Background: Bupivacaine induces central neurotoxicity at lower blood concentrations than cardiovascular toxicity. However, central sensitivity to bupivacaine is poorly understood. The toxicity mechanism might be related to glutamate-induced excitotoxicity in hippocampal cells.

Methods: The intracellular free Ca²⁺ concentration ([Ca²⁺]_i), mitochondrial membrane potential, and reactive oxygen species generation were measured by fluorescence and two-photon laser scanning microscopy in fetal rat hippocampal neurons and astrocytes.

Results: In astrocyte/neuron cocultures, 300 μM bupivacaine inhibited glutamate-induced increases in [Ca²⁺]_i in astrocytes by 40% (P < 0.0001; n = 20) but significantly potentiated glutamate-induced increases in [Ca²⁺]_i in neurons by 102% (P = 0.0007; n = 10). Ropivacaine produced concentration-dependent effects similar to bupivacaine (0.3 to 300 μM). Tetrodotoxin did not mimic bupivacaine’s effects. In pure cell cultures, bupivacaine did not affect glutamate-induced increases in [Ca²⁺]_i in neurons but did inhibit increased [Ca²⁺]_i in astrocytes. Moreover, bupivacaine produced a 61% decrease in the mitochondrial membrane potential (n = 20) and a 130% increase in reactive oxygen species generation (n = 15) in astrocytes. Cyclosporin A treatment suppressed bupivacaine’s effects on [Ca²⁺]_i, mitochondrial membrane potential, and reactive oxygen species generation. When astrocyte/neuron cocultures were incubated with 500 μM dihydrokainic acid (a specific glutamate transporter–1 inhibitor), bupivacaine did not potentiate glutamate-induced increases in [Ca²⁺]_i in neurons but still inhibited glutamate-induced increases in [Ca²⁺]_i in astrocytes.

Conclusions: In primary rat hippocampal astrocyte and neuron cocultures, clinically relevant concentrations of bupivacaine selectively impair astrocytic mitochondrial function, thereby suppressing glutamate uptake, which indirectly potentiates glutamate-induced increases in [Ca²⁺]_i in neurons.