Abstracts

Reversible protein phosphorylation is an important form regulation in neurons. Recently, hippocampal calcineurin immunoreactivity has been shown to be altered in patients with temporal lobe epilepsy. Calcineurin, a calmodulin-stimulated phosphatase is involved in several aspects of gamma-aminobutyric acid (GABA) function, including the regulation of GABA transport and the desensitization of GABA A receptors. Calcineurin immunoreactivity and content have also been shown to increase markedly in kindled rat brain. Additionally, calcineurin inhibitors have been shown to prevent the development of kindling and mossy fiber sprouting in the hippocampus. Because neuronal injury during prolonged seizure activity may occur via mechanisms of increased neurotransmitter release and membrane depolarization, we assessed the protective effect of calcineurin inhibitors against CA1 depolarization-induced neuronal injury in the hippocampal slice.
Using paired rat hippocampal slices, we monitored the CA1 orthodromic and antidromic population spike (PS) amplitude during depolarization injury with and without calcineurin inhibitor treatment. To induce depolarization injury, slices were exposed to 25 mM KCl for 8 mins. Treatment with calcineurin inhibitors was begun 30 minutes prior to KCl exposure and continued for the first 15 mins. of recovery.
Cyclosporin A, a calcineurin inhibitor, provided robust neuroprotection of CA1 PS amplitude in hippocampal slices subjected to depolarization-induced injury. Slices exposed to 25 mM KCl demonstrated rapid loss of evoked response with a mean CA1 orthodromic and antidomic recovery of only 12% [plusmn] 2 and 14% [plusmn] 4, respectively. Treatment with Cyclosporin A (25 uM) provided significant protection against this depolarization with CA1 orthodromic and antidromic PS amplitude recovering to 90% [plusmn] 3 and 92% [plusmn] 2. Treatment with FK-506, a more specific calcineurin inhibitor, provided similar protection of CA1 PS amplitude in hippocampal slices subjected to depolarization injury. At a FK-506 concentration of 1 uM, CA1 orthodromic and antidromic PS amplitude recovered to 93% [plusmn] 3 and 94% [plusmn] 6, compared to unmedicated slices which recovered to only 12% [plusmn] 1 and 15% [plusmn] 2, respectively. Treatment with rapamycin (1uM and 5 uM), an immunosuppressant that has a similar structure to FK-506, but does not inhibit calcineurin failed to provide protection against depolarization injury.
These studies demonstrate that inhibitors of calcineurin provide neuroprotection against CA1 depolarization injury. In addition, these data suggest that the use of calcineurin inhibitors may be a useful strategy in the prevention of brain injury during status epilepticus.
[Supported by: VA Research Service and the UCLA Brain Injury Research Center.]