Primary brain cell isolation and culture.  

1. Cerebella were removed from 7-day-old mice and passed through Nitex nylon netting (80 μm pore size) into primary cell system containing 20% (v/v) fetal calf serum (FCS).

2. The cell suspension was plated at a density of four cerebella per 12-well culture plate.

3. Primary cell culture system was changed two days after plating and twice a week thereafter, gradually decreasing the FCS concentration to 10%.

4. At 14 days in culture, dibutyryl-cAMP was added to the medium for 1 week to promote the morphological differentiation of astrocytes.

5. Experiments were performed on 3-week-old cultures.

6. Cortical astrocytes were prepared from 1-day-old mice following the same procedure used for cerebellar astrocytes plated at a density of two cortices per 12-well culture plate.

7. Cerebellar neurons were isolated and cultured from the cerebellum of 7-day-old mice, after mild trypsinization of the tissue followed by trituration in a DNase solution containing a trypsin inhibitor derived from soybeans.

8. Cells were suspended in a slightly modified primary cell culture containing 50 μM kainic acid and 10% (v/v) FCS and plated at density 2 × 10⁶ cells per well in a 12-well culture plate.

9. The wells had been coated with poly-L-lysine.

10. Cytosine arabinoside (20 μM) was added after 48 h to prevent astrocyte proliferation.

11. Cells were used for experiments after 1 week in culture.

12. For the co-cultures, the neurons and astrocytes were prepared separately and seeded as described earlier.

13. The astrocytes were cultured on inserts for 21 days, and the neurons were cultured in culture plates for 7 days prior to incubation in co-culture.

14. The astrocytes were grown on permeable membranes in tissue culture plate inserts, and the neurons were cultured in standard tissue culture plates.

15. The inserts were transferred to the neuronal plates 24 h prior to incubation.

16. At time of insert transfer, half of the neuronal medium was extracted and replaced with astrocyte cell culture system.