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

Summary

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.

Abstract

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.

Introduction

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 functions^{1,2,3,4,5,6,7}. 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 unclear^8,9. 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 structures^12,13,14. Both sub-cerebral projection neurons^12,13,14 and cortical projection neurons¹⁴ organize into cell-type specific microcolumns with a diameter of 1–2 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 brain^12,14 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 µm and have a spatial periodicity of approximately 40 µm. 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 tissues^15,16. 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.

Protocol

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

1. Imaging chamber¹⁹
   1. 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).
2. Slice spacer
   1. Prepare spacers to hold samples, using silicone rubber sheets with a thickness of 0.5 mm (Figure 1C).

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.

1. Make injections into the pons of adult mice.
   1. Draw 1 µL of fluorescently-labeled cholera toxin subunit B (25 µg/µL in PBS) into a 26G Hamilton syringe.
   2. Place an injector pump on the syringe.
   3. Place the syringe and pump on the tool holder of a manipulator placed on a stereotaxic instrument. Tilt the manipulator 12° posteriorly from the vertical axis.
   4. 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 (2–3%). Wait until the mouse makes no response when its tail is pinched with forceps, indicating that the mouse is fully anesthetized.
   5. Place the mouse on the stereotaxic instrument.
   6. 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.
   7. 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.
   8. 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.
   9. At the marked position, drill a hole with a diameter of approximately 1 mm.
   10. Insert the syringe tip through the hole so that the tip depth is 6.9 mm more than that measured at the bregma.
   11. Inject 1 µL of tracers using the pump at 0.2 µL/min.
   12. Remove the syringe from the brain.
   13. If necessary, cover the exposed brain with small fragments of microfibrillar hemostat and instant adhesive.
   14. Rinse the exposed brain using saline delivered with a pipette to prevent infection and suture the scalp.
   15. Remove the mouse from the stereotaxic instrument. Allow the mouse to recover from anesthesia in an incubator at 30 ˚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.
   16. Maintain the mouse for 3–7 days.
2. Make injections into the superior colliculus of adult mice.
   1. Prepare a glass pipette with a tip diameter of 30–50 µm.
   2. Connect the glass pipette to a Hamilton syringe through a plastic tube (Figure 2A).
   3. Fill the glass pipette, plastic tube, and Hamilton syringe with the paraffin liquid.
   4. Place an injector pump on the syringe.
   5. Place the glass pipette on the manipulator and tilt the manipulator 60° posteriorly from the vertical axis (Figure 2B).
   6. Perform 2.1.4–2.1.9. The position of the manipulator is 1.4 mm posterior, 0.5 mm lateral to the lambda.
   7. Place a small plastic paraffin film on the skull, then put approximately 1 µL of the tracer solution on it. Quickly advance the glass pipette and fill it with at least 0.5 µL of the tracer solution.
   8. Insert the glass pipette so that the tip depth is 3.0 mm from the brain surface.
   9. Inject 0.5 µL of tracers using the pump at 0.2 µL/min.
   10. Remove the glass pipette from the brain.
   11. Perform 2.1.13–2.1.16.

3. Fixation and Trimming

1. 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.
2. Euthanize the mouse humanely by perfusing the mouse transcardially²⁰ with 0.9% saline.
3. Fix the mouse²⁰ by perfusing 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.5).
4. Cut the scalp using a pair of scissors to expose the skull as described²⁰.
   1. Cut the midline of the exposed skull using a pair of scissors. Remove the skull using forceps.
   2. 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 ˚C.
5. To perform antibody staining of inhibitory neurons, cut the brain samples into slices.
   1. Place the brain sample on a vibratome.
   2. Cut slices up to 500 µm thick in PBS at room temperature and proceed to step 5 (the AbScale method).
6. 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 °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.

4. Clearing without Antibody Staining (the SeeDB Method)

1. Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% α-thioglycerol and 20% (w/v) fructose and incubate it for 4 h with gentle shaking at room temperature.
2. Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% α-thioglycerol and 40% (w/v) fructose and incubate it for 4 h with gentle shaking at room temperature.
3. Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% α-thioglycerol and 60% (w/v) fructose and incubate it for 4 h with gentle shaking at room temperature.
4. Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% α-thioglycerol and 80% (w/v) fructose and incubate it for 12 h with gentle shaking at room temperature.
5. Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% α-thioglycerol and 100% (w/v) fructose and incubate it for 12 h with gentle shaking at room temperature.
6. Transfer the sample using a spatula to a 50 mL plastic tube containing 20 mL of 0.5% α-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.
7. 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.
8. 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.

5. Clearing with Antibody Staining (the AbScale Method)

1. Prepare reagents as described in Table 2.
2. 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 °C (Figure 4B).
3. 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 °C.
4. 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 °C.
5. 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 °C.
6. 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.
7. Carefully remove the slices to a 2 mL plastic tube using a spatula.
8. Incubate with primary antibodies (Table 3) in 1 mL of AbScale solution for 48-72 h at 37 °C (Figure 4C) with gentle shaking.
9. Carefully remove the slices to a 5 mL plastic tube using a spatula.
10. Incubate in 4 mL of AbScale solution for 2 h 2 times at room temperature with gentle shaking.
11. Carefully remove the slices to a 2 mL plastic tube using a spatula.
12. Incubate with fluorescently-labeled secondary antibodies (Table of Materials, 1:100) in 1 mL of AbScale solution for 48 h at 37 °C with gentle shaking.
13. 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.
14. 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.
15. 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.
16. 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.
17. 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 °C.
18. 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).
19. 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.

6. Cell Position Determination

1. For each position in the scanned images, calculate correlation values using a three-dimensional image filter¹⁴ (Figure 5A-5D).
2. Determine the positions of the peaks of the correlation value (Figure 5D).
3. Investigate images around the peaks to locate cells (Figure 5E).

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Crym-egfp transgenic mice	MMRRC	012003-UCD
Tlx3-cre transgenic mice	MMRRC	36547-UCD
ROSA-CAG-flox-tdTomato mice	Jackson Laboratory	JAX #7909
Silicone rubber sheet	AS ONE	6-611-01	0.5 mm thickness
Silicone rubber sheet	AS ONE	6-611-02	1.0 mm thickness
Silicone rubber sheet	AS ONE	6-611-05	3.0 mm thickness
Petri dishes	Falcon	351008
Cover glass	Matsunami	C022241
Cholera toxin subunit B (recombinant), Alexa Fluor 488 conjugate	Invitrogen	C22841
Cholera toxin subunit B (recombinant), Alexa Fluor 555 conjugate	Invitrogen	C22843
Cholera toxin subunit B (recombinant), Alexa Fluor 594 conjugate	Invitrogen	C22842
Cholera toxin subunit B (recombinant), Alexa Fluor 647 conjugate	Invitrogen	C34778
26 G Hamilton syringe	Hamilton	701N
Injector pump	KD Scientific	KDS 310	Pons injection
Injector pump	KD Scientific	KDS 100	Superior colliculus injection
Manipulator	Narishige	SM-15
Sodium pentobarbital	Kyoritsu Seiyaku	Somnopentyl
Isoflurane	Pfizer
Lidocaine	AstraZeneca	Xylocaine injection 1% with epinephrine
Drill	Toyo Associates	HP-200
Avitene microfibrillar hemostat	Davol Inc	1010090
Alonalfa	Daiichi-Sankyo	Alonalpha A
Surgical silk	Ethicon	K881H
Incubator	UVP	HB-1000 Hybridizer
Glass pipette	Drummond Scientific Company	2-000-075
Electrode puller	Sutter Instrument Company	P-97
Paraffin Liquid, light	Nacalai tesque	26132-35
Saline	Otsuka	1326
Paraformaldehyde	Nacalai tesque	26126-54
Tungsten needle	Inter medical	Φ0.1 *L200 mm
Vibratome	Leica	VT1000S
50 mL plastic tube	Falcon	352070
α-thioglycerol	Nacalai tesque	33709-62
D(-) Fructose	Nacalai tesque	16315-55
BluTack	Bostik	CKBT-450000
Two-photon microscope	Nikon	A1RMP
Water-immersion long working distance objectives	Nikon	CFI Apo LWD 25XW, NA 1.1, WD 2 mm
Water-immersion long working distance objectives	Nikon	CFI LWD 16XW, NA 0.8, WD 3 mm
Motorized stage	COMS	PT100C-50XY
Filter	Semrock	FF01-492/SP-25
Filter	Semrock	FF03-525/50-25
Filter	Semrock	FF03-575/25-25
Filter	Semrock	FF01-629/56
Filter	Chroma	D605/55m
5 mL plastic tube	AS ONE	VIO-5B
2 mL plastic tube	Eppendorf	0030120094
Urea	Nacalai tesque	35905-35
Triton X-100	Nacalai tesque	35501-15
Glyserol	Sigma-aldrich	191612
D(-)-sorbitol	Wako	191-14735
Methyl-β-cyclodextrin	Tokyo chemical industry	M1356
γ-Cyclodextrin	Wako	037-10643
N-acetyl-L-hydroxyproline	Skin Essential Actives	33996-33-7
DMSO	Nacalai tesque	13445-45
Bovine Serum Albumin	Sigma-aldrich	A7906
Tween-20 (1.1 g/mL)	Nacalai tesque	35624-15
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555	Invitrogen	A21422
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555	Invitrogen	A21428
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	Invitrogen	A21235
Goat anti-Mouse IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488	Invitrogen	A11029
Donkey anti-Rabbit IgG (H+L) Highly CrossAdsorbed Secondary Antibody, Alexa Fluor 488	Invitrogen	A21206
Confocal microscope	Olympus	FV1000
Water-immersion long working distance objectives	Olympus	XLUMPLFLN 20XW, NA 1.0, WD 2 mm
Anti-NeuN	Millipore	MAB377
Anti-NeuN	Millipore	ABN78
Anti-CTIP2	Abcam	ab18465
Anti-Statb2	Abcam	ab51502
Anti-GAD67	Millipore	MAB5406
Anti-GABA	Sigma	A2052
Anti-Parvalbumin	Swant	235
Anti-Parvalbumin	Frontier Institute	PV-Go-Af460
Anti-Parvalbumin	Sigma	P3088
Anti-Parvalbumin	Abcam	ab11427
Anti-Somatostatin	Peninsula Laboratories	T-4103
Anti-c-Fos	CalbioChem	PC38