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....

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. Sl....

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

List of materials used in this article

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