Evaluation of glutathione-sensitive fluorescent dyes in cortical culture

The sensitivity of six fluorophores to glutathione (GSH! was evaluated in l iving rat cortical neuronal/glial mixed cultures during the first 23 days i n vitro (DIV). Four of the dyes require glutathione-S-transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: t hese included t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (CMAC ), 7-amino-4-chloromethylcoumsuin (CMAC-blue), monochlorobimane (MCB), and 5-chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2 ,3-naphthalenedicarboxaldehyde (NDA) and o-phthaldehyde (OPD), do not requi re GST for adduct formation with GSH. To examine the specificity of the dye s for GSH, cultures grown less than 6 DIV were pretreated with diethyl male ate or DL-buthionine-(S,R)-sulfoximine to deplete endogenous GSH. This resu lted in a substantial loss of staining by CMAC, CMAC-blue, and MCB and part ial loss of staining by OPD, indicating specificity for GSH, while staining by CMFDA or NDA was not altered, indicating a lack of specificity for GSH. Neurons experienced a dramatic decline in GSH levels relative to astrocyte s between 5-6 DIV, as shown by a loss of neuronal staining with CMAC, CMAC- blue and MCB. This decrease in staining was not due to a decrease in GST ac tivity, as neurons stained with the GST-insensitive OPD also exhibited a de cline in GSH-sensitive staining. Immunolabeling experiments demonstrated th at CMAC staining co-localized with GFAP-positive astrocytes, but not with M AP-2-positive neurons, in 18 DIV cultures. Finally, CMAC was exploited as a specific morphological marker of astrocytes in cultures aged >5 DIV. CMAC staining was employed to monitor astrocyte proliferation and to resolve ast rocytes in living mixed cultures co-loaded with the Ca2+-sensitive dye, cal cium green 5N-AM. Published 2000 Wiley-Liss, Inc.