
			<td style="width:297px;">CPU: Intel Xeon Gold 5118 
			GPU: Nvidia Quadro P5000 
			RAM: 128 GB 2666 MHz DDR4 
			SSD: 2 TB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Primary antibodies</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:251px;">Goat anti-RABV N</td>
			<td style="width:119px;">Friedrich-Loeffler-Institut</td>
			<td style="width:160px;"></td>
			<td style="width:297px;">Monospecific polyclonal goat anti-RABV N serum, generated by goat immunization with baculovirus-expressed and His-tag-purified RABV nucleoprotein N 
			Dilution: 1:400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Rabbit anti-GFAP</td>
			<td style="width:119px;">Dako</td>
			<td style="width:160px;">Z0334</td>
			<td style="width:297px;">Polyclonal antibody (RRID:AB_10013382) 
			Dilution: 1:100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Rabbit anti-MAP2</td>
			<td style="width:119px;">Abcam</td>
			<td style="width:160px;">ab32454</td>
			<td style="width:297px;">Polyclonal antibody (RRID:AB_776174) 
			Dilution: 1:250</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:251px;">Rabbit anti-RABV P 160-5</td>
			<td style="width:119px;">Friedrich-Loeffler-Institut</td>
			<td style="width:160px;"></td>
			<td style="width:297px;">Monospecific polyclonal rabbit anti-RABV P serum, generated by rabbit immunization with baculovirus-expressed and His-tag-purified RABV phosphoprotein P (see reference 23: Orbanz et al., 2010) 
			Dilution: 1:1,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Secondary antibodies</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-goat IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-mouse IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-rabbit IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Goat anti-mouse IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Goat anti-rabbit IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
	</tbody>
</table>

high-resolution 3d imaging of rabies virus infection in solvent-cleared brain tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

The combination of iDISCO14 and uDISCO13 coupled with high-resolution CLSM provides deep insights into the spatiotemporal resolution and plasticity of RABV infection of brain tissue and the surrounding cellular context.
Using immunostaining of RABV phosphoprotein (P), complex layers of infected neuronal cells can be visualized in thick sections of mouse brains (Figure 3). Subsequently, seamless 3D projections of the ...

Watch this Scientific Journal Video about High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue at JoVE.com

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

high-resolution-3d-imaging-rabies-virus-infection-solvent-cleared

Research

JoVE Journal

Neuroscience

10.8K Views.  Federal Research Institute for Animal Health. Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

Video: High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...