Effects of positive AMPA receptor modulators on calpain-mediated spectrin degradation in cultured hippocampal slices
Introduction
Calpain is a neutral Ca2+-activated cysteine protease present in postsynaptic structures where it has a wide range of substrates (Lynch and Baudry, 1987, Goll et al., 2003). This protease is essential for the induction of synaptic long-term potentiation (LTP) (Oliver et al., 1989, Muller et al., 1995, Bednarski et al., 1995, Vanderklish et al., 1996, Caba et al., 2002). One of the preferred calpain substrates in brain is brain spectrin; brain spectrin, initially referred to as fodrin, is a prominent component of the membrane-associated cytoskeleton. Substantial evidence implicates spectrin in the regulation of cellular morphology in red blood cells and muscle sarcomeres, and in the clustering of acetylcholine receptors at neuromuscular junctions (Bloch and Morrow, 1989). Distinct isoforms of spectrin are expressed in neurons (Hesketh et al., 1983), and calpain-mediated spectrin proteolysis has been associated with marked and lasting effects on the morphology and physiology of synapses (Lynch and Baudry, 1987, Bednarski et al., 1995). Calpain-mediated spectrin degradation results in the formation of 145–150 kDa breakdown products, and represents site-specific and activity-dependent markers of synaptic activity (Lynch and Baudry, 1987, Saido et al., 1993, Dosemeci and Reese, 1995, Vanderklish et al., 2000, Zakharov and Mosevitsky, 2001). It has been proposed that calpain activation and spectrin degradation could reflect rapid and long lasting reorganizations of postsynaptic structures under physiological or pathological conditions (Masliah et al., 1990, Lee et al., 1991, Vanderklish and Bahr, 2000, Neumar et al., 2001, Caba et al., 2002).
Glutamate release in the brain elicits a fast postsynaptic response mediated by the opening of AMPA-type glutamate receptor (AMPAr) channels. This response depends on binding of glutamate to the receptor and can be positively modulated by a group of allosteric effectors of these receptors called ampakines. Ampakines have been shown to bind to the AMPA receptors at a site distinct from the glutamate site and to increase AMPAr-mediated current in acute and cultured hippocampal slices and in vivo, to improve performance on several behavioral tasks when administered to animals, and to enhance memory encoding in humans (Ingvar et al., 1997; Hampson et al., 1998a, Hampson et al., 1998b; Arai et al., 2000, Suppiramaniam et al., 2001). Furthermore, several ampakines have been shown to facilitate LTP induction in hippocampal slices and in vivo, and this effect has been linked to their cognitive enhancing properties. In view of the proposed role of calpain in LTP induction, it was of interest to determine the effect of ampakines on calpain activation and spectrin degradation in hippocampus. Our results indicate that treatment with some, but not all, positive modulators of AMPA receptors results in calpain activation and spectrin degradation.
Section snippets
Hippocampal slice culture
Ten to 13 day-old rat pups were obtained from Jackson Laboratories (Bar Harbor, Maine) and used to prepare hippocampal slice cultures as previously described (Stoppini et al., 1991). Briefly, animals were decapitated and the brains were dissected, trimmed, and 400 μm thick hippocampal slices were cut using a McIllwain tissue slicer (Fotodyne Inc., New Berlin, WI). Slices were plated onto Millipore insert membranes, placed in six-well culture plates, and fed with regular slice culture medium (50%
Treatment of cultured hippocampal slices with CX614 increases spectrin degradation
Calpain is activated by increased intracellular Ca2+ concentrations, and calpain-mediated spectrin degradation specifically produces a doublet band of 145–150 kDa. We monitored this characteristic band as an indicator of calpain activation. Brain membrane fractions treated with 2 mM CaCl2 (see Section 2) were used as a positive control for calpain-mediated spectrin degradation (Massicotte et al., 1990, Lu et al., 2001). Hippocampal slices were incubated with the ampakine CX614 (50 μM for 48 h) or
Discussion
The present results indicate that treatment of cultured hippocampal slices with some but not all positive AMPA receptor modulators was associated with relatively rapid and sustained calpain activation, as evidenced by the accumulation of spectrin breakdown products identical to those generated by calpain-mediated spectrin degradation (Siman et al., 1984, Bahr et al., 1995, Bi et al., 1996). In addition, a calpain inhibitor prevented the accumulation of spectrin breakdown products. CX614 is a
Acknowledgements
We would like to thank Dr. Jihua Liu and Miss Gabriela Saavedra for excellent technical assistance. This research was supported by Grant P01NS045260-01 (PI: Dr. C.M. Gall) from NINDS.
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