Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Summary

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Abstract

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum¹. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra². The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions^3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Protocol

1. Preparation of Rat Tail Collagen

1. Collect 6-10 adult rat tails (it is possible to store the tails at -20 °C until use).
2. Soak the tails in 95% ethanol overnight at room temperature (RT).

Dissection of rat tails (in tissue culture hood):
(keep tools in 70% ethanol when not using them and make sure that all solutions, tools and glassware used throughout this procedure are sterile)

3. To collect tendons from the tails, cut off the tip of the tail. Hold large end of the tail with a pair of forceps. Use another pair of forceps to hold, bend and break the tail close to the other for....

Discussion

The collagen matrix assay described here has been used and improved by many different labs in the past decades to investigate a variety of axon guidance molecules and neuronal systems (e.g. see references 5-8). These studies have shown that this assay is a powerful tool for studying the effects and regulation of axon guidance molecules secreted by different (intermediate) target tissues.

However, it should be noted that the collagen matrix is a substitute for the extracellular environment (EC.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal Calf Serum	BioWhittaker	14-801F
Glutamine (200mM)	PAA Laboratories	M11-004
Hepes	VWR international	441476L
β-Mercapt–thanol	Merck & Co., Inc.	444203
Minimum Essential Media (MEM)	GIBCO, by Life Technologies	61100-087
Neurobasal	GIBCO, by Life Technologies	21103-049
B27	GIBCO, by Life Technologies	17504-044
Leibovitz’s L-15 Medium	GIBCO, by Life Technologies	11415-049
Penicillin-Streptomycin	GIBCO, by Life Technologies	15070-063
Prolong Gold Antifade Reagent	Invitrogen	P36930
Dialysis tubing	Spectrum Labs	132660
Rabbit anti-Tyrosine Hydroxylase	Pel-Freez Biologicals	P40101-0
Rabbit anti-Darpp32 (H-62)	Santa Cruz Biotechnology, Inc.	Sc-11365
Mouse anti-βIII tubulin	Sigma-Aldrich	T8660
Alexa Fluor labeled secondary antibodies	Invitrogen