The use of cultured hippocampal slices for studies of calpainmediated pathogenesis was investigated. Breakdown products (BDPs), which result from proteolysis of spectrin by calpain I, were assayed with BDP-specific antibodies developed against peptide sequences on either side of the calpain cleavage site. The antibodies recognized either amino- or carboxy-terminal BDPs (147-kD BDPN and 152-kD BDPC, respectively). Various pathogenic manipulations, including trimethyltin, certain snake venoms and agonists for excitatory amino acid receptors, were found to cause rapid and pronounced increases in the proteolytic fragments. These effects were selective, ie, chemicals or toxins directed at nonglutamatergic neurons had little effect on BDP concentrations. Transient accumulations of spectrin frag-ments were obtained with brief applications of N-methyl-D-aspartate; longer infusions resulted in lasting increases. Results similar to these have been observed in vivo with ischemic episodes of varying duration. Agonists of the a-amino-3-hy-droxy-5-methylisoxazole-4-propionic acid subclass of glutamate receptors produced significant increases in spectrin proteolysis; however, prolonged exposure of the slices to centrally active drugs that enhance the currents passed by the receptors did not. The sensitivity, selectivity and temporal properties of the proteolytic response support the idea that cultured slices can be used to analyze the events leading to and following from calpain activation in the adult brain.

The membrane cytoskeletal protein spectrin is a preferred substrate ofthe calcium-activated protease calpain(Boivin et at., 1990; Siman et at., 1984), particularly in the presence of calmodulin(Seubert et at., 1987). Moreover, cleavage of spec-trim by calpain results in a large and relatively stable BDP (Siman et at., 1984), which is present in only low concentrations in the normal brain. The presence of the BDP thus provides a marker for sites ofcalpain activation and has been used for this purpose to test the idea that the protease contributes to various of neuropathologic conditions. Biochemical experiments that used antibodies against spectrin showed that BDP accumulates after a variety of pathogenic manipulations, including ischemia(Seubert et at., 1989a), toxins (Seubert et at., 1989b), excitotoxins(Seubert et at., 1988b; Siman and Noszek, 1988), denervation(Seubert et at., 1988a) and genetic abnormalities(Seubert et at., 1990) in