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...

<ol>
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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>26G 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>&#934;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>&#945;-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 colspan="2">CFI Apo LWD 25XW, NA 1.1, WD 2 mm</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&#160;</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-&#946;-cyclodextrin</td>
			<td>Tokyo chemical industry</td>
			<td>M1356</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-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 ...

This method can help answer key questions in the neuroscience field such as the analysis of the structure and the function of the neocortex. The main advantage of this technique is that it can reveal the large-scale three-dimensional structure in the brain. Though this method can provide insight into the neocortex, it can also be applied to other nervous systems such as the spinal cord.To inject the tracer into the pons of the adult mice, first draw one microliter of fluorescently-labeled Cholera toxin subunit B into a 26-gauge Hamilton syringe. Place an injector pump on the syringe, then place the syringe and pump on the tool holder of a manipulator on a stereotaxic instrument. Tilt the manipulator 12 degrees posteriorly from the vertical axis.Afterward, place the mouse on the stereotaxic instrument. Using a razor blade, remove its hair to prevent infection. Then cut 10 millimeters of the scalp so that the bregma and lambda are visible.Administer 0.1 milliliters 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. Next, adjust the position of the manipulator by sliding it onto the stereotaxic instrument so that the tip of the syringe is close to the bregma.Record the position of the manipulator. Retract the syringe by moving the tool holder on the manipulator. Subsequently move the manipulator 5.4 millimeters posteriorly and 0.4 millimeters laterally.Advance the syringe so that the tip is close to the entry point on the skull, then retract the syringe and mark the entry point. At the marked position, drill a hole with a diameter of approximately one millimeter. Insert the syringe tip through the hole so that the tip depth is 6.9 millimeters more than that measured at the bregma.Then, inject one microliter of tracers using the pump at 0.2 microliters per minute. After the injection, remove the syringe from the brain. If necessary, cover the exposed brain with small fragments of microfibrillar hemostat and instant adhesive.Wipe the exposed brain with saline to prevent infection and suture the scalp. Next remove the mouse from the stereotaxic instrument. Allow the mouse to recover from anesthesia in an incubator at 30 degrees Celsius for about an hour.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 is fully recovered. To collect the brain tissue, cut the scalp using a pair of scissors to expose the skull.Next, cut the midline of the exposed skull using a pair of scissors. Then, remove the skull using forceps. If marking the position of the bregma and lambda is necessary, first remove one side of the skull.Then insert thin tungsten needles into the brain at the positions of the bregma and lambda, then remove the remaining skull. Subsequently, place the brain sample on the vibratome platform. Cut the sections up to 500 micrometers thick in PBS at room temperature.In this procedure, transfer the slices to a five-milliliter plastic tube containing four milliliters of Scale S0 solution. Then incubate them for 12 hours with gentle shaking at 37 degrees Celsius. After 12 hours remove the solution from the tube using a pipette and add four milliliters of Scale A2 solution.Incubate the slices for 36 hours with gentle shaking at 37 degrees Celsius. After 36 hours, remove the solution from the tube and add four milliliters of Scale B4 solution. Incubate the slices for 24 hours with gentle shaking at 37 degrees Celsius.After 24 hours, remove the solution from the tube and add four milliliters of Scale A2 solution. Incubate the slices for 12 hours with gentle shaking at 37 degrees Celsius. After 12 hours, remove the solution from the tube and add four milliliters of PBS.Incubate the slices for six hours with gentle shaking at room temperature. Subsequently, carefully remove the slices to a two-milliliter plastic tube. Incubate them with a primary antibody in one milliliter of AB Scale solution for 48 to 72 hours at 37 degrees Celsius with gentle shaking.Afterward, carefully remove the slices to a five-milliliter plastic tube. Incubate them in four milliliters of AB Scale solution for two hours two times at room temperature with gentle shaking. Afterward, carefully remove the slices to a two-milliliter plastic tube.Incubate with a fluorescently-labeled secondary antibody in one milliliter of AB Scale solution for 48 hours at 37 degrees Celsius with gentle shaking. Carefully remove the slices to a five-milliliter plastic tube containing four milliliters of the antibody Scale solution and incubate the slices for six hours with gentle shaking at room temperature. Remove the solution and add four milliliters of the AB Scale solution and incubate the slices for two hours two times with a gentle shaking at room temperature.Next, remove the solution and add four milliliters of 4%PFA and incubate the slices for one hour with gentle shaking at room temperature. Then, remove the solution and add four milliliters of PBS and incubate the slices for one hour with gentle shaking at room temperature. Remove the solution and add four milliliters of the Scale S4 solution and incubate the slices for 12 hours with gentle shaking at 37 degrees Celsius.Subsequently, place the spacer on a glass slide and place the slices in the spacer and immerse the slices in Scale S4 solution. Seal the spacer with a cover glass. Put some water on the cover glass and image the slices using confocal or two-photon microscopy with a water-immersion long-working-distance objective.In this study, cortical projection neurons were labeled in green by tdTomato expression in Tlx3-cre AI9 transgenic mice and sub cerebral projection neurons in magenta were visualized by injecting the retrograde tracer CTB488 into the pons at seven weeks of age. This is the top view to show the approximate positions of cortical areas. Here are the oblique views of CTB488 fluorescence showing sub cerebral projection neurons and tdTomato fluorescence showing cortical projection neurons and this is the merged image.The cell bodies of the two cell types were visible over a wide range of brain areas and optical sections showed periodic micro columns. This image shows the cell bodies and apical dendrites of eGFP-expressing sub cerebral projection neurons in optical section of a Crym-eGFP mouse and this image shows the parvalbumin-expressing cells labeled in green and sub cerebral projection neurons labeled in magenta by injecting CTB488 into the pons of a C57 Black 6 mouse by the antibody Scale method. Following this procedure, other methods like 3D image processing can be performed in order to answer additional questions like quantitative characterization of the cerebral organization.After its development, this technique paved the way for researchers in the field of cortical biology to explore novel modular organization in the mouse brain.

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Research

JoVE Journal

Neuroscience

8.2K visualizações.  RIKEN Brain Science Institute. This method can help answer key questions in the neuroscience field such as the analysis of the structure and the function of the neocortex. The main advantage of this technique is that it can reveal the large-scale three-dimensional structure in the brain. Though this method can provide insight into the neocortex, it can also be applied to other nervous systems such as the spinal cord.To inject the tracer into the pons of the adult mice, first draw one microliter of fluorescently-labe...