1. Preparation of Rat Tail Collagen
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)
2. Dissection of the Dopaminergic Midbrain7,8
All subsequent dissection steps are performed in L15 medium on ice.
3. Dissection of the Striatum
All dissection steps are performed in L15 medium on ice.
4. Assembly Collagen Matrix Assays
Preparation of the collagen (all steps on ice, use cooled pipette tips)
Setup co-culture in collagen gel
5. Analysis by Immunohistochemistry and Quantification
Immunohistochemistry
Quantification by calculating P/D ratio
6. Representative Images
After successful culture of dopaminergic and striatal explants a large number of axons are visible using bright field microscopy or as visualized by anti-βIII tubulin immunocytochemistry (not shown). A subset of these axons will be dopaminergic or striatal axons as visualized using immunocytochemistry (Figure 3). By dividing the explants into quadrants as described and determining P/D ratios, a putative axon attractive or repulsive effect can be quantified (Figure 3E).
Figure 1. Photos illustrating the preparation of collagen from rat tail tendons. A) Pulling apart the rat tail exposes the tendons. B) Tendons are visible as rope-like bundles. C) The tendons are easy to distinguish from veins by their shiny white appearance. D) After dissolving the tendons in acetic acid, the solution is transferred to dialysis tubing. To keep the dissolved collagen cold, tubes are placed on sterile aluminum foil on ice.
Figure 2. Schematic indicating the different steps of the procedure. Appropriate brain regions are dissected and cut to generate explants. A single midbrain and striatal explant are positioned in close proximity in a collagen matrix and left to grow for 48-72 hours at 37 °C. Axons are visualized by fluorescence immunohistochemistry and a P/D ratio is calculated to quantify chemotropic responses.
Figure 3. Representative results showing axon growth in a collagen matrix culture. A) Midbrain explant stained with anti-tyrosine hydroxylase antibody revealing axon outgrowth. Dotted line indicates adjacent explant. B) Magnification of A showing individual neurites and growth cones (arrows). C) Striatal explant stained with anti-DARPP32. D) Magnification of C showing individual neurites. E) Example of P/D ratio quantification. Explants are divided into equal quadrants. The proximal quadrant is facing the adjacent explant while the distal quadrant is facing away from it. Scale bars indicate 100 μm.
Name of the reagent | Company | Catalogue number | Comments |
Fetal Calf Serum | BioWhittaker | 14-801F | |
Glutamine (200mM) | PAA | M11-004 | |
Hepes | VWR International | 441476L | |
β-Mercaptoethanol | Merck | 444203 | |
Minimum Essential Media (MEM) | Gibco | 61100-087 | |
Neurobasal | Gibco | 21103-049 | |
B27 | Gibco | 17504-044 | |
Leibovitz’s L-15 Medium | Gibco | 11415-049 | |
Penicillin-Streptomycin | Gibco | 15070-063 | |
Prolong Gold Antifade Reagent | Invitrogen | P36930 | |
Dialysis tubing | Spectrum Labs | 132660 | |
Rabbit anti-Tyrosine Hydroxylase | Pel-Freez | P40101-0 | |
Rabbit anti-Darpp32 (H-62) | Santa-Cruz | Sc-11365 | |
Mouse anti-βIII tubulin | Sigma | T8660 | |
Alexa Fluor labeled secondary antibodies | Invitrogen |
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 regions3,4. 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.
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 regions3,4. 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.
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 regions3,4. 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.