248 Brain Research, 567 (1991) 248-252 (~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$03.50 BRES 17259 Increased synthesis of two polypeptides in area CA1 of the hippocampus in response to repetitive electrical stimulation Noriaki Uenishi, Tracey J. Shors, Caleb E. Finch, Nancy R. Nichols and Richard F. Thompson Neurosciences Program and the Neurogerontology Division, University of Southern California, Los Angeles, CA 90089 (U.S.A.) (Accepted 30 July 1991) Key words: Protein synthesis; Electrophoresis; Polyacrylamide gel; CA1; Long-term potentiation; Hippocampal slice; Polypeptide Changes in the pattern of newly synthesized polypeptides were investigated in the in vitro hippocampal slice following exposure to repetitive stimulation with and without the induction of long-term potentiation. Using [35S]methionine labeling of polypeptides and two-dimensional gel electrophoresis, we detected an increase in the rate of synthesis of two polypeptides (48 kDa and 89 kDa) in CA1 in response to repetitive stimulation of the Schaffer collaterals. The synthesis of the 48 kDa polypeptide (pI - 6.6) increased 240% in response to high-frequency stimulation (100 I-Iz) relative to the same protein from unstimulated slices (n = 14), and increased 220% in response to low-frequency stimulation (1 Hz) (n = 5). Blockade of the N-methyl-r~-aspartate (NMDA) receptor induced the protein 180%, with no further increase following tetanic stimulation. An 89 kDa doublet (pI - 6.8) increased 150% following high-frequency and 140% following low-frequency stimulation. Blockade of the NMDA receptor increased this protein as well (180% of the unstimulated control) and no further increase was observed following high-frequency stimulation. Based on physicochemical and electrophysioiogicalproperties, these proteins are not identi- fiable as any of those previously associated with long-term potentiation or repetitive electrical stimulation. INTRODUCTION Repetitive stimulation delivered to afferent in the hip- pocampus produces a transient increase in synaptically- mediated responses 1. In 1973, Bliss and Lomo 5 deter- mined that acute stimulation delivered at high frequencies resulted in an enduring increase in synaptic efficacy: long-term potentiation (LTP). Because it is long- lasting, associative in nature 19, and strengthened by rep- etition 4, the phenomenon possesses several synaptic properties presumed necessary for long-term storage of information 16. Although most studies focus on mechanisms through which high-frequency (HF) stimulation induces potenti- ation, others have addressed cellular mechanisms under- lying long-term maintenance of the response. Of these mechanisms, structural changes have been given strong consideration and support 2'7'12A7'3°, as well as long-term modifications in existing subcellular components or the possible synthesis of new ones 16'22. Since de novo protein synthesis is required for maintenance of LTP in both the dentate gyrus and CA111'26'29, it has been hypothesized that tetanic stimulation increases the synthesis of already existing proteins and/or induces previously silent genes. To test this hypothesis, we examined protein synthesis in response to LTP as evoked by tetanic stimulation. Using in vitro hippocampal slices, area CA1 was exposed to repetitive stimulation and radiolabeled. Protein patterns were compared by two-dimensional electrophoresis be- tween hippocampal slices with and without the induction and expression of LTP. MATERIALS AND METHODS Long-Evans male rats (n = 14, 275-325 g) were obtained from Simonsen (San Francisco, CA) and kept for at least 7 days under standard laboratory conditions. Following decapitation at 10 00 h, in vitro hippocampal slices were prepared according to previously reported methods28. Following 1 h of incubation, a glass mieropi- pette containing 2 M NaC1 was positioned in the dendritic region of the CA1 pyramidal cells to record excitatory post-synaptic poten- tials (EPSPs) and a concentric bipolar stimulating electrode was positioned in the Schaffer collaterals (Fig. 1). Once a stable EPSP of approximately 1-2 mV was obtained, pulse duration was in- creased from 0.1 ms until a slight depolarization was observed (usually 0.18 ms). Slices were then exposed to either 1 s of HF tetanization at 100 Hz or 100 s of low-frequency (LF) stimulation at 1 Hz and monitored for 30 rain. The response to single-pulse stim- ulation was monitored for 30 min. The stimulating electrode was then repositioned at 9 adjacent locations along the trajectory of the Schaffer collaterals, each time delivering an HF tetanus. Frequency, intensity, and pulse width were the same as the initial tetanus. Slices remained in the chamber for an additional 30 rain and were then removed for incubation in [35S]methionine. For each animal, addi- tional slices were incubated in the slice chamber and removed simultaneously as unstimulated controls (UC). For each of 5 rats, Correspondence: T.J. Shors, Department of Psychology, Green Hall, Princeton University, Princeton, NJ 08544-1010, U.S.A.