| Abstract | The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione‐S‐transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t‐butoxycarbonyl‐Leu‐Met‐7‐amino‐4‐chloromethylcoumarin (CMAC), 7‐amino‐4‐chloromethylcoumarin (CMAC‐blue), monochlorobimane (MCB), and 5‐chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2,3‐naphthalenedicarboxaldehyde (NDA) and o‐phthaldehyde (OPD), do not require GST for adduct formation with GSH. To examine the specificity of the dyes for GSH, cultures grown less than 6 DIV were pretreated with diethyl maleate or DL‐buthionine‐(S,R)‐sulfoximine to deplete endogenous GSH. This resulted in a substantial loss of staining by CMAC, CMAC‐blue, and MCB and partial 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 astrocytes 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 activity, as neurons stained with the GST‐insensitive OPD also exhibited a decline in GSH‐sensitive staining. Immunolabeling experiments demonstrated that CMAC staining co‐localized with GFAP‐positive astrocytes, but not with MAP‐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 astrocytes in living mixed cultures co‐loaded with the Ca2+‐sensitive dye, calcium green 5N‐AM. |
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