Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Summary

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Abstract

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Introduction

The neocortex is the major site of cognitive, emotional, and sensorimotor functions. It is composed of six horizontal layers oriented in parallel to the surface of the brain. During development progenitor cells in the lateral wall of the dorsal telencephalon give rise to projection neurons that migrate radially towards the pial surface and acquire a layer type-specific neuronal identity. After being generated in the ventricular/subventricular zones (VZ/SVZ) these neurons become transiently multipolar and slow their migration. After a short stay in the intermediate zone (IZ) they switch to a bipolar morphology, attach to the radial glial scaffold, and continue radially....

Protocol

All experimental procedures were approved by the Animal Welfare Committee (Regierungspräsidium Tübingen) and carried out in accordance by the German Animal Welfare Act and the EU Directive 2010/63/EU.

1. In Utero Electroporation

1. Microinjection needles
   1. Pull borosilicate glass capillaries (outer diameter: 1.0 mm, inner diameter: 0.58 mm, length: 100 mm) into microinjection needles using a micropipette puller with a box filament (2.5 mm x 2.5 mm) and the following program: HEAT: 540, PULL: 125, VELOCITY: 20, and DELAY: 140. Determine the HEAT value for every individual filament by per....

Results

Previously, we have shown that genetic deletion of Bcl11a by in utero electroporation impairs radial migration of late-born upper-layer projection neurons¹⁰. Electroporation of a DNA plasmid vector containing Cre-IRES-GFP efficiently deleted Bcl11a in conditional Bcl11a^flox/flox brains¹¹. When we analyzed E14.5 electroporated brains three days after the electroporation, most control neu.......

Discussion

Radial migration is a key process in neocortex development. Mutations in genes affecting different steps of this process can cause severe cortical malformations, including lissencephaly and white matter heterotopia^1,2. We recently showed that Bcl11a, which is expressed in young migrating cortical projection neurons, plays a role in radial migration. We used time-lapse confocal imaging of migrating neurons in acute cortical slices of electroporated brains to direc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
isoflurane	Abbott Laboratories	506949	Forene
6-well plate	Corning	351146
12-well plate	Corning	351143
non-absorbable surgical suture	Ethicon	K890H	3/8 circle, 13 mm, taper point
Micro Adson Forceps	Fine Science Tools	11018-12	serrated, length: 12 cm
fine scissors	Fine Science Tools	14063-09	angled to side, length: 9 cm
Mathieu Needle Holder	Fine Science Tools	12510-14	tungsten carbide, length: 14 cm
fine tipped forceps	Fine Science Tools	11370-40	straight, 11 cm
Vannas Tübingen Spring Scissors	Fine Science Tools	15005-08	angled up, 9.5 cm
ring forceps	Fine Science Tools	11103-09	OD: 3mm, ID, 2.2 mm, length: 9 cm
HBSS (10X)	Gibco	14180046
L-Glutamine	Gibco	25030081
Penicillin/Streptomycin	Gibco	15140122
horse serum	Gibco	26050088
BME	Gibco	41010026
borosilicate glass capillaries	Harvard Apparatus	30-0016	1.0 OD x 0.58 ID x 100 L mm
anesthsesia system	Harvard Apparaus	72-6471
anesthetizing chamber	Harvard Apparaus	34-0460
fluosorber filter canister	Harvard Apparaus	34-0415
low melting point agarose	Invitrogen	16520100
vibrating blade microtome	Leica	VT1200 S
fluorescence stereo microscope	Leica	M205 FA
stereo microscope	Leica	M125
inverted fluorescence tissue culture microscope	Leica	DM IL LED
confocal laser scanning microscope	Leica	TCS SP5II
hybrid detector	Leica	HyD
objective, 40x/0.60 NA	Leica	11506201
microscope temperature control system	Life Imaging Services	Cube, Brick & Box
cell culture insert	Millipore	PICM0RG50
microgrinder	Narishige	EG-45	use 38° angle for beveling
microinjector	Parker Hannifin	052-0500-900	Picospritzer III
carprofen	Pfizer Animal Health	NDC 61106-8507	Rimadyl
emdedding mold	Polysciences	18986-1
endotoxin-free plasmid maxi kit	Qiagen	12362
fast green	Sigma	F7252
laminin	Sigma	L2020
poly-L-lysine	Sigma	P5899
HEPES	Sigma	H4034
D-glucose	Sigma	G6152
calcium chloride	Sigma	C7902
magensium sulfate	Sigma	M2643
sodium bicarbonate	Sigma	S6297
square wave electroporator	Sonidel	CUY21EDIT
tweezers with 5 mm platinum disk electrodes	Sonidel	CUY650P5
micropipette puller	Sutter Instrument	P-97
box filament	Sutter Instrument	FB255B	2.5 mm x 2.5 mm
micro-spoon spatula	VWR	231-0191	185 mm x 5 mm
glass bottom dish, 50 mm	World Precision Instruments	FD5040-100