We deeply thank the technical staff in Yamamori lab and animal facilities (RRD) for their help. We thank the RIKEN CBS-Olympus Collaboration Center for the technical assistance with confocal image acquisition. This work was supported by the program for Scientific Research on Innovative Areas (grant number 22123009) from MEXT, Japan, by Brain/MINDS and Brain/MINDS2.0 from AMED, Japan (JP15dm0207001, JP23wm0625001, and JP24wm0625218), and by JSPS KAKENHI Grant Number 24K09678 to A.W.

&#8203;All experimental procedures were carried out following the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996 and the Japanese Physiological Society&#39;s &#34;Guiding Principles for the Care and Use of Animals in the Field of Physiological Science,&#34; and were approved by the Experimental Animal Committee of RIKEN (W2020-2-009(2)).
1. Tracer injection
<ol>
	<li>Perform injections of fluorescent and non-fluorescent tracers to the marmoset brain according to previously reported procedures14.
	<ol>
		<li>Regarding the combination with the STPT method, ensure that the fluorescence of the tracers is strong enough to be detected without enhancement. Adopt a double-vector TET-Off system10 for enhanced expression. 
		NOTE: Non-fluorescent tracers can also be injected and detected after STPT is done using the produced slices. Examples of non-fluorescent tracers include Biotinylated Dextran Amine (BDA), cre-expressing AAV in AAV2 retro for retrograde detection of the input cell nuclei10, as well as GFP-based smFP-tag AAVs (see below).</li>
	</ol>
	</li>
	<li>Perfusion fix and obtain the marmoset brain after 4 weeks.
	<ol>
		<li>To anesthetize the marmoset, administer Medetomidine (0.04 mg/kg), midazolam (0.4 mg/kg), and butorphanol (0.4 mg/kg), called MMB, intramuscularly, followed by intraperitoneal injection of Thiopental Sodium (100 mg/kg).</li>
		<li>After confirming that the pain reflex is lost, cut open the chest cavity to expose the heart and cut the right atrium to allow the blood and fixative to exit.</li>
		<li>Then cut the left ventricle to insert the perfusion needle for first flushing the blood with the prefix solution (250 mM sucrose, 5 mM MgCl2 in 0.02 M phosphate buffer [PB, pH 7.4]) for a few minutes (~50-100 mL), followed by fixation with 4% paraformaldehyde/0.1 M phosphate buffer (PB; 2-3 times the weight of the animal over ~20 min).</li>
		<li>Good perfusion is critical for a good result. When inserting the perfusion needle, advance it along the septum toward the aorta to secure a good flow of the fixative. The tip of the needle becomes visible when it reaches the aorta. Retract until the tip is barely visible so it does not bypass the carotid artery.</li>
	</ol>
	</li>
	<li>Keep the brain in 4% paraformaldehyde/0.1 M PB for 48 h at 4 &#176;C and transfer to 50 mM PB. If not used immediately, store the brain in 0.75% Glycine/0.1 M PB to prevent autofluorescence due to overfixation.</li>
</ol>
2. Sample preparation (Figure 1)
<ol>
	<li>Incubate the fixed brain with collagenase (1 mg/mL in 3 mM CaCl2 in 10 mL of tris buffered saline [TBS])at 37 &#176;C for 1 h. 
	NOTE: Prewarm the brain at 37 &#176;C for 5 min before incubation.</li>
	<li>Carefully rub the brain surface with a cotton swab to peel off the pia matter and other meninges (Figure 1A, Supplementary video). Use fine forceps to peel the detached meninges. 
	NOTE: It is important to remove the meninges surrounding the midbrain, including the superior colliculus or those at the top of the thalamus. It is difficult to expose these structures that are hidden deep inside. However, remove meninges as much as possible. Otherwise, they often remain uncut, float up, and interfere with imaging and slicing (Figure 2A).</li>
	<li>Take extra caution when the brain is subjected to ex vivo MRI before embedding, during which the brain is immersed in fluorine solution15. The leftover fluorine, if embedded together, could form tiny air bubbles under the objective while imaging. To avoid this, keep the brain in PB for 1 week before embedding. 
	NOTE: This waiting step is usually not necessary but is critical when combined with ex vivo MRI.</li>
	<li>Prepare the NaBH4 buffer by dissolving 0.2 g of NaBH4 in 100 mL of 50 mM borate buffer (pH 9.2) warmed to 40&#176;C. Leave the cap loose to let the CO2 gas out. Keep the solution overnight with the cap loosened.&#160;Wrap the bottle in aluminum foil to protect it from light until use.</li>
	<li>Make oxidized agarose by stirring 2.25 g of agarose and 0.21 g of NaIO4 in 100 mL of PB for 2-3 h. Filter the solution with vacuum suctioning and wash it with three changes of the PB. Resuspend the agarose in 50 mL of PB.</li>
	<li>Completely melt the agarose in a microwave oven and cool to 60-65 &#176;C.</li>
	<li>Place the brain into a custom-made chamber and embed the brain in agarose. Take care to introduce the agarose into the cavity below the corpus callosum (Figure 1B). 
	NOTE: Prewarm the brain first at room temperature and then at 65 &#176;C for 5 min to allow the agarose to settle on the brain surface without solidifying.</li>
	<li>Disassemble the chamber and immerse the agarose block in the NaBH4 buffer overnight at 4 &#176;C.</li>
	<li>Change the buffer to PB several times over 1-2 weeks at 4 &#176;C. 
	NOTE: The tissue autofluorescence becomes very weak if the buffer exchange is incomplete.</li>
	<li>Make a slide glass stage by attaching four Neodymium magnets with epoxy instant mix.</li>
	<li>Mount the agarose block onto the stage with a strong adhesive (e.g., super glue) (Figure 1C).</li>
</ol>
3. Tissue processing
<ol>
	<li>Operate the whole tissue imaging system according to the manufacturer&#39;s instructions. A detailed protocol is also published16. The current procedure is mainly used for TissueCyte1000 but can be applied to other models, such as TissueCyte 1600FC.</li>
	<li>The operation is slightly different between the models, but follow the points below that are commonly important.
	<ol>
		<li>The entire marmoset brain can be processed coronally from the anterior to the posterior ends without any changes in the hardware. Ensure that the stage moving limits are not exceeded when placing the brain.</li>
		<li>Fine-tune the angle of the blades to ensure that the surface depths at both ends of the blade are within 10 &#181;m. Because of dense myelination, even a 10 &#181;m difference in depth could lead to different laser penetrations through adult marmoset white matter. For the same reason, set the imaging plane at about 25-35 &#181;m from the surface.</li>
		<li>Use a ceramic blade (recommended) to cut the entire brain at 50 &#181;m intervals (~over 650 slices).</li>
		<li>The meninges remain uncut and interfere with slicing if not properly removed (Figure 2). Some meninges (e.g., those surrounding the hippocampus or pulvinar) are difficult to remove. Manually extract these meninges by fine forceps when noticed.</li>
	</ol>
	</li>
</ol>
4. Auxiliary histological techniques
<ol>
	<li>Section retrieval
	<ol>
		<li>Recover the tissue slices to perform various histological staining. Accurately align these sections in order of slicing. Trace the blood vessels across cortical layers for alignment (Figure 3).</li>
	</ol>
	</li>
	<li>Cut off excess agarose from each section for better handling. 
	NOTE: This process needs not to be excessive. The agarose serves to hold tissue segments in place without interfering with the histological processing.</li>
	<li>Backlit imaging
	<ol>
		<li>Mount the sections onto a slide glass and dry.</li>
		<li>Rehydrate the section with PBS and place the coverslip for imaging.</li>
		<li>Observe a myelination pattern with no staining when the section is imaged by a fluorescence microscope (Figure 4) in a brightfield mode. Adjust the exposure time so that the light reflection can be visible. 
		NOTE: Any microscope can be used if it can use dark field illumination.</li>
		<li>Remove the coverslip and proceed to Nissl staining or any other staining. Any standard staining procedure is sufficient.</li>
		<li>Use this method to directly compare different staining patterns with the myelination patterns (Figure 4).</li>
	</ol>
	</li>
	<li>BDA imaging
	<ol>
		<li>See Table 1 for a detailed procedure.</li>
		<li>First, treat the sections with Dent&#39;s Solution (20% DMSO, 80% Methanol). Methanol included in Dent&#39;s solution dramatically enhances the BDA signals in the axons.</li>
		<li>Use the tyramide signal amplification (TSA) method to enhance the BDA signal to be fluorescently detected17. 
		NOTE: TSA-biotin solution can be purchased from the vendor, but the In-House reagent is much cheaper and more effective.</li>
		<li>To register to the STPT image, take a backlit image after mounting the section to a slide glass with the mounting media.</li>
	</ol>
	</li>
	<li>Spaghetti monster fluorescent protein (smFP) staining 
	NOTE: The &#39;spaghetti monster&#39; fluorescent proteins (smFPs) are a family of non-fluorescent GFP variants with multiple epitope tags18. They provide excellent fluorescent signals upon immunodetection and are suitable as companion tracers to fluorescent tracers. See Table 2 for the AAV constructs available from Addgene.
	<ol>
		<li>Follow the detailed procedure provided in Table 1.</li>
	</ol>
	</li>
</ol>
5. Post-imaging data processing
<ol>
	<li>Data integrity test 
	NOTE: STPT generates a vast number of image files. When the brain of an adult marmoset is coronally sectioned at 50 &#181;m intervals, more than 650 sections are typically required to cover the entire brain. Each section&#39;s data consists of a series of tiling images captured in three channels. These image files are stored in a single folder along with a metadata file that records the position of each tile. To minimize the total amount of data and processing time, imaging is conducted in blocks of runs, during which the number of X and Y steps for tiling gradually changes. Typically, 20-30 blocks of runs, each comprising the imaging of 30-50 sections, constitute the complete dataset for a single brain. To utilize this data, several image processing steps are required, including stitching of the tiled images, segmentation of fluorescent signals, and registration of the volumetric data to a standard template, among others.
	<ol>
		<li>Perform the image processing steps individually using various software tools or through dedicated pipelines tailored to the researchers&#39; specific needs. Details of the image processing are beyond the scope of this paper and can be found in other publications11,19.</li>
		<li>Regardless of the procedure used, ensure that the data is organized consistently. 
		NOTE: Since the entire imaging process involves many blocks of runs and spans several days, it is not uncommon for a run to be canceled midway and restarted anew. In such cases, the data structure can become disrupted, hindering the proper execution of the image processing pipeline.</li>
		<li>To minimize the risk of disruption of the data structure, run a Python script to check the integrity of the data structure (kn_pipeline_check_mosaic.py) available at GitHub (github.com/watkarbey/STPT_depo).</li>
	</ol>
	</li>
	<li>Registration of the stained image to the STPT image 
	NOTE: Non-linear transformation of the stained image to the STPT image can be performed using the bUnwarpJ plugin of ImageJ.
	<ol>
		<li>Adjust the size of the images of the target (STPT) and the source (stained) images so that they are approximately equal.</li>
		<li>Binarize each image to visualize the shapes of the sections.</li>
		<li>Run bUnwarpJ. Remember to check the Save Transformations option.</li>
		<li>Start bUnwarpJ again. Select the original image before binarization as the source image. Then click on Load Elastic Transformation and choose the saved transformation.</li>
	</ol>
	</li>
</ol>

Volumetric Imaging and Axonal Projection Mapping in the Marmoset Brain

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The authors have nothing to disclose.

This article explained the practical solutions to handle marmoset brains for whole-brain processing as well as auxiliary histological techniques that enhance the utility of the STPT technique. The strength of &#34;whole brain neuroanatomy&#34; using STPT is that you can obtain the 3D coordinates of any region of interest, whether it is anatomically annotated or not. By high-precision 3D-to-3D registration, it is possible to transform these coordinates into a standard template for the overlay of multiple datasets. This way, the standard template serves as the medium of data integration. This was an essential aspect of our prefrontal cortex (PFC) mapping project10, where data obtained from many individuals were analyzed. Furthermore, the information mapped to the standard template can be compared with various data that have already been mapped, whether it is Nissl, myelin patterns, tracer data, MRI (including diffusion MRI) data, or anatomical annotations11. Importantly, it can also be compared with future data that will be gained by yet-to-emerge technology. There currently exist multiple templates for the marmoset brain, which are based on Nissl staining23, MRI23,24,25,26,27,28, and STPT11. But they can be transformed to each other&#39;s coordinates based on image contrasts and precalculated parameters11. Data integration at the whole brain scale across studies contributes to a better understanding the brain as a system. The prerequisite for this strategy to work is reliable data acquisition across the brain. Below, critical steps and potential problems associated with the current protocol are discussed.
One of the most vulnerable processes of STPT imaging is tissue slicing. As mentioned above, the meninges often remain uncut and can interfere with the slicing. In particular, the pulvinar nucleus and the superior colliculus are the two most affected brain regions: depending on the thoroughness of meninge removal and embedding in agarose, they can be stripped off from the tissue block during slicing. These deep regions are difficult to approach from the outside and can easily break during meninge removal. A careful but thorough meninge removal is critical for successful imaging. Another concern is the flip back of sliced sections onto the tissue block, which sometimes occurs when the sections remain attached even after slicing. It can be minimized by shaping the block so that the blade cuts obliquely at the very end.
By imaging deep into the tissue block, STPT avoids the bumpiness of its surface. Whereas the fluorescent signals can pass through the cortical region rather easily, they are highly diminished in the myelinated regions. Therefore, the imaging depth needs to be carefully determined to balance between the consistent imaging across the entire block surface and the brightness of the signals in the myelin-rich region, such as the white matter. In the setup used here, we usually aim at 25-35 &#181;m from the surface. It also needs to be cautioned that the cut surface may become unevenly shrunk after long hours of storage. To minimize the imaging area, we split the imaging session into 20-30 runs with different stage settings for 5-6 days. We either make the interval between runs less than 2 h or confirm the surface depth and adjust the stage height before each run.
In this protocol, the BDA signal was amplified by the TSA method. This method is highly effective and can detect anterogradely transported BDA signals even at relatively low resolution (e.g., Figure 6B). TSA biotin is commercially available from Akoya Biosciences, but the homemade solution shows much better enhancement. On the other hand, the dilution of the antibody and solution requires careful adjustment to obtain the optimal result. Pretreatment of the section with a methanol solution is critical. Without pretreatment, the BDA signals are barely detected in the myelinated axons.
When an anterograde tracer is used, it is often difficult to identify the exact site of injection because of the saturation of the fluorescence signals. In the whole tissue imaging system setup used in this study, we use the blue channel to identify the infected cells (Figure 5I). Even when the red and green channels are saturated, each individual infected neuron is usually detectable in the blue channel. This is also true with the Allen Mouse Brain Connectivity Atlas1. Examination of the cells of origin is important because the infection sometimes involves only particular layers. We encountered such partial infections rather frequently for the Mouse Brain Connectivity Atlas, perhaps because of the utilization of the iontophoresis method29. The lateral spread of the viral tracers could be more or less variable depending on the injections. This variability can potentially affect the result of tracing and needs careful normalization.
The successful registration of the obtained 3D image to the standard template is a key process of the whole brain neuroanatomy. Registration of the STPT image to the STPT template can be pretty accurate, and we observed only deviances of a few voxels (50 &#181;m isocubic) for the borders with a high image contrast10. Still, there is a limit to what registration can do. Because of the meninge removal process, the STPT samples generally have gaps between the hemispheres and between the cortex and midbrain/hindbrain, whereas brain tissues are tightly packed in the in vivo MRI images. Such differences are difficult to adjust by registration. The Marmoset cortex is mostly devoid of sulci, but the intraparietal sulcus is very deep in some Individuals. Such a sulci will be lost (top-to-top fusion occurs) upon registration. Although registration is a powerful technique, it is necessary to go back to the raw data for confirmation of the obtained result.
The generation of massive image data is both a strength and a limitation of this technique. While it enhances the completeness of the dataset, it necessitates careful management of the acquired data and the development of an automated image processing pipeline for efficient data interpretation. In the future, the application of generative artificial intelligence (AI) in constructing image processing pipelines may significantly simplify this process. Systematic whole-brain imaging has also been performed using slide scanner-based methods21,30. Compared to such methods, STPT does not require additional computation for 3D reconstruction. Combined with optical sectioning, we have demonstrated that STPT has the potential to reconstruct even axon segments across sections31. With the further combination of tissue clearance techniques, Economo et al. developed a method to image the entirety of sparsely labeled neurons32,33. The latest versions of TissueCyte now offer options for an additional laser to enhance the excitation of red fluorescent proteins or a section capture unit for the automatic recovery of sections. With these advancements, the whole-brain approach will become more efficient, providing a foundation for a comprehensive understanding of the primate brain, including that of humans.

In neuroanatomical studies, researchers are faced with the need to observe micrometer-order structures (e.g., axons and boutons) in the context of the whole brain. This difficult task has been typically approached by visually inspecting the serial sections and looking for the region of interest for detailed imaging, analyses, and photo recording. With technological advancement, however, it is becoming possible to image the entire brain at high resolution for whole-brain analysis. In a ground-breaking study by Oh et al.1, hundreds of mouse brains received tracer injections for connectomic analysis, which were processed by serial two-photon tomography (STPT) imaging technique2. This study&#39;s characteristic was that they selected anterograde tracers to quantify &#34;connectivity&#34;. While anterograde tracers provide very detailed spatial information on axonal distribution, neuroanatomists have relied on laborious manual segmentation for its analysis. By automating various analytical procedures, including this segmentation step, they succeeded in the &#34;mass production&#34; of ready-to-use high-resolution tracer data for multi-purpose uses. The effectiveness of their approach is obvious, given a variety of studies that used their tracer data3,4,5 and their standard brain6.
Historically, the neural connectivity of non-human primate brains has attracted the attention of many neuroanatomists since the development of the degeneration method in the 1950s, to anterograde/retrograde substance transportation methods in the 1970s to the present viral strategy7,8. As such, vast pieces of literature exist that investigate primate neural connections. In particular, many researchers have investigated the complex corticocortical connectivity of the macaque brain, and their results have been curated for tabulation (e.g., CoCoMac9). Although useful, these classic studies had several limitations. First, because each study focuses only on limited brain regions, the obtained information inevitably becomes fragmental. Second, each study uses different methods and conditions. Thus, the quantitative evaluation across studies becomes complicated. Third, the connectivity is usually shown either as a camera lucida of representative sections or as a semi-quantitative table/graph for author-defined brain regions. In other words, only very limited information from a complex brain structure is extracted for presentation in the published literature. With the development of magnetic resonance imaging (MRI) techniques, whole-brain studies at low resolution became prominent. However, there is a large gap between the levels of detail between what we know about neural connections in mice and those in primates.
With such background in mind, we set out to perform comprehensive anterograde tracing of the common marmoset brains10,11. Although much smaller and smoother than the macaque brain, the marmoset counterpart exhibits clear signs of primates, such as the presence of area MT and the granular prefrontal areas, neither of which is clearly defined in rodents12,13. Here, the small size was a big advantage because even the marmoset brain weighs ten times greater than the mouse brain. Fortunately, we could image the whole marmoset brain with minimal updates of the original version of TissueCyte1000 (henceforth referred to as a whole tissue imaging system) operating software, the commercially available microscope for STPT imaging2. This update was to allow extra movement of the stage before slicing. The current version is now sufficient for processing the marmoset brain. This article shares a protocol to handle the marmoset brain for STPT imaging. The post-imaging protocol that further enhances the utility of this method is also provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose type I</td>
			<td>&#160;Sigma</td>
			<td>A6013</td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-one fluorescence microscope</td>
			<td>Keyence</td>
			<td>BZ-X series</td>
			<td>This study used BZ-X710</td>
		</tr>
		<tr>
			<td>anti-mouse Cy3</td>
			<td>Jackson Immuno Research Laboratories, Inc.</td>
			<td>115-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-myc antibody</td>
			<td>MBL</td>
			<td>M192</td>
			<td>mouse monoclonal</td>
		</tr>
		<tr>
			<td>Butorphanol&#160;</td>
			<td>Meiji Animal Health</td>
			<td>Vetorphale</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic blade</td>
			<td>TissueVision&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Wako Chemical</td>
			<td>#031-17601</td>
			<td>Aliquot into 100 &#181;L x 10 of 100 mg/mL in PBS&#160;</td>
		</tr>
		<tr>
			<td>Cotton swab (HUBY-340)</td>
			<td>HUBY</td>
			<td>BB-013SP</td>
			<td>micro cotton swabs</td>
		</tr>
		<tr>
			<td>Custom-made chamber</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>The chamber consist of four side plates and one bottom plates, all made of 1 cm thick acrylic plates, which can be securely fastened together using screw bolts. An additional acrylic plate is placed inside the chamber, with three metal poles&#160; inserted through it to hold the marmoset brain in an inverted position.</td>
		</tr>
		<tr>
			<td>Cy3-Streptavidin&#160;</td>
			<td>Jackson Immuno Research Laboratories, Inc.</td>
			<td>016-160-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps, Inox</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy instant mix</td>
			<td>Locktite</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td>NIH</td>
			<td colspan="2">https://imagej.net/software/fiji/downloads</td>
		</tr>
		<tr>
			<td>kn_pipeline_check_mosaic.py</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>github.com/watkarbey/STPT_depo; This is a checking script for TissueCyte data.</td>
		</tr>
		<tr>
			<td>Medetomidine&#160;</td>
			<td>Zenoaq</td>
			<td>Domitor</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Sandoz</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>&#160;Sigma</td>
			<td>#452882</td>
			<td></td>
		</tr>
		<tr>
			<td>NaIO4</td>
			<td>&#160;Sigma</td>
			<td>S1878</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion needle&#160;</td>
			<td>Natsume Seisakusho Co., Ltd</td>
			<td>&#160;KN-348, 20G-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Slideglass (76 mm x 52 mm)</td>
			<td>Matsunami</td>
			<td>S9111</td>
			<td></td>
		</tr>
		<tr>
			<td>StAvHRP&#160;</td>
			<td>Jackson Immuno Research Laboratories, Inc.</td>
			<td>016-030-084</td>
			<td>HRP-streptavidin (titration needs to be carefully determined)</td>
		</tr>
		<tr>
			<td>TissueCyte1000</td>
			<td>TissueVision&#160;</td>
			<td>https://www.tissuevision.com/tissuecyte</td>
			<td></td>
		</tr>
		<tr>
			<td>TSA blocking reagent</td>
			<td>Kiko Tech</td>
			<td>FP1012</td>
			<td>Refer to TSA-biotin kit (Akoya Biosciences)</td>
		</tr>
		<tr>
			<td>TSA-biotin</td>
			<td>House-made</td>
			<td>N/A</td>
			<td>See Okamoto et al (ref. 16)</td>
		</tr>
		<tr>
			<td>VECTASHIELD HardSet Mounting Medium</td>
			<td>Vector laboratories Inc</td>
			<td>N/A</td>
			<td>Antifade mounting medium</td>
		</tr>
	</tbody>
</table>

serial two-photon tomography of the whole marmoset brain for neuroanatomical analyses

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. We successfully implemented this technique for tracing the axonal projections in the marmoset brain. Here, the detailed experimental procedures that resulted in reliable volumetric imaging of the whole marmoset brain are described. A key process for successful imaging was the removal of meninges surrounding the brain, which interferes with slicing. A big advantage of this methodology is that the sliced sections can be used for additional staining. In the original set-up, the sliced sections are scrambled in the water bath. These sections can be correctly aligned in their original order according to the blood vessel patterns in the cortex. An example of effective histology is the visualization of myelin structure by simple light reflection, which can be combined with Nissl staining to define anatomical borders. These sections can also be used for immunological detection of non-fluorescent anterograde and retrograde tracers, which can be registered to the STPT data for layering of multiple data.

In the typical set-up used here, the whole brain of an adult marmoset can be imaged (Figure 5) at the resolution of ~1.3 x 1.3 &#181;m/pixel with a 50 &#181;m section interval in about 1 week. This amounts to ~650 coronal images in three channels after image stitching. With a 16x objective lens (Nikon 16xW CFI75 LWD; NA = 0.80), the field of view of a single shot is about 1 x 1 mm. The image for the entire coronal surface is obtained by stitching these shots (Figure 5A). The alignment in the Z direction is excellent and a good 3D image is obtained by simply stacking the coronal image data (Figure 5B,E). For standardization, the STPT template for the marmoset brain11 can be used for 3D-3D registration (Figure 5F). This data transformation is one of the key aspects of the whole brain neuroanatomy, in which a region of interest is assigned an absolute space coordinate independent of anatomical annotation. Once the sample brain is registered to the standard space, one can easily take out the subregions of interest for further analysis (Figure 5F,G). In particular, the cortical regions can be transformed into a flat map using a predetermined parameter (Figure 5H).
The sections generated during the imaging can be used for various histological purposes. As shown in Figure 4A,B, the backlit imaging with no staining provides a very similar pattern to the authentic myelin staining. This can be an excellent alternative to myelin staining. Furthermore, if the backlit image is obtained before Nissl staining, the same section can be used to obtain the patterns of both myelin and Nissl staining, thus providing useful information to identify areas and layers (Figure 5C-E). These sections can also be used for immunological staining. In Figure 5J, the section around the injection center was counterstained with NeuN antibody to estimate the transduction efficiency of the AAV virus. It is also a good strategy to inject non-fluorescent tracers in addition to fluorescent tracers and detect them histologically after section retrieval. In our previous study, we combined anterograde green tracers with retrograde &#34;cre&#34; vector, which was later detected by anti-cre antibody10. In an example of Figure 6A-E, BDA was injected into the contralateral side of the green tracer (clover) and fluorescently detected it. Note that the red BDA signals can be registered to the TissueCyte image to be localized in the whole brain coordinate. In another example of Figure 6F-I, smFP-myc was injected into the parietal area (Figure 6G), while the green tracer was injected into the frontal area. This way, multiple tracers can be injected into the same animal without interfering with the imaging. A big advantage of using the STPT sections for additional staining is that the relationship between the fluorescent and non-fluorescent tracers can be determined for the same brain. As such, we were able to determine the reciprocity of corticocortical projections at high-precision10. Another advantage is that the 3D coordinates of the stained sections can be mapped back to the STPT data and then to the standard template. Thus, it may not be necessary to use all the retrieved sections for staining. For better interpretation, sections can be selected for staining to add further context to the STPT data.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67694/67694fig1.jpg" /> 
Figure 1: Sample preparation for STPT. (A) Meninge removal using cotton swabs. The meninges surrounding the brainstem can be removed by fine-tipped forceps. The meninges surrounding the marmoset brain are removed manually by rubbing with cotton swabs. The photo shows the meninges pealed from within the lateral sulcus. (B) The acryl box used for agarose embedding. The pins are movable and used to adjust the angle of the brain to be close to the stereotaxically fixed position20. (C) A magnetic slide made of 76 mm x 52 mm slide glass and four Neodymium magnets attached by epoxy adhesive. The agarose block is attached to the magnetic stage with super glue. <a href="https://www.jove.com/files/ftp_upload/67694/67694fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67694/67694fig2.jpg" /> 
Figure 2: The effect of meninges on imaging. (A) Meninges that remain uncut float up, as shown by the white arrow. In this example, extensive protrusion of the meninges is seen because they were not removed before agarose embedding. Usually, meninges remain uncut only for several difficult regions. (B) An example of bad slicing due to the presence of meninges between the corpus callosum and the upper part of the thalamus. In this case, bad slicing led to the alternation of a deep slice (329) and a relatively normal slice (330). #78-329 stands for sample no. 78 section no. 329 in the Brain/MINDS data portal (C) Another example of bad imaging. In a worse case, the pulvinar nucleus shown by the red arrow may entirely come off. Scale bars: 0.5 mm (upper panel), 0.5 &#181;m (lower panel). (D) Another example of bad imaging. The meninges deep within the calcarine sulcus is difficult to remove. The shadows seen in sections 469 and 470 are caused by the floating meninges that came under the objective. Scale bars: 0.5 mm (upper panel), 0.5 &#181;m (lower panel). <a href="https://www.jove.com/files/ftp_upload/67694/67694fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67694/67694fig3.jpg" /> 
Figure 3: Alignment of tissue sections in order. (A) Up to 50 marmoset coronal sections can be correctly aligned in order in a plastic container (31 cm x 22.5 cm). To visualize the detailed structures, the container is placed on black paper and lighted from the side. These sections are first roughly aligned in order and then subject to precise alignment. (B) The precise alignment uses the blood vessel as the marker. The cerebral cortex contains many blood vessels that run vertically across cortical layers. They are identified as elongated holes that systematically change their positions within the cortical layers (white arrows). Using this method, even sections with 50 &#181;m intervals can be accurately aligned. These blood vessel holes can be identified in the SPTP section images for confirmation. Scale bar: 5 mm (upper panel), 1 mm (lower panel). <a href="https://www.jove.com/files/ftp_upload/67694/67694fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67694/67694fig4.jpg" /> 
Figure 4: Backlit image as a substitute for the myelin staining. (A,B) The identical section was used for backlit imaging and myelin staining. First, the section was mounted onto the slide glass, dried, rehydrated with PBS, coverslipped, and imaged using a light&#160;microscope (Table of Materials). After removing the coverslip, the same section was used for myelin staining21 and imaged using a fluorescence microscope. The backlit image was registrated to the myelin image using the bUnwarpJ plugin of ImageJ. The green boxes show the magnified views of each image. Note that these images show almost identical patterns, except that the myelin staining visualizes fibrous structures better. (C-E) The identical section was used for backlit imaging and Nissl staining. The low-threshold mask for the backlit image was first registered with the low-threshold mask for the Nissl image using the bUnwarpJ plugin, and the original image was transformed using the same parameter. Note that the blood vessels (white arrowheads) are matched well between the two images. Because these two images are for the same section, the matching is almost perfect, and one can directly compare the myelin and Nissl patterns for the identification of cortical layers. Scale bars: 5 mm (panels A-E). <a href="https://www.jove.com/files/ftp_upload/67694/67694fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67694/67694fig5.jpg" /> 
Figure 5: Typical result of STPT imaging. (A) A simple tiling of Ch2 (green) images of an example section (section 310 of sample #21). Without background correction, the borders for each tile are visible. In the middle column, the tiles were stitched with background correction for Ch1 (red) and Ch2 (green), respectively. To reduce the signals of lipofuscin (see panel C), Ch1 signals were subtracted from Ch2 and shown green. To reduce the lipofuscin signals of Ch1, the &#34;Remove Outilers..&#34; command was used before stitching by ImageJ. In the right column, the tracer signals were segmented by image processing pipeline11. The arrowheads (c, d) show the locations where the magnified views are shown in panels C and D. Scale bar: 2 mm. (B) Overview of serial sections for sample #21. STPT generated 635 high-resolution coronal images for this sample. (C) A high magnification view shown by arrowhead c in panel A. This is a simple overlay of Ch1 (red) and Ch2 (green) with no further processing. The triangles show lipofuscin fluorescence signals, which show a widespread spectrum. The tracer segmentation algorithm accurately distinguishes the tracer signals from the lipofuscin background despite a very similar shape (right panel). Scale bar: 100 &#181;m. (D) Another example of a high magnification view. Note that fine axon fibers in layer 1 can be well visible. Scale bar: 100 &#181;m. (E) The 3D reconstruction of original STPT images. The 635 low-resolution coronal images as shown in panel B were used as the tiff-stack for 3D visualization using fluorender22. Scale bar: 5 mm. (F) The 3D reconstruction of the segmented tracer signals registered to the STPT template (grey). The tracer signals in different brain regions were shown by different colors. These regions were cut out by using the annotation shown in panel G. Scale bar: 5 mm. (G) STPT template overlaid with annotation of different brain regions. Scale bar: 5 mm. (H) The cortical tracer signal shown in panel F was shown in the form of a flatmap. (I)&#160;Identification of the injection site by the Ch3 fluorescence, which is less sensitive to the tracer fluorescence and remains unsaturated. Scale bar: 1 mm. (J) Staining the section around the injection center with NeuN antibody showed that approximately 30 % of the neurons show strong expression of Clover green fluorescence. Scale bar: 40 &#181;m. Abbreviations: Cx; cortex, St; striatum, Th; thalamus, SC; superior colliculus. Amy; amygdala, Hip; hippocampus, Cb; cerebellum. <a href="https://www.jove.com/files/ftp_upload/67694/67694fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67694/67694fig6.jpg" /> 
Figure 6: Post-STPT histology showing multiple non-fluorescent tracers. (A-C) Comparison of STPT image with BDA-stained image. In this sample, BDA is injected into the contralateral side of Clover injection. Panels A and C show the Ch2 image and tracer segmentation of the STPT data. Panel B shows the post-STPT staining for BDA. Clover fluorescence is diminished due to the methanol treatment of the section. Scale bar: 2 mm. (D,E) The dotted boxes in panels A and B are magnified. Scale bar: 100 &#181;m. (F,G) Comparison of STPT image with anti-myc tag antibody staining. In this sample, Clover injection is to the PFC, whereas the AAV-smFP-myc is injected into the contralateral parietal cortex. The white rectangles are magnified in panels H and I. Scale bar: 4 mm. (H) Tracer segmentation is shown in green. Scale bar: 200 &#181;m. (I) The myc staining is shown in red. The Clover fluorescence is shown in green. Scale bar: 200 &#181;m. The green signals in panels H and I are present in a similar position but not identical because STPT retrieves only ~10 &#181;m optical section. <a href="https://www.jove.com/files/ftp_upload/67694/67694fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BDA fluorescent staining protocol</td>
			<td></td>
		</tr>
		<tr>
			<td>Remove agarose</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS wash</td>
			<td>10 min (2x)</td>
		</tr>
		<tr>
			<td>1% H2O2 in Dent&#39;s solution&#160;</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>TBS wash</td>
			<td>Brief</td>
		</tr>
		<tr>
			<td>0.5% TNB&#160; blocking</td>
			<td>1 h</td>
		</tr>
		<tr>
			<td>StAvHRP (1:4000) in TNB</td>
			<td>2 overnight</td>
		</tr>
		<tr>
			<td>TNT wash</td>
			<td>10 min (3x)</td>
		</tr>
		<tr>
			<td>TSA Biotin (1:4000) in 0.1 M borate (pH8.5) + 0.003% H2O2</td>
			<td>2 h</td>
		</tr>
		<tr>
			<td>TNT wash</td>
			<td>10 min (3x)</td>
		</tr>
		<tr>
			<td>Cy3-streptavidin&#160; (1:1000) in TNT</td>
			<td>3 h</td>
		</tr>
		<tr>
			<td>TNT wash</td>
			<td>10 min (2x)</td>
		</tr>
		<tr>
			<td>TBS wash</td>
			<td>Keep the section until mounting</td>
		</tr>
		<tr>
			<td colspan="2">Mount section onto slideglass using an antifade mounting medium</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">anti-myc fluorescent staining protocol</td>
		</tr>
		<tr>
			<td>Remove agarose</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS wash</td>
			<td>10 min (2x)</td>
		</tr>
		<tr>
			<td>blocking in IB</td>
			<td>1 h</td>
		</tr>
		<tr>
			<td>Anti-Myc (1:4000) in IB&#160;</td>
			<td>2 over night</td>
		</tr>
		<tr>
			<td>TNT wash</td>
			<td>10 min (3x)</td>
		</tr>
		<tr>
			<td>Anti-mouse Cy3 (1:1000) in TNT</td>
			<td>3 h</td>
		</tr>
		<tr>
			<td>TNT wash</td>
			<td>10 min (2x)</td>
		</tr>
		<tr>
			<td>TBS wash</td>
			<td>Keep the section until mounting</td>
		</tr>
		<tr>
			<td colspan="2">Mount section onto slideglass using an antifade mounting medium</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buffers/solutions</td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>0.5% TNB</td>
			<td>0.5% TSA Blocking Reagent in TS7.5</td>
		</tr>
		<tr>
			<td>Dent&#39;s Solution</td>
			<td>20% DMSO, 80% Methanol</td>
		</tr>
		<tr>
			<td>Immersion buffer (IB)&#160;</td>
			<td>10% FBS, 2% BSA 0.5% TritonX100 in TBS</td>
		</tr>
		<tr>
			<td>TBS (Tris-buffered saline)</td>
			<td>25 mM Tris, 137 mM NaCl, 2.7 mM KCl (pH 7.4)</td>
		</tr>
		<tr>
			<td>TNT</td>
			<td>0.05 % Tween20&#160; in TS7.5</td>
		</tr>
		<tr>
			<td>TS7.5</td>
			<td>0.1 M TRIS-HCl, pH 7.5, 0.15 M NaCl&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: BDA fluorescent staining and anti-myc fluorescent staining protocol
<table border="1" fo:font-size="7px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plasmid for AAV tracer</td>
			<td>Addgene No.</td>
			<td>Recommended antibody</td>
			<td>Suggested dilution</td>
			<td>Expected result</td>
		</tr>
		<tr>
			<td>pAAV-EF1_Cre&#160;</td>
			<td>201198</td>
			<td>Millipore clone 2D8</td>
			<td>1:1000</td>
			<td>good for cells</td>
		</tr>
		<tr>
			<td>pAAVCam1.3_smFP_Myc&#160;</td>
			<td>201205</td>
			<td>MBL M192 My3 (mouse)</td>
			<td>1:4000</td>
			<td>excellent</td>
		</tr>
		<tr>
			<td>pAAVCam1.3_smFP_HA&#160;</td>
			<td>201206</td>
			<td>CST C29F4 (rabbit)</td>
			<td>1:1000</td>
			<td>good for cells</td>
		</tr>
		<tr>
			<td>pAAVCam1.3_smFP_FLAG&#160;</td>
			<td>201207</td>
			<td>MBL PM020B (rabbit)</td>
			<td>1:1000</td>
			<td>OK</td>
		</tr>
		<tr>
			<td>AAVTRE3_smFP_Myc&#160;</td>
			<td>201208</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAVTRE3_smFP_HA&#160;</td>
			<td>201209</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAVTRE3_smFP_FLAG&#160;</td>
			<td>201210</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: List of Addgene plasmids available for the production of non-fluorescent tracers. The cre construct targets the nucleus and is suitable for retrograde tracing enveloped by AAV2 retro. smFP_HA construct is good for cell body detection (and retrograde).
Supplementary Video: Microscope view of meninges removal. <a href="https://www.jove.com/files/ftp_upload/67694/movieS1.zip" target="_blank">Please click here to download this Video.</a>

Serial Two-Photon Tomography of the Whole Marmoset Brain for Neuroanatomical Analyses

Serial two-photon tomography (STPT) imaging is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. Here we describe a protocol for implementing it for marmoset brains to better understand their structural features.

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic ...

serial-two-photon-tomography-whole-marmoset-brain-for-neuroanatomical

Research

JoVE Journal

Neuroscience

321 Views.  RIKEN Center for Brain Science. Ons onderzoek verkent de structurele basis van hersenfuncties, waaronder die bij mensen. Door penseelaap als model te gebruiken, willen we de hersenen van primaten beter begrijpen door neurale verbindingen te identificeren die worden gedeeld tussen soorten en die uniek zijn voor het geslacht van primaten. We hebben de prefrontale projecties van de zijdeaapjes uitgebreid in kaart gebracht.Met behulp van seriële twee-fotontomografietechniek slaagden we ...

Video: Seriële twee-fotontomografie van het hele zijdeaapje brein voor neuroanatomische analyses

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Serial Two-Photon Tomography of the Whole Marmoset Brain for Neuroanatomical Analyses

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