Preconditioning with 4-aminopyridine protects cerebellar granule neurons against excitotoxicity.

Preconditioning by excitatory stimuli such as N-methyl-d-aspartate (NMDA) offers good neuroprotection against excitotoxic insults, but is potentially limited by the risk of damage associated with the treatment. We report here the potential of an alternative strategy, tested on rat neonatal cerebellar granule neurons, which involves a 48-hour preconditioning step using the potassium channel blocker 4-aminopyridine (4-AP), at a low (50 microM) and at a higher (2500 microM) concentration (in the presence or absence of the GABA(A) receptor antagonist, bicuculline). 4-Aminopyridine gave extensive protection against a number of stressors (glutamate, NMDA and 3-nitropropionic acid) applied 24 h following the end of the preconditioning period. Blockade of neuronal depolarisation by tetrodotoxin during preconditioning attenuated but did not eliminate protection, whilst co-application with the NMDA receptor blocker MK-801 increased protection. Western blot analysis showed that CREB phosphorylation was significantly increased by the 4-AP preconditioning, although bcl-2 expression was not stimulated. Glutamate induced cell death without significant activation of caspase-3, suggesting that 4-AP preconditioning is effective primarily against necrotic excitotoxicity. Since 4-AP preconditioning affords extensive protection against a range of neurotoxic insults we propose that it could provide the basis for a novel neuroprotective therapy worthy of further investigation.