The present study evaluated the neurotoxicity of various gangliosides against dopaminergic neurons in mesencephalic cultures. Among them, GD1a and GD1b but not GD3 and GQ1b were found to be neurotoxic against dopaminergic neurons as determined by TH immunocytochemistry and [(3)H]DA uptake. When quantified and expressed as a percentage of control values, treatment with 60-200 microg/ml GD1a and GD1b attenuated the number of TH-ip neurons by 31-47% and 37-55%, respectively, compared with non-treated control cultures. Consistent with the results of the TH immunocytochemistry, treatment with 60-200 microg/ml GD1a and GD1b reduced [(3)H]DA uptake levels by 27-56% and 41-60%, respectively, compared with non-treated control cultures. This neurotoxicity was almost completely abolished in the presence of neuraminidase, which removes the sialic acid residues from ganglioside, or in the treatment of insulin or IGF-1. Additional immunostaining also showed a significant loss of GABAergic neurons in GD1a or GD1b-treated cultures, indicating non-selective neurotoxicity of GD1a and GD1b. Moreover, these gangliosides had little effect on nitric oxide (NO) production in mesencephalic or microglia cultures. Together, these data suggest that GD1a and GD1b exert a direct neurotoxicity against dopaminergic neurons independent of NO and/or microglia.