The effect of glia-conditioned medium on dopamine neurons in culture. Modulation of apoptosis, tyrosine hydroxylase expression and 1-methyl-4-phenylpyridinium toxicity

Summary.

Recent experiments have shown that glia-conditioned medium (GCM) protects against L-3,4-Dihydroxyphenylalanine (L-DOPA) toxicity for dopamine neurons in culture. In this study we have investigated the effect of GCM on the number of tyrosine hydroxylase (TH) immunoreactive neurons, levels of dopamine, number of high affinity dopamine uptake sites, and percentage of apoptotic cells in midbrain neuronal cultures, before and after exposure to 1-methyl-4-phenylpyridinium (MPP⁺).

Fetal midbrain neuronal cultures were treated with vehicle, MPP⁺, 10⁻⁵ M, mesencephalic GCM, or MPP⁺ plus GCM. GCM was administered a) simultaneously, b) 24 hours before MPP⁺, and c) 24 and d) 72 hours after MPP⁺, respectively. In the absence of GCM, MPP⁺ reduced the number of TH immunoreactive neurons and increased apoptosis. GCM increased the number of TH+ neurons and the levels of dopamine and decreased apoptosis. In the cultures treated with GCM and MPP⁺, GCM counteracted the effects of MPP⁺ and increased the length and arborization of TH+ neurites. The protective effect of GCM was maximal in cultures co-treated with GCM and MPP⁺ simultaneously, but it also restored dopamine parameters in cultures receiving GCM 1 or 3 days after MPP⁺. The protective effect of GCM was negligible in cultures pretreated with GCM and receiving MPP⁺ 24 hours later. In neuronal cultures, grown for 8 days in vitro untreated with MPP⁺, short term exposure to GCM reversed the effect of aging and restored the number of TH+ neurons to levels higher than those observed at the time of seeding. Therefore, GCM does not only protect against MPP⁺ but does also induce de novo expression of dopamine phenotype in midbrain cultures.