M.S. is grateful for the support from all of the staff in the medical information engineering course in Graduate School of Medicine and Faculty of Medicine, and wants to thank Prof. Tetsuya Takakuwa, Prof. Nobukatsu Sawamoto, and Doris Zakian for their helpful comments. This study was supported by a Grant-in-Aid for Challenging Exploratory Research and by the Leading Initiative for Excellent Young Researchers (LEADER) program to M.S. from MEXT (The Ministry of Education, Culture, Sports, Science, and Technology). The MRI experiments in this work were performed in the Division for Small Animal MRI, Medical Research Support Center, Graduate School of Medicine, Kyoto University, Japan.

All experimental procedures described here have been approved by the Kyoto University Animal Care Committee.
1. Animals (Day 1)
<ol>
	<li>Prepare female C57BL/6J mice (n = 6, aged 3&#8722;5 weeks). 
	NOTE: This protocol is applicable to all rodent species.</li>
</ol>
2. MRI Settings (Day 1)
<ol>
	<li>Put the mouse in an acrylic anesthesia box placed on a heater mat adjusted to 37 &#176;C using a heater mat controller.</li>
	<li>Anesthe...

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We developed a new protocol called the 3D-NEO protocol to bridge macroscopic and microscopic spatial scales by overlapping two brain surfaces more accurately than before. Originally, there were two challenges in creating this protocol which made possible the accurate overlapping of two brain surface images and recording healthy neuronal activities from living organisms. First, it was necessary to effectively wipe cutting solution surrounding the extracted brain after extracting it from the skull without injuring the brai...

Nonuniform multiscale architectures at various physical and biological organizations are commonly found1,2. The brain is also a very nonuniform and multiscale network organization3,4. Various cognitive functions are coded in such network organizations, holding temporal changes of electrical spike patterns of neuronal populations in submillisecond temporal resolutions. Historically, the complex networks among neurons were structurally observed in detail using the staining techniques by Santiago Ram&#243;n y Cajal from over 150 years ago5. To observe group behaviors of active neurons, researchers have developed various recording technologies6,7,8, and the recent significant developments of such technologies have enabled us to record electrical activities from huge numbers of neurons simultaneously. Furthermore, from such functional activities, scientists have succeeded to reconstruct networks of causal interactions among huge numbers of neurons and have declared the topological architecture of their complex interactions &#8216;microconnectome&#8217;9. Macroscopic observations of the brain also allow for regarding a whole brain as a network organization because many brain regions are connected by multiple fiber-bundles. The embedding of microconnectomes into the global brain map still has clear limitations within current technological advances, which is why this embedding protocol is so important. However, there are many challenges to the development of the embedding protocol. For example, in order to observe activities of living local neuronal circuits in purely isolated brain regions, brain slices need to be produced for in vitro recordings. Additionally, recordings from brain slices for in vitro recordings are still an important choice for at least two reasons. First, it is still not easy to observe activities of many living individual neurons simultaneously from brain regions deeper than ~1.5 mm and in high temporal resolution (&#60;1 ms). Second, when we hope to know the internal architecture of a local neuronal circuit, we need to stop all inputs coming from external brain regions to eliminate confounding factors. In order to identify the directions and positions of produced brain slices, it will be further necessary to integrate the spatial positions of these produced brain slices using coordinates. There are, however, a few systematic and reliable ways to make brain slices in an organized way10,11. Here, a new coregistration protocol is introduced, using 3D scanning technology for neuroscientific research in order to provide an integrative protocol. This protocol acts to coordinate micro- and macroscales and embed multielectrode array (MEA) microdata12,13 and staining data onto a macroscopic MRI space through 3D scan surfaces of extracted brains, as well as of noninvasively recorded brains. Surprisingly, this showed a distance error of only ~50 &#956;m as the mode value of the histogram. As a result, the mode values of minimum distances between two surfaces between the MRI surface and the scanned 3D surface were nearly 50 &#181;m for all six mice, which is a suitable number when checking for commonality among individuals. The typical slice width had a recorded spike activity of around 300 &#181;m.

<table>
	<tbody>
		<tr>
			<td>Air compressor</td>
			<td>Kimura Medical</td>
			<td>KA-100</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>All-in-one fluorescence microscope</td>
			<td>KEYENCE</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia box</td>
			<td>Bio Research Center</td>
			<td>RIC-01</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Anesthesia system</td>
			<td>ACOMA Medical Industry</td>
			<td>NS-5000A</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Anti-GAD67, clone 1G10.2</td>
			<td>Merk Millipore</td>
			<td>MAB5406</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Calcium Chrolide</td>
			<td>nacalai tesque</td>
			<td>06729-55</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Choline Chloride</td>
			<td>nacalai tesque</td>
			<td>08809-45</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>curved blunt forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal scalpel</td>
			<td>Kai</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(-) without Ca and Mg, liquid(10x)</td>
			<td>nacalai tesque</td>
			<td></td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Wako</td>
			<td>049-31165</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>nacalai tesque</td>
			<td>16605-42</td>
			<td>re-secctioning</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td>
			<td>Invitrogen</td>
			<td>A32723</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555</td>
			<td>Invitrogen</td>
			<td>A32732</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Heater mat</td>
			<td>Bio Research Center</td>
			<td>HM-10</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Heater mat controller</td>
			<td>Bio Research Center</td>
			<td>BWT-100A</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Heater system</td>
			<td>SA Instruments</td>
			<td>MR-compatible Small Animal Heating System</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>AbbVie</td>
			<td></td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>ACOMA Medical Industry</td>
			<td>MKIIIai</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Linear Slicer</td>
			<td>DOSAKA</td>
			<td>Neo Linear Slicer MT</td>
			<td></td>
		</tr>
		<tr>
			<td>L(+)-Ascorbic Acid Sodium Salt</td>
			<td>Wako</td>
			<td>196-01252</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Magnesium Chrolide Hexahydrate</td>
			<td>Wako</td>
			<td>135-00165</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>MaxOne Single-Well MEA</td>
			<td>MaxWell Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal Spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monitoring system</td>
			<td>SA Instruments</td>
			<td>Model 1025</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Monitoring software</td>
			<td>SA Instruments</td>
			<td>PC-SAM V.5.12</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>MRI compatible cradle</td>
			<td>Bruker BioSpin</td>
			<td>T12812</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>MRI coil</td>
			<td>Bruker BioSpin</td>
			<td>T9988</td>
			<td>For MRI</td>
		</tr>
		<tr>
			<td>MRI operation software</td>
			<td>Bruker BioSpin</td>
			<td>ParaVision 5.1</td>
			<td>For MRI</td>
		</tr>
		<tr>
			<td>Neo LinearSlicer MT</td>
			<td>D.S.K.</td>
			<td>NLS-MT</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuN (D4G40) XP Rabbit mAb</td>
			<td>Cell Signaling</td>
			<td>24307</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Wako</td>
			<td>143-06561</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Wako</td>
			<td>163-03545</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol Mono-p-isooctylphenyl Ether</td>
			<td>nacalai tesque</td>
			<td>12967-45</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Pressure-sensitive respiration sensor</td>
			<td>SA Instruments</td>
			<td>RS-301</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Preclinical MRI scanner</td>
			<td>Bruker BioSpin</td>
			<td>BioSpec 70/20 USR</td>
			<td>For MRI</td>
		</tr>
		<tr>
			<td>Pyruvic Acid Sodium Salt</td>
			<td>nacalai tesque</td>
			<td>29806-54</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>SCAN in a BOX</td>
			<td>Open Technologies srl</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sieve bottle</td>
			<td>TIGERCROWN</td>
			<td>81</td>
			<td>For 3D scan</td>
		</tr>
		<tr>
			<td>SlowFade Gold Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>S36937</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Sodium Dihydrogenphosphate</td>
			<td>Wako</td>
			<td>197-09705</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Sodium Hydrogen Carbonate</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td>aCSF</td>
		</tr>
		<tr>
			<td>Sodium Hydrogensulfite</td>
			<td>nacalai tesque</td>
			<td>31220-15</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Thermistor temperature probe</td>
			<td>SA Instruments</td>
			<td>RTP-101-B, PLTPC-300</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Tooth bar</td>
			<td>Bruker BioSpin</td>
			<td>T10146</td>
			<td>Animal preparation for MRI</td>
		</tr>
		<tr>
			<td>Winged intravenous needle</td>
			<td>TERUMO</td>
			<td>SV-23CLK</td>
			<td>For perfusion</td>
		</tr>
		<tr>
			<td>1 mol/l-Tris-HCl Buffer Solution</td>
			<td>nacalai tesque</td>
			<td>35436-01</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>1 mol/l-Hydrochloric Acid</td>
			<td>nacalai tesque</td>
			<td>37314-15</td>
			<td>For pH adjustment of solution</td>
		</tr>
		<tr>
			<td>16%-Paraformaldehyde Aqueous Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>For immunostaining</td>
		</tr>
	</tbody>
</table>

3d scanning technology bridging microcircuits and macroscale brain images in 3d novel embedding overlapping protocol

The human brain, being a multiscale system, has both macroscopic electrical signals, globally flowing along thick white-matter fiber bundles, and microscopic neuronal spikes, propagating along axons and dendrites. Both scales complement different aspects of human cognitive and behavioral functions. At the macroscopic level, MRI has been the current standard imaging technology, in which the smallest spatial resolution, voxel size, is 0.1&#8211;1 mm3. Also, at the microscopic level, previous physiological studies were aware of nonuniform neuronal architectures within such voxels. This study develops a powerful way to accurately embed microscopic data into a macroscopic map by interfacing biological scientific research with technological advancements in 3D scanning technology. Since 3D scanning technology has mostly been used for engineering and industrial design until now, it is repurposed for the first time to embed microconnectomes into the whole brain while preserving natural spiking in living brain cells. In order to achieve this purpose, first, we constructed a scanning protocol to obtain accurate 3D images from living bio-organisms inherently challenging to image due to moist and reflective surfaces. Second, we trained to keep speed to prevent the degradation of living brain tissue, which is a key factor in retaining better conditions and recording more natural neuronal spikes from active neurons in the brain tissue. Two cortical surface images, independently extracted from two different imaging modules, namely MRI and 3D scanner surface images, surprisingly show a distance error of only 50 &#956;m as mode value of the histogram. This accuracy is comparable in scale to the microscopic resolution of intercellular distances; also, it is stable among different individual mice. This new protocol, the 3D novel embedding overlapping (3D-NEO) protocol, bridges macroscopic and microscopic levels derived by this integrative protocol and accelerates new scientific findings to study comprehensive connectivity architectures (i.e., microconnectome).

We evaluated distances between cortical surfaces, produced by stripping MRI volume, and surfaces obtained from 3D scans of extracted brains. The mode values of the histogram of the distances are only 55 &#956;m (Figure 3a). Additionally, when accumulating the histogram from the point where the distance equals zero, the accumulated value reaches 90% of total sample numbers at ~300 &#956;m (Figure 3b). The final histogram of the di...

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3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol

This article introduces an experimental protocol using 3D scanning technology bridging two spatial scales: the macroscopic spatial scale of whole-brain anatomy imaged by MRI at &#62;100 &#956;m and the microscopic spatial scale of neuronal distributions using immunohistochemistry staining and a multielectrode array system and other methods (~10 &#956;m).

The human brain, being a multiscale system, has both macroscopic electrical signals, globally flowing along thick white-matter fiber bundles, and ...

This protocol is the first to introduce 3D scanning technology in the anatomical, specifically neuroanatomical research field, and has achieved highly accurate overlapping performance. The 3D neuro embedding overlap, or 3D neuro protocol, embeds micro-scale sterile sockets into macro-scale brain images, and bridges these different spatial scales seamlessly. We also have to apply the 3D neuro protocol to human MRIs and post-mortem brain.The overlapping enables us to identify known MRI contrast patterns relating to disease. 3D scan systems are sensitive to water surrounding the extracted bio organism, so it is critical to wipe the water very carefully. Furthermore, the procedure must be performed as quickly as possible.Begin this procedure with preparation of a mouse, as well as MRI acquisitions and equipment preparations as described in the text protocol. On day two, cut the scalp of a previously euthanized mouse along the sagittal suture using surgical scissors. Use surgical scissors to cut the skull in diagonal directions from the lambda point to a point a little in front of the bregma.Then, gently peel off the skull from the brain using curved tweezers. Lift the brain with a spatula and transfer it to a Petri dish with ice-cold cutting solution. Using an external gas bomb with a controlled rotating pump speed, flow air containing 95%oxygen and 5%carbon dioxide to generate small bubbles in the ice-cold cutting solution.After one to two minutes, put the brain on a microfiber cloth whose surface is slightly covered by a flour sieve. Gently wipe the fluid from the brain surface using the microfiber cloth. Put the brain, with its dorsal aspect up, onto the sample stand.Then, place the sample stand at the center of the automatic turntable. Darken the room during the 3D scan and perform a 3D scan on the turntable. To test whether the 3D scan works well, click 3D Scan by selecting the angle between two shots as 22.5, the starting angle as zero, and the final angle as 360.Move the brain to a Petri dish and bubble the brain in the cutting solution for approximately 10 seconds. Cut the brain into two blocks at the middle of the coronal plane, using a scalpel. Then, move the two brain blocks gently to the flat surface using a surgical spatula.Attach the brain blocks of the brain block base using instant glue. Softly and carefully wipe the fluid from the brain surface within one to two minutes, using the microfiber cloth. Then, perform a 3D scan again.Attach a blade to the blade holder of the vibratome. Attach the black tape covering the center of the brain block base to the center of the cutting stage for a vibratome. Pour cutting solution into the cutting stage and set the cutting stage on the vibratome.Adjust the cutting speed and amplitude. Make two to five coronal slices of 300 microns thick from the two brain blocks. Keep the solution in the cutting stage bubbling if possible.Optimize the cutting speed, frequency, and vibration amplitude of the system by setting them as 12.7 millimeters per minute for the speed, 87 to 88 hertz for the frequency, and 0.8 to 1.0 millimeters for the swing width. While cutting the brain slices, record the sliced coordinate in the format, including the anterior-posterior coordinate, hemisphere, and other conditions. Gently transfer the brain slices to a beaker filled with pre-warmed ACSF using a thick plastic pipette.Incubate the brain slices in the beaker at approximately 34 degrees Celsius for one hour. During this time, perform a 3D scan of the remaining brain blocks on the cutting stage. Now, set a multielectrode array, or MEA chip, on a recording unit.Connect the chip to a peristaltic pump using two tubes. Use one tube to guide the same ACSF into the MEA chip and the other tube to guide the ACSF out of the MEA chip. Attach two needles, connected at the tips of the two tubes, to the top of the wall of the MEA chip.Fix their positions with their tips following the inner wall of the MEA chip. Set the flow rate of the ACSF to 4.1 RPM. Perform MRI data processing to extract cortical volumes as described in the text protocol.To perform MRI image processing, download the 3D Slicer free software. Open the MR images of the extracted brains that were produced using the volume rendering and editor modules in the 3D Slicer. Change the mode from editor to volume rendering, and click a target button to make the image of the brain come to the center of the screen.Select the MRT-2 brain mode and tune the thresholds by moving the shift bar. Then, move back from volume rendering to editor and click the threshold effect button. Apply the label 41, cerebral cortex.Then, save the brain surface data as an STL file by changing the file format from VTK to STL on the form checklist. Perform an automatic coregistration among eight or 16 images taken from eight or 16 different angles within a sequence of a scan to correct small mismatches. To do so, click Global Registration included in the alignment option and integrate the images.Repeat scans from different angles to obtain the whole brain surface. If the integration among images scanned from different angles was not successful, click Manual Alignment and select a pair of fixed image and a moving image. Start the manual alignment of the images by selecting three or four common points in different images.Then, click OK.The optimization algorithm is the iterative closest-point algorithm and does not include a non-linear deformation. Make a mesh of all the aligned images by selecting all images and clicking the mesh generation button. Then, select the option, small artistic object, to get the mesh at the highest resolution.Save the image as STL Binary or an ASCII format. Now, open the MRI surface and merge it with the 3D scan surface using the surface processing software. Perform a manual alignment process as before.Then, save these coregistered surface images again. If necessary, clean the individual surface data by erasing small noises surrounding the brain region, especially in the case of the MRI data, and by filling any holes, especially in the case of the 3D scan data. Finally, open the surface data with data analysis software such as MATLAB.Generate and evaluate the histograms of the minimum distances between the two surfaces. Distances were evaluated between cortical surfaces produced by stripping MRI volume and surfaces obtained from 3D scans of extracted brains. The mode values of the histogram of the distances are only 55 microns.Additionally, when accumulating the histogram from the point where the distance equals zero, the accumulated value reaches 90%of total sample numbers at approximately 300 microns. The final histogram of the distances between two surfaces showed a typical peak around 50 microns. From a macroscopic viewpoint, this mode value corresponds to the geometrical limitation, which was 100 microns from the voxel size of MRIs.This point indirectly suggests that the overlapping algorithm between the MRI and 3D scan worked superbly well, and that the noise levels of both the MRI and the 3D scan were suppressed as a low value. This protocol is the first to uprise through the scanning technology to bio-organism. The technology was originally utilized for purely engineering demands.Applying this technology to medicine can answer new questions. Following 3D scanning, we can use calcium-imaging wall patch gram recordings to get complementary knowledge in terms of temporal and the spacial resolution and recordable numbers of cells. The highly accurate overlapping performance this protocol provides will make a seamless association between the anatomical spatial scale and the zero-spatial scale more realistic than before.

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Research

JoVE Journal

Neuroscience

7.1K visualizações.  Kyoto University. Este protocolo é o primeiro a introduzir a tecnologia de digitalização 3D no campo de pesquisa anatômica, especificamente neuroanatomical, e tem alcançado um desempenho altamente preciso sobreposto. A neuro 3D incorporando sobreposição, ou protocolo neuro 3D, incorpora tomadas estéreis em microescamas em imagens cerebrais em macroesponíveis, e conecta essas diferentes escalas espaciais perfeitamente. Também temos que aplicar o protocolo neuro 3D às ressonâncias magnéticas humanas...