Effects of AF64A on [3H]acetylcholine synthesis in neuron-enriched primary brain cell cultures

[3H]Acetylcholine (ACh) synthesis was measured in primary neuronal cultures from neonatal rat brains. Neuronal [3H]ACh synthesis was blocked by hemicholinium-3 and depended on the age of the cultures, increasing for ca. 10 days, and eventually declining. The irreversible inhibitor AF64A (10 or 30 microM) inhibited [3H]ACh synthesis from [3H]choline at concentrations (10 or 30 microM) with affecting choline acetyltransferase activity. Nine-day-old cultures recovered 90% of their [3H]ACh synthesis within 7 days after AF64A, while 13-day-old cultures never recovered. These results suggest that the turnover of neuronal choline transporters is age-related.