Quantification of zinc toxicity using neuronal networks on microelectrode arrays

Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 microM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min and consisting of increased spiking and enhanced, coordinated bursting, followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Above 100 microM total zinc acetate, the activity loss was associated with massive cell swelling, blebbing, and even vigorous neuronal cell lysing. Glia showed stress, but did not participate in the extensive cell swelling. Network activity loss generally preceded morphological changes. Cultures pretreated with the GABA(A) receptor antagonists bicuculline (40 microM) and picrotoxin (1mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 microM). However, recovery was not complete and slow deterioration of network activity continued over 6-h periods. Removal of zinc by early medium changes showed irreversible, catastrophic network failure to develop in a concentration-dependent time window between 50% and 90% activity loss.