INVOLVEMENT OF MULTIPLE PHOSPHATIDYLINOSITOL 3-KINASE-DEPENDENT PATHWAYS IN THE PERSISTENCE OF LATE-PHASE LONG TERM POTENTIATION EXPRESSION A. KARPOVA, a P. P. SANNA b AND T. BEHNISCH a * a Leibniz Institute for Neurobiology, Brennecke Strasse 6, 39118 Mag- deburg, Germany b Molecular and Integrative Neuroscience Department, The Scripps Re- search Institute, 10550 N Torrey Pines Road, La Jolla, CA 92037, USA Abstract—The mechanisms responsible for the stabilization and persistence of synaptic plasticity remain largely un- known. In this study, we investigated the time course of the dependence of late-phase long term potentiation of field ex- citatory post-synaptic potential on phosphatidylinositol 3- kinase and its downstream effectors mTOR and AKT. In agreement with our previous results obtained on an early- phase long-term potentiation paradigm we observed that application of a nanomolar concentration of wortmannin (100 nM) 1 h after late-phase long term potentiation induction reversed potentiation completely. However, application of wortmannin 4 h after late-phase long term potentiation induc- tion resulted in a more limited reduction of field excitatory post-synaptic potential suggesting that the dependence of late-phase long term potentiation expression on phosphati- dylinositol 3-kinase decreases over time. Application of a nanomolar concentration of rapamycin (200 nM) during the tetanization paradigm prevented the induction of late-phase long term potentiation consistent with our earlier results. Application of rapamycin 1 h after late-phase long term po- tentiation induction resulted in a less pronounced though significant decline of field excitatory post-synaptic potential. Immunohistological analysis demonstrated that the concen- tration of rapamycin used was effective in inhibiting the phosphorylation of p70S6K at Thr389, the main determinant of its pro-translational activity, and that Thr389 phosphoryla- tion recovered after washout. Lastly, a transient application of Akt inhibitor I (10 M) one hour after late-phase long term potentiation induction also induced a partial although signif- icant reduction of potentiated field excitatory post-synaptic potential that stabilized at a level of approximately 114% of baseline three hours after application, suggesting that AKT also contributes to the stabilization of late-phase long term potentiation expression. These results confirm and extend previous observations that the expression of long term poten- tiation in the CA1 of rat hippocampus involves several elements of the phosphatidylinositol 3-kinase signaling pathway. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: synaptic plasticity, late-phase LTP, phosphatidyl- inositol 3-kinase, hippocampus, CA1. A variety of signal transduction pathways has been shown to be involved in long-term potentiation (LTP)-induction (Bliss and Collingridge, 1993; Sweatt, 2001). However, it remains unclear what intracellular signaling pathways are responsible for the persistence of LTP expression. Sus- tained activation of protein kinases has long been believed to be a key mechanism for LTP maintenance (Malinow et al., 1988; Ling et al., 2002). Recently, the phosphatidylino- sitol 3-kinase (PI3K) has been shown to be required for amygdala fear conditioning (Lin et al., 2001) and for LTP expression in the CA1 area of the hippocampus (Sanna et al., 2002), but see (Opazo et al., 2003). The atypical protein kinase M (PKM) has also been shown to be involved in LTP expression in the CA1 area of the hip- pocampus and in memory formation (Ling et al., 2002). The PI3K family consists of at least four different isozymes that initiate intracellular signal cascades by phos- phorylating the D-3 position of the inositol ring of phosphoi- nositides and are distinguishable by their extracellular activa- tion signals, substrates and expression pattern (Toker, 2000; Cantrell, 2001). An important effector of PI3K is the serine- threonine kinase mTOR (mammalian target of rapamycin; also known as FRAP, RAFT and RAPT) whose activation in turn leads to the phosphorylation and activation of the 70 kDa ribosomal S6 kinase (p70S6K), which promotes the transla- tion of a specific subset of mRNAs (Raught et al., 2001) that increase the translational capacity of neurons and are be- lieved to be involved in the induction of protein synthesis- dependent forms of LTP (Cammalleri et al., 2003). Addition- ally, p70S6K also interacts with the actin cytoskeleton through neurabin (neuronal tissue-specific F actin binding proteins) (Burnett et al., 1998) which might also contribute to its role in synaptic plasticity (Sanna et al., 2002; Raymond et al., 2002; Cammalleri et al., 2003). PI3K-dependent activa- tion of mTOR involves the serine/threonine protein AKT (also known as protein kinase B (Franke et al., 1997)) that is activated by the convergent action of D-3 phosphorylated phosphoinositides and the phosphoinositide-dependent ki- nase-1 (PDK1) (Thomas and Hall, 1997; Scheid et al., 2002). AKT is persistently activated during LTP (Sanna et al., 2002) and thus, we reason that it could also be involved in the expression of LTP. These signal transduction elements rep- resent branch points for other downstream effector mole- cules, so that for example the activity of atypical protein kinases has been shown to depend on PDK1 (Chou et al., 1998; Le Good et al., 1998), which operates both as an on/off switch for the phosphorylation of downstream elements and to prime the activation of other protein kinases (Cantrell, 2001). *Corresponding author. Tel: +49-39-1626-3425; fax: +49-39-1626-3421. E-mail address: behnisch@ifn-magdeburg.de (T. Behnisch). Abbreviations: ACSF, artificial cerebrospinal fluid; fEPSP, field excita- tory postsynaptic potential; L-LTP, late-phase long-term potentiation; LTP, long-term potentiation; mTOR, mammalian target of rapamycin; PBS, phosphate-buffered saline; PDK1, phosphoinositide-dependent kinase-1; PI3K, phosphatidylinositol 3-kinase; p70S6K, 70 kDa ribo- somal S6 kinase; Thr389, threonine 389. Neuroscience 137 (2006) 833– 841 0306-4522/06$30.00+0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.10.012 833