Glutamate-induced excitotoxicity contributes to neuronal damage during a cerebral ischaemic event such as stroke. Physiological levels of glutamate and proinflammatory cytokines such as tumour necrosis factor-α (TNFα) play a role in the regulation of synaptic plasticity within the hippocampus. During a stroke pathophysiological levels may cause dysregulation of these processes, enhancing vulnerability of these cells to an ischaemic insult. Previous studies suggest that a mild transient ischaemic attack (TIA) within 72 h of a stroke may result in attenuation of its clinical severity (Castillo et al. (2003)). We have developed an in vitro model of TIA using organotypic hippocampal cultures (modified technique of Stoppini et al. (1991)). Hippocampal slices (400 µm) were cultured from male Wistar rats at post-natal day 7 (humanely killed – decapitated). At 6 days in vitro (DIV) cultures were pretreated for 30 min with 30 µM glutamate or 5 ng/ml TNFα. They were then placed in fresh media for 24 h (recovery period). As Ca²⁺ is a well-established mediator of glutamate-induced excitotoxicity, we investigated whether glutamate/TNFα preconditioning altered glutamate-induced Ca²⁺ influx. At 7 DIV pretreated cultures were loaded with a Ca²⁺ indicator dye, Fluo-4 (3 µM) and imaged with Zeiss laser scanning confocal microscope. Relative changes in fluorescence of Ca²⁺-bound dye correlates with relative changes in [Ca²⁺]_i. After 20 s of baseline recording, 30 µM glutamate was applied and the Ca²⁺ response was recorded for 70 s. Data analysis was carried out with Zeiss Image Examiner software and statistics were compiled using one-way ANOVA followed by Bonferroni post-test. Results are expressed as mean±S.E.M. Pretreatment with 5 ng/ml TNFα/30 µM glutamate resulted in a reduction in glutamate-induced Ca²⁺ influx after 24 h (TNFα; 1293±28, n=3340, glutamate; 1088±23, n=2439, Vs. control; 2679±48, n=3524, p<0.001). Changes in baseline Ca²⁺ levels due to pretreatments were analysed using a ratiometric Ca²⁺ dye, Indo-1 (5 µM). Preliminary data suggest that TNFα/glutamate pretreatment significantly lowered baseline [Ca²⁺]_i compared to control. Thus, an acute mild TNFα/glutamate exposure may alter both basal [Ca²⁺]_i within the cell and its responsiveness to glutamate-induced Ca²⁺ influx. Pretreatment with 5 ng/ml TNFα in the presence of the mGluR5 metabotrophic glutamate receptor antagonist, MPEP (10 µM), significantly enhanced TNFα’s preconditioning effect (1125±21, n=2298, p<0.05). The NMDA receptor antagonist, D-AP5 (100 µM) did not significantly alter the preconditioning effect of glutamate when co-applied (1072±27, n=1764, p>0.05). We conclude that TNFα’s preconditioning effects may be TNFR and mGluR5 receptor mediated, whereas NMDAR activation during glutamate preconditioning may be less important.