Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Summary

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Abstract

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Introduction

The hippocampus plays an important role in memory and learning as well as emotional behavior. One main function consists of the consolidation of short-term memory into long-term memory, which requires high plasticity of the nervous system. The dentate gyrus of the hippocampus acts as the primary gateway for input information and is also one of two brain regions with ongoing neurogenesis throughout adulthood^1,2. The development of the hippocampal structure occurs during late embryogenesis and particularly during the first 3 to 4 weeks postnatal³. During early development of the dentate gyrus a stem cell pool is established required for postnatal a....

Protocol

NOTE: All animal experiments were carried out in accordance with the German law and were approved by the government offices in Tübingen.

1. Preparation of Micropipettes, Solutions and Membranes

1. Preparation of Micropipettes
   1. Pull glass micropipettes using a micropipette puller with the following program: Heat: 540, Pull: 125, Velocity: 20 and Delay: 140. The needle length amounts to 5.5 cm.
   2. Bevel needles using a microgrinder to obtain a suitable tip size of 4 mm. Store the needles in a box or 15 cm Petri dish to prevent damaging of the tips.
2. Preparation of Solutions
   1. P....

Results

Ablation of the transcription factor Bcl11b causes the impairment of progenitor cell proliferation and neuronal differentiation resulting in a reduced dentate gyrus size and cell number. Furthermore mutant neurons fail to integrate into the hippocampal circuitry causing learning and memory impairment⁸. To answer questions concerning the regulatory mechanism(s) of Bcl11b in these processes ex utero electroporation was employed.

Addressing the question whether Bcl11b cell-aut.......

Discussion

The hippocampus has an important function in learning and memory. The dentate gyrus is also one of two brain regions where neurogenesis occurs not only during development but also throughout adulthood. Postnatal and adult hippocampal neurogenesis proceeds in a similar way involving many common factors. Defining the regulatory mechanisms of these factors will be very helpful in understanding neurodegenerative diseases which in turn will lead to new therapies and preventive measures. To obtain this information one requires.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Flaming/ Brown Micropipette Puller	Sutter Instruments Company (USA)	P-97
Fine Glass Pipettes	Warner Instruments	G100F-4
Microgrinder	Narishige, Japan	EG-44
Anesthetic Bracket unit	Harvard Apparatus	PY2 34-0412
Halovet Vaporizer	Harvard Apparatus	PY2 34-0398
Fluovac System	Harvard Apparatus	PY2 34-0387
IMS Fluosorber	Harvard Apparatus	PY2 34-0415
Anesthetizing Chamber	Harvard Apparatus	PY2 34-0460
Electroporator	BEX Company	CUY21 EDIT
Tweezers with disk electrodes	BEX Company	LF650P3	3 mm electrodes for E15.5
Tweezers with disk electrodes	BEX Company	LF650P5	5 mm electrodes for E18.5
Picospritzer III	Parker Hannifin Corporation	P/N 052-0500-900
HM 650 V Vibrating Blade Microtome, 230 V	Thermo Scientific	920120
Dissection Microscope	Carl Zeiss Microscopy Gmbh	Stemi SV8
Inverted Microscope	Leica	Leica DM IL LED
Confocal Microscope	Leica	Sp5II
6 well dish	BD Falcon	#353502
6 well dish	CELLSTAR	#657160
Tissue culture inserts	BD Falcon	#353090
Fast Green	Sigma	F7252
Laminin	Sigma	#L2020
Poly-L-lysine	Sigma	#P5899
Spring scissors	Fine Science Tools	15003-08
Extra Fine Bonn Scissors	Fine Science Tools	14084-08
Forceps	Dumont #55	11255-20 Inox
HBSS 10x	Life Technology	14180-046
BME	Life Technology	41010-26