Journal of Ni’,,,orlie;i,,.s nv I.ippi ncoti—-Ras en I’ub!isher~. Phitadetpilia 1997 tnteniationa! Society ir Neuroclieniistry NMDA Receptor—Mediated Regulation of AMPA Receptor Properties in Organotypic Hippocampal Slice Cultures David M. Gellerman, Xiaoning Bi, and Michel Baudry Neuroscience Progratn, Unir’ersitv ~?t Southern California, Los Angeles, California, U.S.A. Abstract: Activation of the calcium-dependent protease calpain has been proposed to be a necessary step in the formation of long-term potentiation (LTP) in the hip- pocampus, and stimulation of N-methyl-D-aspartate (NMDA) receptors leads to an increase in intracellular calcium concentration, calpain activation, proteolysis of cytoskeletal elements, and modification of ci-amino-3-hy- droxy-5-methyl-4-isoxazole propionic acid (AMPA) re- ceptor properties. In the present study, we evaluated the effects of NMDA treatment of cultured hippocampal slices on the properties of AMPA receptors. Cultured hip- pocampal slices were treated with NMDA (100 pM) for 15 mm and [ 3H]AMPAbinding to membrane fractions was measured. NMDA-treated slices exhibited an in- crease in both “high-affinity” and “low-affinity” [3H]- AMPA binding, with smaller changes in 6-cyano-7- nitro[3H]quinoxaline-2,3-dione binding. The increase in [3HIAMPA binding was significantly reduced by preincu- bation of cultures with calpain inhibitor or calpeptin (100 pM). Furthermore, NMDA exposure decreased the num- ber of GIuR1 subunits of AMPA receptors detected by an antibody against the C-terminal domain of the subunit in western blots and resulted in the formation of a lower molecular weight species detected by an antibody against the N-terminal domain. Both effects were com- pletely prevented by calpain inhibitors. These results indi- cate that NMDA receptor activation produces calpain ac- tivation and complex modifications of AMPA receptor properties, which could be involved in NMDA receptor— mediated changes in synaptic efficacy. Key Words: Hip- pocampus — Calpain — Receptor — Plasticity — Gluta- mate—Calcium. J. Neurochem. 69, 131—136 (1997). Long-term potentiation (LTP) is a long-lasting in- crease in synaptic transmission that has been suggested to represent a cellular mechanism of learning and memory (for review, see Bliss and Collingridge, 1993). In CAl and dentate gyrus of the hippocampus, LTP induction is dependent on N-methyl-D-aspartate (NMDA) receptor stimulation that leads to an influx of calcium ions and the activation of a cascade of biochemical events (Lisman, 1989; Baudry and Massi- cotte, 1992; Artola and Singer, 1993; Bliss and Col- lingridge, 1993; Bear and Malenka, 1994; Maren and Baudry, 1995). Whereas LTP induction requires acti- vation of NMDA receptors. LTP expression and main- tenance are presumed to be due in part to modifications of r-arnino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In particular, the waveform of extracell ularly recorded excitatory postsynaptic po- tentials (EPSPs) is modified in a way best interpreted by a change in AMPA receptor/channel kinetics (Ambros-Ingerson et al., 1991; Ambros-Ingerson and Lynch, 1993). The effects on EPSPs of the chaotropic ion, perchlorate, which enhance agonist affinity for the AMPA receptors. are also decreased after LTP induc- tion (Shahi and Baudry, 1992): in a similar manner, the enhancing effects of aniracetam on AMPA recep- tor—mediated EPSPs are decreased after LTP induction (Staubli et al., 1990). Finally, the recent discovery that a significant fraction of synapses are functionally silent due to the lack of functional AMPA receptors clearly supports that LTP could be due to the un- masking or ‘‘activation” of~’’s ilent’’ AMPA receptors (Isaac et al., 1995; Liao ci al., 1995). Consistent with this idea, Maren et al. (1993) showed that [1H]AMPA binding is increased in the molecular layer of the den- tate gyrus after LTP induction and that the increased binding correlates positively with the increase in syn- aptic response (for a different result, however, see Kessler et al., 1991 ). Received January 7. 1997: revised manuscript received February 20, 1997, accepted February 20, 1997. Address correspondence and reprint requests to Dr. M. Baudry at HNB 309. University ot Southern California. Los AngeIe~, CA 90089-2520, U.S.A. Preliminary results of this siLidy were presented at the Society for Neuroscience Meeting, 1995, San Diego. CA. U.S.A. Abbreviations used: C—Ab and N-Ah. antibody against C- and N- terminal domains, respectively. al GluR I subunits of AMPA re- ceptors: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid: CNQX. 6-cyano-7-nitroquinoxaline-2.3-dione: DMSO. di- methyl sulfoxide: EPSP. excitatory postsynaptic potential: KSCN. potassium thiocyanate: LTP. long-term potcntiation: MEM. mini- niulli essential medium: NMDA. N—rnethyl—o—aspartate: PAGE, poly- acrylamide gel electrophoresis: .SBDP. spectrin breakdown product: SDS. sodium dodecyl sulfate: TBS. Tris-huffercd saline. 131