Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Summary

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Abstract

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages ^1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain ^3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase ⁵. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system ⁶.

Protocol

Parts of this protocol are modified from Daza et al., 2007 ⁷.

1. Preparations

1. Can be prepared one day in advance
   1. Prepare 1X Krebs buffer (1.5 L): 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄:H₂O, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 11 mM glucose, 25 mM NaHCO₃; adjust pH to 7.4. Filter sterilize (0.22 μm pore size) and store at 4°C.
   2. Prepare the culture medium (20 mL): 5 mL HBSS, 9 mL DMEM high glucose, 850 μL of 30 % glucose, 5 mL horse serum (25 %). Add 200 μL 100X Penicllin/Streptomycin. Store at 4°C.

Discussion

The organotypic slice culture method presented here provides a system for the short-term in vitro analysis of developing dopaminergic neurons and their migratory and projection routes in the embryonic ventral midbrain. We found that there are a number of critical steps in the protocol that should be carefully attended to in order to obtain slices that allow the normal development of ventral midbrain dopaminergic neurons. The most critical step is the dissection of the embryonic brain, which has to be both fast a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Table of specific reagents and equipment
DMEM	Sigma-Aldrich	D6429
Glucose 30%	Sigma-Aldrich	G7528-250
Horse Serum	Invitrogen	26050-088
DMEM (4,5g/L Glc., with L-Gln, Na Pyr, NaHCO3)	Sigma-Aldrich	D6429-500
Penicillin/Streptomycin 100x	Sigma-Aldrich	P4333-20
L-ascorbic acid	Sigma-Aldrich	A4403	prepare 200mM stock and store at -20°C
UltraPure LMP agarose	Invitrogen	15517-022
Millicel inserts	EMD Millipore	PICMORG50
μ-dish 35 mm, low	Ibidi	80136
Vibratome	Microm International	HM 650V
Razor Blade	Plano GmbH	121-6
Histoacryl glue	BRAU9381104	Braun Aesculap
Perforated Spoon Dia diameter 15 mm	Fine Science Tools	10370 -18
Forceps 5 Dumoxel	Fine Science Tools	11252 - 30
Antibodies used for immunostainings:
Rabbit anti-tyrosine hydroxylase	EMD Millipore	AB152	Dilution 1:500
Mouse anti-tyrosine hydroxylase	EMD Millipore	MAB318	Dilution 1:500
Mouse anti-BrdU	BD Biosciences	555627	Dilution 1:200
Rabbit anti-cleaved caspase 3	Cell Signaling Technology	9661	Dilution 1:200
Donkey anti-rabbit IgG-Alexa 488	Invitrogen	A21206	Dilution 1:500
Donkey anti-mouse IgG-Cy3	Jackson ImmunoResearch	715-165-150	Dilution 1:200