We thank Atsushi Miyawaki and Hiroshi Hama for their advice on the AbScale experiments, Charles Yokoyama for editing of the manuscript, Eriko Ohshima and Miyuki Kishino for their technical assistance. This work was supported by research funds from RIKEN to T.H. and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to T.H. (Innovative Areas &#34;Mesoscopic Neurocircuitry&#34;; 22115004) and S.S. (25890023).

All experimental procedures were approved by the RIKEN Wako Animal Experiments Committee and RIKEN Genetic Recombinant Experiment Safety Committee and performed according to the institutional guidelines of the animal facilities of the RIKEN Brain Science Institute.
1. Preparation of Imaging Chambers
<ol>
	<li>Imaging chamber19
	<ol>
		<li>Using silicone rubber sheets, prepare a chamber with a thickness of approximately 5 mm and floor plates of various thicknesses. Also, prepare Petri dishes with and without a glass bottom (Figure 1A).</li>
	</ol>
	</li>
	<li>Slice spacer
	<ol>
		<li>Prepare spacers to hold samples, using silicone rubber sheets with a thickness of 0.5 mm (Figure 1C).</li>
	</ol>
	</li>
</ol>
2. Tracer Injection
NOTE: Make injections into either the pons (2.1) or superior colliculus (2.2). Injection into the pons label sub-cerebral projection neurons in a wide brain region including the visual and motor areas, while injection into the superior colliculus labels sub-cerebral projection neurons in the visual area. For control experiments, inject saline instead of fluorescently-labeled cholera toxin subunit B. For the maintenance of sterile condition use sterilized equipment and plastic gloves cleaned with ethanol.
<ol>
	<li>Make injections into the pons of adult mice.
	<ol>
		<li>Draw 1 &#181;L of fluorescently-labeled cholera toxin subunit B (25 &#181;g/&#181;L in PBS) into a 26G Hamilton syringe.</li>
		<li>Place an injector pump on the syringe.</li>
		<li>Place the syringe and pump on the tool holder of a manipulator placed on a stereotaxic instrument. Tilt the manipulator 12&#176; posteriorly from the vertical axis.</li>
		<li>Anesthetize a male or female adult mouse (C57BL/6J or Tlx3-cre/Ai9) by injecting sodium pentobarbital intraperitoneally (60 mg/kg body weight) or by administering isoflurane (2&#8211;3%). Wait until the mouse makes no response when its tail is pinched with forceps, indicating that the mouse is fully anesthetized.</li>
		<li>Place the mouse on the stereotaxic instrument.</li>
		<li>Carefully remove hair using a razor blade to prevent infection and cut 10 mm of the scalp so that the bregma and lambda are visible. Administer 0.1 mL of 1% lidocaine using a pipette. Set the angle of the head by adjusting the vertical position of the mouthpiece on the stereotaxic instrument so that the bregma and lambda have the same z-level.</li>
		<li>Adjust the position of the manipulator by sliding it on the stereotaxic instrument so that the tip of the syringe is close to the bregma and record the position of the manipulator. Retract the syringe by moving the tool holder on the manipulator.</li>
		<li>Move the manipulator 5.4 mm posteriorly and 0.4 mm laterally. Advance the syringe so that the tip is close to the entry point on the skull. Retract the syringe and mark the entry point.</li>
		<li>At the marked position, drill a hole with a diameter of approximately 1 mm.</li>
		<li>Insert the syringe tip through the hole so that the tip depth is 6.9 mm more than that measured at the bregma.</li>
		<li>Inject 1 &#181;L of tracers using the pump at 0.2 &#181;L/min.</li>
		<li>Remove the syringe from the brain.</li>
		<li>If necessary, cover the exposed brain with small fragments of microfibrillar hemostat and instant adhesive.</li>
		<li>Rinse the exposed brain using saline delivered with a pipette to prevent infection and suture the scalp.</li>
		<li>Remove the mouse from the stereotaxic instrument. Allow the mouse to recover from anesthesia in an incubator at 30 &#730;C, typically for 1 h. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. Return the mouse to the company of other animals after it has fully recovered.</li>
		<li>Maintain the mouse for 3&#8211;7 days.</li>
	</ol>
	</li>
	<li>Make injections into the superior colliculus of adult mice.
	<ol>
		<li>Prepare a glass pipette with a tip diameter of 30&#8211;50 &#181;m.</li>
		<li>Connect the glass pipette to a Hamilton syringe through a plastic tube (Figure 2A).</li>
		<li>Fill the glass pipette, plastic tube, and Hamilton syringe with the paraffin liquid.</li>
		<li>Place an injector pump on the syringe.</li>
		<li>Place the glass pipette on the manipulator and tilt the manipulator 60&#176; posteriorly from the vertical axis (Figure 2B).</li>
		<li>Perform 2.1.4&#8211;2.1.9. The position of the manipulator is 1.4 mm posterior, 0.5 mm lateral to the lambda.</li>
		<li>Place a small plastic paraffin film on the skull, then put approximately 1 &#181;L of the tracer solution on it. Quickly advance the glass pipette and fill it with at least 0.5 &#181;L of the tracer solution.</li>
		<li>Insert the glass pipette so that the tip depth is 3.0 mm from the brain surface.</li>
		<li>Inject 0.5 &#181;L of tracers using the pump at 0.2 &#181;L/min.</li>
		<li>Remove the glass pipette from the brain.</li>
		<li>Perform 2.1.13&#8211;2.1.16.</li>
	</ol>
	</li>
</ol>
3. Fixation and Trimming
<ol>
	<li>Inject sodium pentobarbital (60 mg/kg body weight) intraperitoneally into a mouse (C57BL/6J or Tlx3-cre/Ai9, with or without the tracer injection as described in step 2. Wait until the mouse makes no response when its tail is pinched with forceps, indicating that the mouse is fully anesthetized.</li>
	<li>Euthanize the mouse humanely by perfusing&#160;the mouse transcardially20 with 0.9% saline.</li>
	<li>Fix the mouse20 by perfusing 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.5).</li>
	<li>Cut the scalp using a pair of scissors to expose the skull as described20.
	<ol>
		<li>Cut the midline of the exposed skull using a pair of scissors. Remove the skull using forceps.</li>
		<li>If marking the position of the bregma and lambda is necessary, first remove one hemisphere of the skull. Insert thin tungsten needles into the brain at the positions of the bregma and lambda on the skull remaining on the brain, then remove the remaining skull. 
		NOTE: The brain can be stored in PBS at 4 &#730;C.</li>
	</ol>
	</li>
	<li>To perform antibody staining of inhibitory neurons, cut the brain samples into slices.
	<ol>
		<li>Place the brain sample on a vibratome.</li>
		<li>Cut slices up to 500 &#181;m thick in PBS at room temperature and proceed to step 5 (the AbScale method).</li>
	</ol>
	</li>
	<li>If antibody staining is unnecessary, trim the brain sample to blocks (up to 3 mm thick) using a razor blade (Figure 3) and proceed to step 4 (the SeeDB method). 
	NOTE: The brain can be stored in PBS at 4 &#176;C after cutting or trimming. The SeeDB method is preferable when antibody staining is not necessary because it requires less time than the AbScale method.</li>
</ol>
4. Clearing without Antibody Staining (the SeeDB Method)
<ol>
	<li>Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% &#945;-thioglycerol and 20% (w/v) fructose and incubate it for 4 h with gentle shaking at room temperature.</li>
	<li>Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% &#945;-thioglycerol and 40% (w/v) fructose and incubate it for 4 h with gentle shaking at room temperature.</li>
	<li>Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% &#945;-thioglycerol and 60% (w/v) fructose and incubate it for 4 h with gentle shaking at room temperature.</li>
	<li>Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% &#945;-thioglycerol and 80% (w/v) fructose and incubate it for 12 h with gentle shaking at room temperature.</li>
	<li>Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% &#945;-thioglycerol and 100% (w/v) fructose and incubate it for 12 h with gentle shaking at room temperature.</li>
	<li>Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% &#945;-thioglycerol and 80.2% (w/w) fructose and incubate it for 24 h with gentle shaking at room temperature. 
	NOTE: Handle the sample carefully to keep deformations as small as possible. Do not incubate samples longer than indicated, as samples can quickly become opaque.</li>
	<li>Embed the sample in an imaging chamber filled with the 80.2% fructose solution (Figure 1B). If necessary, fix the sample by putting small pieces of rubber adhesive around. If the chamber is too deep, put a floor plate in the chamber before placing the samples.</li>
	<li>Place the Petri dish with a glass cover on the imaging chamber and put water in the dish, and image using confocal or two-photon microscopy with a water-immersion long working distance objective. Excitation wavelengths and emission filters are described in Table 1. If necessary, use a motorized stage.</li>
</ol>
5. Clearing with Antibody Staining (the AbScale Method)
<ol>
	<li>Prepare reagents as described in Table 2.</li>
	<li>Transfer the slices using a spatula to a 5 mL plastic tube containing 4 mL of Sca/eS0 solution and incubate them for 12 h with gentle shaking at 37 &#176;C (Figure 4B).</li>
	<li>Remove the solution from the tube using a pipette and add 4 mL of Sca/eA2 solution, and incubate the slices for 36 h with gentle shaking at 37 &#176;C.</li>
	<li>Remove the solution from the tube using a pipette and add 4 mL of Sca/eB4 solution, and incubate the slices for 24 h with gentle shaking at 37 &#176;C.</li>
	<li>Remove the solution from the tube using a pipette and add 4 mL of Sca/eA2 solution, and incubate the slices for 12 h with gentle shaking at 37 &#176;C.</li>
	<li>Remove the solution from the tube using a pipette and add 4 mL of PBS and incubate the slices for 6 h with gentle shaking at room temperature.</li>
	<li>Carefully remove the slices to a 2 mL plastic tube using a spatula.</li>
	<li>Incubate with primary antibodies (Table 3) in 1 mL of AbScale solution for 48-72 h at 37 &#176;C (Figure 4C) with gentle shaking.</li>
	<li>Carefully remove the slices to a 5 mL plastic tube using a spatula.</li>
	<li>Incubate in 4 mL of AbScale solution for 2 h 2 times at room temperature with gentle shaking.</li>
	<li>Carefully remove the slices to a 2 mL plastic tube using a spatula.</li>
	<li>Incubate with fluorescently-labeled secondary antibodies (Table of Materials, 1:100) in 1 mL of AbScale solution for 48 h at 37 &#176;C with gentle shaking.</li>
	<li>Carefully remove the slices using a spatula to a 5 mL plastic tube containing 4 mL of the AbScale solution and incubate the slices for 6 h with gentle shaking at room temperature.</li>
	<li>Remove the solution from the tube using a pipette and add 4 mL of the AbScale solution and incubate the slices for 2 h 2 times with gentle shaking at room temperature.</li>
	<li>Remove the solution from the tube using a pipette and add 4 mL of 4% PFA and incubate the slices for 1 h with gentle shaking at room temperature.</li>
	<li>Remove the solution from the tube using a pipette and add 4 mL of PBS and incubate the slices for 1 h with gentle shaking at room temperature.</li>
	<li>Remove the solution from the tube using a pipette and add 4 mL of the Sca/eS4 solution, and incubate the slices for 12 h with gentle shaking at 37 &#176;C.</li>
	<li>Place the spacer on a glass slide and place the slices in the spacer and immerse the slices with Sca/eS4 solution. Seal the spacer with a cover glass (Figure 1D).</li>
	<li>Put water on the cover glass and image using confocal or two-photon microscopy with a water-immersion long working distance objective. If necessary, use a motorized stage. 
	NOTE: Check whether the deep parts of the sample are labeled similarly to the superficial parts to confirm the absence of a significant labeling bias. 
	NOTE: Penetration of the tested antibodies is described in Table 3.</li>
</ol>
6. Cell Position Determination
<ol>
	<li>For each position in the scanned images, calculate correlation values using a three-dimensional image filter14 (Figure 5A-5D).</li>
	<li>Determine the positions of the peaks of the correlation value (Figure 5D).</li>
	<li>Investigate images around the peaks to locate cells (Figure 5E).</li>
</ol>

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</ol>

The authors have nothing to disclose.

We have presented procedures to obtain large-scale three-dimensional images of the cell type-specific organization of the major cell types in mouse neocortical layer 5. Compared to the conventional slice staining, the method is more useful in determining the three-dimensional organization of the neocortex. The method enables image acquisition from the wider and the deeper brain regions compared to the typical in vivo 2-photon microscopy or conventional confocal microscopy and, thus, can allow the comprehensive a...

The mammalian neocortex is composed of a large number of cell types, each with the specific gene expression patterns, electrophysiological and biochemical properties, synaptic connections, and in vivo functions1,2,3,4,5,6,7. Whether these cell types are organized into repeated structures has been unclear. Cortical columns, including visual orientation columns and somatosensory barrels, have repeated structures, but their cellular organization remains unclear8,9. These are present in the specific cortical areas and are not a brain-wide system.
In neocortical layer 5, the large majority of neurons are classified into four major types. A major type of excitatory neurons, sub-cerebral projection neurons, projects axons to subcortical targets including the pons, spinal cord, and superior colliculus, and, therefore, represents the major cortical output pathway10. Cortical projection neurons, another major type of excitatory neurons, innervate the cortex10. Inhibitory neurons also contain two major classes: parvalbumin-expressing and somatostatin-expressing cells11.
Recent analyses indicate that the four cell types are organized into repeated structures12,13,14. Both sub-cerebral projection neurons12,13,14 and cortical projection neurons14 organize into cell-type specific microcolumns with a diameter of 1&#8211;2 cells. Parvalbumin-expressing and somatostatin-expressing cells align specifically with microcolumns of sub-cerebral projection neurons but not with microcolumns of cortical projection neurons14. Microcolumns themselves periodically align to form a hexagonal lattice array14 and are present in multiple cortical areas including visual, somatosensory, and motor areas in mouse brain12,14 and in language areas of human brain13. Neurons in the individual microcolumn exhibit synchronized activity and have similar sensory responses14. These observations indicate that layer 5 cell types organize into a microcolumn lattice structure representing the first known brain-wide organization of repeating functional modules.
Microcolumns have a radius of approximately 10 &#181;m and have a spatial periodicity of approximately 40 &#181;m. In addition, the orientation of microcolumns is parallel to their apical dendrites and changes depending on their position in the cortex14. Therefore, the microcolumn system is difficult to analyze using conventional cortical slices with a typical thickness of a few tens of micrometers. In addition, the analysis of periodicity requires three-dimensional data from a wide-range of brain areas, and, therefore, the typical imaging area of confocal microscopy or in vivo 2-photon imaging is too narrow.
Recently, techniques have been developed to clear thick tissues15,16. Here we describe the application of these methods to obtain large-scale, three-dimensional images of the major cell types in mouse neocortical layer 5 that comprise the microcolumn system. Subcerebral projection neurons are labeled by the retrograde labeling or by the expression of the enhanced green fluorescent protein in Crym-egfp transgenic mice12, and cortical projection neurons are labeled by either the retrograde labeling or by the tdTomato expression in Tlx3-cre/Ai9 mice17. Parvalbumin-expressing and somatostatin-expressing cells are labeled by immunohistochemistry. The (Antibody Scale S) AbScale method18 is used for the antibody staining experiments, while the (See Deep Brain) SeeDB method19 is used for other experiments. These methods overcome the above-mentioned difficulties of the conventional imaging methods and reveal the accurate cellular organization of layer 514.

<table><tbody><tr><td>Crym-egfp transgenic mice</td><td>MMRRC</td><td>012003-UCD</td><td></td></tr><tr><td>Tlx3-cre transgenic mice</td><td>MMRRC</td><td>36547-UCD</td><td></td></tr><tr><td>ROSA-CAG-flox-tdTomato mice</td><td>Jackson Laboratory</td><td>JAX #7909</td><td></td></tr><tr><td>Silicone rubber sheet</td><td>AS ONE</td><td>6-611-01</td><td>0.5 mm thickness</td></tr><tr><td>Silicone rubber sheet</td><td>AS ONE</td><td>6-611-02</td><td>1.0 mm thickness</td></tr><tr><td>Silicone rubber sheet</td><td>AS ONE</td><td>6-611-05</td><td>3.0 mm thickness</td></tr><tr><td>Petri dishes</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>Cover glass</td><td>Matsunami</td><td>C022241</td><td></td></tr><tr><td>Cholera toxin subunit B (recombinant), Alexa Fluor 488 conjugate</td><td>Invitrogen</td><td>C22841</td><td></td></tr><tr><td>Cholera toxin subunit B (recombinant), Alexa Fluor 555 conjugate</td><td>Invitrogen</td><td>C22843</td><td></td></tr><tr><td>Cholera toxin subunit B (recombinant), Alexa Fluor 594 conjugate</td><td>Invitrogen</td><td>C22842</td><td></td></tr><tr><td>Cholera toxin subunit B (recombinant), Alexa Fluor 647 conjugate</td><td>Invitrogen</td><td>C34778</td><td></td></tr><tr><td>26 G Hamilton syringe</td><td>Hamilton</td><td>701N</td><td></td></tr><tr><td>Injector pump</td><td>KD Scientific</td><td>KDS 310</td><td>Pons injection</td></tr><tr><td>Injector pump</td><td>KD Scientific</td><td>KDS 100</td><td>Superior colliculus injection</td></tr><tr><td>Manipulator</td><td>Narishige</td><td>SM-15</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Kyoritsu Seiyaku</td><td>Somnopentyl</td><td></td></tr><tr><td>Isoflurane</td><td>Pfizer</td><td></td><td></td></tr><tr><td>Lidocaine</td><td>AstraZeneca</td><td>Xylocaine injection 1% with epinephrine</td><td></td></tr><tr><td>Drill</td><td>Toyo Associates</td><td>HP-200</td><td></td></tr><tr><td>Avitene microfibrillar hemostat</td><td>Davol Inc</td><td>1010090</td><td></td></tr><tr><td>Alonalfa</td><td>Daiichi-Sankyo</td><td>Alonalpha A</td><td></td></tr><tr><td>Surgical silk</td><td>Ethicon</td><td>K881H</td><td></td></tr><tr><td>Incubator</td><td>UVP</td><td>HB-1000 Hybridizer</td><td></td></tr><tr><td>Glass pipette</td><td>Drummond Scientific Company</td><td>2-000-075</td><td></td></tr><tr><td>Electrode puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Paraffin Liquid, light</td><td>Nacalai tesque</td><td>26132-35</td><td></td></tr><tr><td>Saline</td><td>Otsuka</td><td>1326</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Nacalai tesque</td><td>26126-54</td><td></td></tr><tr><td>Tungsten needle</td><td>Inter medical</td><td>&amp;Phi;0.1 *L200 mm</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td>50 mL plastic tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>&amp;alpha;-thioglycerol</td><td>Nacalai tesque</td><td>33709-62</td><td></td></tr><tr><td>D(-) Fructose</td><td>Nacalai tesque</td><td>16315-55</td><td></td></tr><tr><td>BluTack</td><td>Bostik</td><td>CKBT-450000</td><td></td></tr><tr><td>Two-photon microscope</td><td>Nikon</td><td>A1RMP</td><td></td></tr><tr><td>Water-immersion long working distance objectives</td><td>Nikon</td><td>CFI Apo LWD 25XW, NA 1.1, WD 2 mm</td><td></td></tr><tr><td>Water-immersion long working distance objectives</td><td>Nikon</td><td>CFI LWD 16XW, NA 0.8, WD 3 mm</td><td></td></tr><tr><td>Motorized stage</td><td>COMS</td><td>PT100C-50XY</td><td></td></tr><tr><td>Filter</td><td>Semrock</td><td>FF01-492/SP-25</td><td></td></tr><tr><td>Filter</td><td>Semrock</td><td>FF03-525/50-25</td><td></td></tr><tr><td>Filter</td><td>Semrock</td><td>FF03-575/25-25</td><td></td></tr><tr><td>Filter</td><td>Semrock</td><td>FF01-629/56</td><td></td></tr><tr><td>Filter</td><td>Chroma</td><td>D605/55m</td><td></td></tr><tr><td>5 mL plastic tube</td><td>AS ONE</td><td>VIO-5B</td><td></td></tr><tr><td>2 mL plastic tube</td><td>Eppendorf&amp;nbsp;</td><td>0030120094</td><td></td></tr><tr><td>Urea</td><td>Nacalai tesque</td><td>35905-35</td><td></td></tr><tr><td>Triton X-100</td><td>Nacalai tesque</td><td>35501-15</td><td></td></tr><tr><td>Glyserol</td><td>Sigma-aldrich</td><td>191612</td><td></td></tr><tr><td>D(-)-sorbitol</td><td>Wako</td><td>191-14735</td><td></td></tr><tr><td>Methyl-&amp;beta;-cyclodextrin</td><td>Tokyo chemical industry</td><td>M1356</td><td></td></tr><tr><td>&amp;gamma;-Cyclodextrin</td><td>Wako</td><td>037-10643</td><td></td></tr><tr><td>N-acetyl-L-hydroxyproline</td><td>Skin Essential Actives</td><td>33996-33-7</td><td></td></tr><tr><td>DMSO</td><td>Nacalai tesque</td><td>13445-45</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-aldrich</td><td>A7906</td><td></td></tr><tr><td>Tween-20 (1.1 g/mL)</td><td>Nacalai tesque</td><td>35624-15</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td><td>Invitrogen</td><td>A21422</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td><td>Invitrogen</td><td>A21428</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647</td><td>Invitrogen</td><td>A21235</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A11029</td><td></td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A21206</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Water-immersion long working distance objectives</td><td>Olympus</td><td>XLUMPLFLN 20XW, NA 1.0, WD 2 mm</td><td></td></tr><tr><td>Anti-NeuN</td><td>Millipore</td><td>MAB377</td><td></td></tr><tr><td>Anti-NeuN</td><td>Millipore</td><td>ABN78</td><td></td></tr><tr><td>Anti-CTIP2</td><td>Abcam</td><td>ab18465</td><td></td></tr><tr><td>Anti-Statb2</td><td>Abcam</td><td>ab51502</td><td></td></tr><tr><td>Anti-GAD67</td><td>Millipore</td><td>MAB5406</td><td></td></tr><tr><td>Anti-GABA</td><td>Sigma</td><td>A2052</td><td></td></tr><tr><td>Anti-Parvalbumin</td><td>Swant</td><td>235</td><td></td></tr><tr><td>Anti-Parvalbumin</td><td>Frontier Institute</td><td>PV-Go-Af460</td><td></td></tr><tr><td>Anti-Parvalbumin</td><td>Sigma</td><td>P3088</td><td></td></tr><tr><td>Anti-Parvalbumin</td><td>Abcam</td><td>ab11427</td><td></td></tr><tr><td>Anti-Somatostatin</td><td>Peninsula Laboratories</td><td>T-4103</td><td></td></tr><tr><td>Anti-c-Fos</td><td>CalbioChem</td><td>PC38</td><td></td></tr></tbody></table>

large-scale three-dimensional imaging of cellular organization in the mouse neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

We labeled cortical projection neurons by expression of tdTomato in Tlx3-cre/Ai9 transgenic mice and visualized sub-cerebral projection neurons by injecting the retrograde tracer CTB488 into the pons. The left hemisphere of the brain was subjected to the SeeDB method and scanned using a two-photon microscope equipped with a water-immersion long working distance objective (25X, N.A. 1.1, working distance 2 mm) and a motorized stage. A stack of 401 images (512 x 512 pixels...

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Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

large-scale-three-dimensional-imaging-cellular-organization-mouse

Research

JoVE Journal

Neuroscience

8.3K Views.  RIKEN Brain Science Institute. Hier beschrijven we een procedure voor het weefsel wissen, fluorescerende benoemen, en grootschalige beeldvorming van muis hersenweefsel, waardoor, zodat, visualisatie van de driedimensionale organisatie van celtypes in de neocortex.

Video: Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Behavior

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light3 do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.4 Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Biology

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard method for soft tissues, although its low spatial resolution poses some limits for small animals. Here, we describe a protocol for obtaining high-resolution three-dimensional (3D) information on mouse neonate brains and skulls using micro-computed tomography (micro-CT). The protocol includes those steps needed to dissect the samples, stain and scan the brain, and obtain morphometric measurements of the whole organ and regions of interest (ROIs). Image analysis includes the segmentation of structures and the digitization of point coordinates. In sum, this work shows that the combination of micro-CT and Lugol's solution as a contrast agent is a suitable alternative for imaging the perinatal brains of small animals. This imaging workflow has applications in developmental biology, biomedicine, and other sciences interested in assessing the effect of diverse genetic and environmental factors on brain development.

Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains &#8211; A Micro-CT Protocol with Lugol&#39;s Solution Contrast Agent

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

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