Organotypic Culture of Developing Striatum - Drugs of Abuse: Neurological Reviews and Protocols - page 396

A key feature of the organotypic culture system that we developed is that
it makes the identification of the striatal compartment-specific cell types
possible in vitro, because the maintenance of the structural architecture of
the striatal slices permits the identifi cation of compartment-specifi c cells in
culture condition. Specifi cally, as we have demonstrated in our previous study,
the striosomal and matrix compartments can be recognized, respectively,
as dopamine and adenosine 3 -monophosphate-regulated phosphoprotein

(DARPP-32)-positive neuronal clusters and matrix of calbindin-D 28KDa -positive
neurons in the cultured striatal slices (7) .
We have taken advantage of this system to study induction of different
proteins with different kinetics of induction in the developing striatum.
In our previous work, we have found that the phosphorylation of cAMP
response element-binding protein (CREB), with induction times of minutes,
and the expression of Fos-like protein, with induction times of hours, are com-
partmentally correlated (7 , 8) . They both occur differentially in striosomal cells
of the cultured striatum at their respective peak induction times. The successful
identifi cation of compartment-specifi c cells in vitro itself is signifi cant, as
it opens a new avenue for cellular and molecular characterization of striatal
compartmentation in vitro (9 , 10) .
To give one example of the value of this approach, I describe in the present
chapter the experimental protocols for compartmental induction of Fos, the
protein product of immediate-early gene c- fos by dopamine agonists in slice
cultures of developing striatum ( Fig. 1 ) (7) .

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2. Materials
1. Postnatal day 0-1 rats (Sprague-Dawley, Taconic Farms, Germantown, NY).
2. 70% Alcohol.
3. D -(+)-Glucose (Sigma, St. Louis, MO).
4. Gey's Balanced Salt Solution (GIBCO BRL, Grand Island, NY) supplemented
with 5.5 mg/mL of D -(+)-glucose (GBSS).
5. SF21 serum-free medium (11) .
6. SKF-81297 HCl (SmithKline Beecham Pharmaceuticals, King of Prussia, PA).
7. R (+)-SCH-23390 HCl (Research Biochemicals Inc., Natick, MA).
8. Low melting temperature agarose (FMC BioProducts, Rockland, ME).
9. 0.1 M Phosphate buffer (PB, pH 7.4).
10. 0.1 M Phosphate-buffered saline (PBS, pH 7.4).
11. 4% Paraformaldehyde in 0.1 M PB.
12. Rabbit polyclonal anti-c-Fos antiserum (Ab2, Oncogene Science Inc., Union-
dale, NY).
13. 30% H 2 O 2 .