Embed Size (px)
Citation preview
Phosphatidylinositol 3-Kinase Activation Is Required forStress Protocol-induced Modification of HippocampalSynaptic Plasticity*□S
Received for publication, August 20, 2007, and in revised form, December 4, 2007 Published, JBC Papers in Press, December 5, 2007, DOI 10.1074/jbc.M706954200
Ping-Chun Yang‡, Chih-Hao Yang‡, Chiung-Chun Huang‡, and Kuei-Sen Hsu‡§1
From the ‡Department of Pharmacology, College of Medicine, and the §Center for Gene Regulation and Signal TransductionResearch, National Cheng Kung University, Tainan 701, Taiwan
Stress dramatically affects the induction of hippocampal syn-aptic plasticity; however, the molecular details of how it does soremain unclear. Phosphatidylinositol 3-kinase (PI3K) signalingplays a crucial role in promoting neuronal survival and neuro-plasticity, but its role, if any, in stress-induced alterations of longterm potentiation (LTP) and long term depression (LTD) isunknown. We found here that inhibitors of PI3K signalingblocked the effects of acute restraint-tail shock stress protocolon LTP and LTD. Therefore, the purpose of the present study isto explore the signaling events involvingPI3K in terms of its rolein mediating stress protocol-induced alterations of LTP andLTD. We found that stress protocol-induced PI3K activationcan be blocked by various inhibitors, including RU38486 forglucocorticoid receptors, LY294002 for PI3K, and DL-2-ami-no-5-phosphonopentanoic acid for N-methyl-D-aspartatereceptors or brain-derived neurotrophic factor antisense oli-gonucleotides. Also, immunoblotting analyses revealed thatstress protocol induced a profound and prolonged phospho-rylation of numbers of PI3K downstream effectors, including3-phosphoinositide-dependent protein kinase-1, protein kinaseB, mammalian target of rapamycin (mTOR), p70 S6 kinase, andeukaryotic initiation factor 4B in hippocampal CA1 homoge-nate, which was prevented by the PI3K inhibitor pretreatment.More importantly, we found that stress protocol significantlyincreased the protein expression of dendritic scaffolding pro-tein PSD-95 (postsynaptic density-95), which is known to beinvolved in LTP and LTD, in an mTOR-dependent manner.These results identify a key role of PI3K signaling in mediatingthe stress protocol-induced modification of hippocampal syn-aptic plasticity and further suggest that PI3K may do so byinvoking the protein expression of PSD-95.
Stress has long been recognized to interfere with manyaspects of brain functions through its activation of the hypo-
thalamus-pituitary-adrenal axis. The hippocampal formation,an important structure involved in forming declarative, spatial,and contextual memory (1, 2), is a brain area particularly sensi-tive and vulnerable to stress and stress hormones (3). Stressdramatically affects hippocampal long term synaptic plasticity(3–7).Work in our laboratory and other laboratories has shownthat a brief experience of acute restraint-tail shock stress pro-tocol can impair high frequency stimulation (HFS)2-inducedlong term potentiation (LTP) (8–14), whereas low frequencystimulation (LFS)-induced long term depression (LTD) can befacilitated in the CA1 region of the hippocampus (11–14).However, the molecular mechanisms underlying the alter-ations of the inducibility of LTP and LTD by a stress protocolhave only begun to be elucidated.Phosphatidylinositol 3-kinases (PI3Ks) are a large family of
intracellular signal transducers that catalyze the phosphoryla-tion of theD-3 position of the inositol ring of phosphoinositides(15). The PI3K pathway is classically implicated in the regula-tion of cell growth, survival, proliferation, and movement (16–18). In the mammalian brain, in addition to its functions inneuronal survival and differentiation (19), a number of recentstudies have implicated PI3K in synaptic plasticity and learningand memory. For example, it has been shown that activation ofPI3K is required for the expression of LTP in the dentate gyrus(20) and CA1 region of the hippocampus (21, 22). Moreover,pharmacological inhibition of PI3K significantly impairedinhibitory avoidance (23), spatial learning (24–25), consolida-tion (26), retrieval, and extinction of fear-associated memory(27). PI3Kmay contribute to regulate LTP andmemory forma-tion by facilitating AMPA receptor insertion into the postsyn-aptic membrane (28), activating mitogen-activated proteinkinase/extracellular signal-related kinase (MAPK/ERK) (26,29) and initiating de novo protein synthesis (30).It has been proposed that stress may affect subsequent hip-
pocampal synaptic plasticity by sharing some aspects of thesame mechanisms underlying the support of LTP (4, 7, 10, 31,32). Because PI3K is critical for the expression of LTP, wehypothesized that PI3K might also play a role in mediating the
* This work was supported by National Health Research Institute Grant NHRI-EX96-9618NI and National Science Council, Taiwan, Grant NSC96-2752-B-006-002-PAE. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S4.
1 To whom correspondence should be addressed: Dept. of Pharmacology,College of Medicine, National Cheng Kung University, 1 University Rd.,Tainan 701, Taiwan. Tel.: 886-6-2353535 (ext. 5498); Fax: 886-6-2749296;E-mail: [email protected].
2 The abbreviations used are: HFS, high frequency stimulation; LTP, long termpotentiation; LTD, long term depression; LFS, low frequency stimulation; PI3K,phosphatidylinositol 3-kinase; S6K, S6 kinase; MAPK, mitogen-activated pro-tein kinase; ERK, extracellular signal-regulated kinase; aCSF, artificial cerebro-spinal fluid; TBS, Tris-HCl buffer solution; BDNF, brain-derived neurotrophicfactor; APV, DL-2-amino-5-phosphonopentanoic acid; mTOR, mammalian tar-get of rapamycin; eIF4B, eukaryotic translation initiation factor 4B; MEK, mito-gen-activated protein kinase/extracellular signal-regulated kinase kinase.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 5, pp. 2631–2643, February 1, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2631
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
effects of stress on hippocampal synaptic plasticity. In thisstudy, we show that PI3K signaling is activated in the hip-pocampal CA1 region following an acute restraint-tail shockstress protocol, and its activity is required for the effects of sucha stress protocol on subsequent LTP and LTD induction.
EXPERIMENTAL PROCEDURES
Animals—Healthy adult male Sprague-Dawley rats weighing250–300 g were used in these experiments. Animal care wasconsistent with the guidelines set by the Laboratory AnimalCenter of National Cheng Kung University. All experimentswere approved by the National Cheng Kung University Institu-tional Animal Care and Use Committee governing the partici-pating laboratories. Animals were housed in groups of four in avivarum with a 12-h light-dark cycle (lights on at 7:00 a.m.),50–60% humidity, and free access to food and water. Ambienttemperaturewas controlled at 24 °C. All experimentswere con-ducted during the light phase of the cycle.Cannulation—Rats were cannulated as described previously
(13, 14). Briefly, rats were anesthetized with sodium pentobar-bital (50mg/kg, intraperitoneally), and 22-gauge cannulas wereimplanted bilaterally toward 1.0 mm above the stratum pyra-midale of the dorsal CA1 region of the hippocampi (coordi-nates: anterior, �4.0; lateral, �4.0; ventral, 2.6), in accordancewith the description by Paxinos andWatson (33). The cannulaswere fixed to the skull with dental cement. The animals wereallowed to recover from surgery for 7 days before the experi-ments started. To deliver the various drugs, we used a 26-gaugeinfusion cannula connected by a polyethylene tube to a 5-�lmicrosyringe. Bilateral injections were performed using aninfusion pump (CAM/100; CAM Microdialysis, Solna, Swe-den). A total volume of 0.5 �l was infused into each side over 1min, and the infusion cannula was kept in place for an addi-tional 2 min to minimize backflow of the injectant. Only slicesfrom animals with accurate cannula placements, as verifiedmicroscopically by the cannula tips located within the hip-pocampal CA1 region, were taken for electrophysiologicalrecordings or biochemical measurements. Although infusionsspread with a radius of �1.2–1.4 mm (as estimated by pilotexperiments using a 0.5-�l solution of 4% methylene blue insaline), wemade analyses fromneuronswithin a radius 0.6–0.8mm from the cannula tips to obtain better effects of infuseddrugs. We never observed any effects of cannulation itself orvehicle artificial CSF (aCSF; 10% Me2SO) injection.Stress Protocol—Animals were allowed to acclimate to the
laboratory for 7 days before any experimental manipulation.Physical stresswas evoked by 60 tail shocks (1mA for 1 s, 30–90s apart) while restrained in a Plexiglas tube, as described previ-ously (11, 13). This restraint-tail shock stress protocol, adaptedfrom the “learned helplessness” paradigm (i.e. animals areexposed to an unpredictable and uncontrollable aversive stim-ulus) (34), has been shown previously to reliably induce behav-ioral (freezing immobility, piloerection, urination, and defeca-tion) and endocrine (elevated serum corticosterone levels)signs of stress in rats (11, 13). Control animals remained in theirhome cages. Control and stressed animals did not have avail-able food and water during the experiments.
Plasma Corticosterone Assay—Blood samples were obtainedfrom the tail before and after stress and immediately centri-fuged at 1,000 � g, and plasma was separated and stored at�20 °C. Plasma corticosterone levels were determined byradioimmunoassay, as described previously (13).Slice Preparation and Electrophysiology—Immediately after
the stress protocol, animals were deeply anesthetized with hal-othane and decapitated with a guillotine, and hippocampalslices (400�mthick)were preparedwith a vibrating blade slicer(VT1000S; Leica Microsystems, Wetzlar, Germany). The sliceswere placed in a humidified interface-type holding chamber ofaCSF oxygenated with 95% O2, 5% CO2 and kept at room tem-perature for at least 1 h before electrophysiological recording.For extracellular recordings, a single slice was transferred to asubmersion-type recording chamber continually perfused with30–32 °C oxygenated aCSF solution (117 mM NaCl, 4.7 mMKCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 1.2 mMNaH2PO4, 11 mM glucose). Extracellular field potential record-ings were carried out using an Axoclamp-2B amplifier (AxonInstruments, Union City, CA). The responses were low pass-filtered at 2 kHz, digitally sampled at 5–10 kHz, and analyzedusing pCLAMP software (version 8.0; Axon Instruments). Theevoked postsynaptic responses were induced in CA1 stratumradiatum by stimulation (0.02 ms duration) of Schaffer collat-eral/commissural afferents at 0.033 Hz with a bipolar stainlesssteel stimulating electrode. The stimulation strength was set toelicit a response having an amplitude that was 30–40% of themaximum spike-free response. Field excitatory postsynapticpotentials (fEPSPs) were recorded with a glass pipette filledwith 1 MNaCl (2–3megaohms resistance), and the fEPSP slopewas measured from �20–70% of the rising phase using a leastsquares regression (13). The LTPwas induced byHFS at the testpulse intensity, consisting of two 1-s trains of stimuli separatedby an intertrain interval of 20 s at 100 Hz. LTD was inducedusing a standard protocol of 900 stimuli at 1 Hz (LFS). Thestimulation intensity during LFS application was the same asthe test pulse intensity.Preparation of Synaptoneurosomes—Synaptoneurosome
fractionswere prepared as described previously (35, 36). Briefly,the microdissected CA1 regions were homogenized in ice-coldCa2�-, Mg2�-free buffer (50 mM HEPES, 100 mM NaCl, and 3mM KAc, pH 7.4) and was centrifuged at 2,000 � g for 1 min.Supernatants were passed through two 100-�m nylon meshfilters, followed by a 5-�m pore filter. The filtrate was thencentrifuged at 1,000 � g for 10 min and then was gently resus-pended with the same buffer at a protein concentration of 2mg/ml.Immunohistochemistry—Animals were deeply anesthetized
with sodium pentobarbital (100 mg/kg, intraperitoneally) andtranscardially perfused with physiological saline and subse-quently with fresh 4% paraformaldehyde with 1.5% picric acidin 0.1 M phosphate buffer, pH 7.4 (4 °C). After the perfusion,brains were removed and postfixed in the same fixative for 4 hand then transferred into 30% sucrose in 0.1 M phosphate bufferovernight. Serial free floating sections (15-�m thickness) wereobtained using a sliding microtome. For immunostaining, freefloating sectionswere incubated for 30minwith 3%H2O2 in 0.1M phosphate buffer to remove endogenous peroxidase activity,
PI3K in Stress Protocol Effects
2632 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 5 • FEBRUARY 1, 2008
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
washed in phosphate buffer three times, and then kept byblocking solution containing 5% normal goat serum and 0.1%Triton X-100 for 30 min at room temperature. The sectionswere incubated overnight at 4 °C with primary antibodies thatrecognize phosphorylated Akt at Ser473 (1:1000; Cell SignalingTechnologies, Beverly, MA), MAP-2 (microtubule-associatedprotein-2; 1:100; Cell Signaling Technologies), or NeuN (neu-ron-specific nuclear protein; 1:100; Chemicon, Temecula, CA).After washing, secondary antibodies conjugated to the fluores-cent markers Alexa-488 and Alexa-568 (1:400; MolecularProbes, Inc., Eugene, OR) were applied to sections for 2 h. Sec-tions were then washed, mounted on slides, and coverslippedwithVectashieldmountingmedium (Vector Laboratories, Bur-lingame, CA), and images were taken using an upright fluores-cence microscope (BX51; Olympus, Tokyo, Japan). Specificstaining was abolished by omission of primary antibody.Western Blotting and Immunoprecipitation—For each
experimental group, homogenates from at least three sliceswere pooled. The microdissected subregions were lysed in ice-cold Tris-HCl buffer solution (TBS; pH 7.4) containing a mix-ture of protein phosphatase and proteinase inhibitors (50 mMTris-HCl, 100 mMNaCl, 15 mM sodium pyrophosphate, 50 mMsodium fluoride, 1 mM sodium orthovanadate, 5 mM EGTA, 5mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 �M micro-cystin-LR, 1 �M okadaic acid, 0.5% Triton X-100, 2 mM benza-midine, 60 �g/ml aprotinin, and 60 �g/ml leupeptin) to avoiddephosphorylation and degradation of proteins and groundwith a pellet pestle (Kontes Glassware, Vineland, NJ). Sampleswere sonicated and spun down at 15,000 � g at 4 °C for 10 min.The supernatant was then assayed for total protein concentra-tion using a Bio-Rad Bradford protein assay kit. Each samplewas separated in 7.5 or 10% SDS-polyacrylamide gel. Followingthe transfer onnitrocellulosemembranes, blotswere blocked inTBS containing 3% bovine serum albumin and 0.01% Tween 20for 1 h and then blotted for 2 h at room temperature with anti-bodies that recognize PSD-95 (1:2000; Upstate Biotechnology,Inc., Lake Placid, NY), phosphorylated Akt at Ser473 (1:1000;Cell Signaling Technologies), phosphorylated Akt at Thr308(1:1000; Cell Signaling Technologies), phosphorylatedERK1/2 MAPK (1:1000; Cell Signaling Technologies), phos-phorylated PDK1 (1:1000; Cell Signaling Technologies),phosphorylated mTOR (1:1000; Cell Signaling Technologies),phosphorylated p70 S6 kinase (S6K) (1:2000; Cell SignalingTechnologies), or phosphorylated eukaryotic translation initi-ation factor 4B (eIF4B) (1:1000; Cell Signaling Technologies). Itwas then probed with horseradish peroxidase-conjugated sec-ondary antibody for 1 h and developed using the ECL immuno-blotting detection system.The immunoblots using phosphoryl-ation site-specific antibodies were subsequently stripped andreprobed with the following antibodies: anti-Akt antibody(1:1000), anti-ERK1/2 MAPK antibody (1:1000), anti-PDK1antibody (1:1000), anti-mTOR (1:1000), anti-p70S6K (1:500),or anti-eIF4B antibody (1:1000) that were purchased from CellSignaling Technologies. Immunoprecipitation was carried outessentially as previously reported (37). Homogenates (�500 �gof protein) from control or 20 min after LTP induction sliceswere incubated with anti-NR2B antibody (1:500; Santa CruzBiotechnology, Inc., Santa Cruz, CA) in TBS for 4 h at 4 °C. The
antibody-protein complexes were then pelleted with proteinA-Sepharose beads. The complex was isolated by centrifuga-tion and washed three times. Proteins eluted from the beadswere subjected to 7.5% SDS-polyacrylamide gel and immuno-blotting for anti-phosphotyrosine antibody (1:2000; Cell Sig-naling Technologies). For sequential probing of the samemem-brane, the membrane was stripped of antibody and reprobed.Immunoblots were analyzed by densitometry using Bio-profilBioLight PC software. Only film exposures that were in thelinear range of the ECL reaction were used for quantificationanalysis.Brain-derived Neurotrophic Factor (BDNF) Immunoassay—
The BDNF levels were measured with a conventional two-siteenzyme-linked immunosorbent assay system according to themanufacturer’s protocol (Promega, Charbonniere, France)(13). Briefly, each hippocampuswasweighted and then homog-enized in 20 volumes of ice-cold lysis TBS, and the homoge-nates were centrifuged for 20 min at 15,000 � g at 4 °C. Tissuesamples were acidified for 15 min and neutralized, because thistreatment has been reported to aid dissociation of boundtrophic factors from their receptors (38). BDNF concentrationswere calculated from regression analysis of human recombi-nant BDNF standard curve run in each assay and expressed asnormalized per gram of tissue wet weight.Drugs—The sequences of BDNF antisense and sense oligo-
nucleotides were 5�-TCTTCCCCTTTTAATGGT-3� and5�-ACCATTAAAAGGGGAAGA-3�, respectively, which cor-respond to all splice variants of BDNF (39). LY294002,LY303511, wortmannin, RU38486, RU28318, DL-2-amino-5-phosphonopentanoic acid (APV), and U0126 were purchasedfrom Tocris Cookson (Bristol, UK); K252a, K252b, and U0124were obtained from Calbiochem; rapamycin and ascomycinwere obtained from Sigma.Drug Treatment—Stock solutions of LY294002, LY303511,
wortmannin, K252a, K252b, rapamycin, ascomycin, U0126,and U0124 were prepared by initially dissolving in Me2SO andthen diluting in aCSF before infusion. Drug and vehicle solu-tions had a final concentration of 10%Me2SO. Drug doses ofLY294002 (10 mM), LY303511 (10 mM), wortmannin (7.3mM), K252a (0.5 �g/�l), K252b (0.5 �g/�l), rapamycin (1ng/�l), ascomycin (1 ng/�l), U0126 (2 �g/�l), and U0124 (2�g/�l) were selected on the basis of published studies (13, 27,40, 41) and pilot experiments in our laboratory. APV was dis-solved in ACSF to a concentration 25 mM, which has beenshown to be effective to completely abolish the novelty-inducedretrograde amnesia (42). The BDNF antisense and sense oligo-nucleotides were dissolved in ACSF and infused into the lateralventricle at a dose of 50 pmol twice per day for 5 days (13).RU38486 was administered 30min before stress by intraperito-neal injectionwith a dose of 40mg/kg, which has been shown tobe effective to completely abolish the glucocorticoid receptor-mediated functions (43). Control animals were injected withthe vehicle propylene glycol for RU38486.Data Analysis—Average data are presented as mean � S.E.
Student’s t tests were used to calculate significance when twogroups were compared. When more than two groups werecompared, a one-way analysis of variance was used, and Bon-ferroni multiple comparisons were used to compare the signif-
PI3K in Stress Protocol Effects
FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2633
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
icance between groups. The number of animals used is indi-cated by n. Probability (p) values of less than 0.05 wereconsidered statistically significant.
RESULTS
PI3K Is Activated after the Restraint-Tail Shock StressProtocol—In response to stress protocol, significantly higherlevels of circulating corticosterone concentrations (126.5 � 7.1ng/ml versus 9.2 � 2.3 ng/ml in basal conditions, n � 10; p �0.05) were observed, confirming that our experimentalrestraint-tail shock stress protocol activates hypothalamus-pi-tuitary-adrenal responses as described previously (11, 13). Toinvestigate whether such a stress protocol affects PI3K activity,we indirectly monitored the activation of PI3K by measuringthe phosphorylation of its downstream target Akt on Ser473using Western blot analysis (44). We found that phosphoryla-ted Akt was readily detected in slices from control rats, andlevels were greatly increased �70% in slices from stressed rats(Fig. 1A). Stress protocol showed no effect on total levels of Akt(supplemental Figs. S1A and S3A). Bilateral injection of twoselective and structurally distinct PI3K inhibitors, LY294002(10 mM, 0.5 �l/side; 30 min before stress) or wortmannin (7.3mM, 0.5 �l/side), into the CA1 region of the hippocampus sig-nificantly reduced basal levels of phosphorylated Akt and com-pletely prevented the increases inAkt phosphorylation inducedby stress protocol (Fig. 1,A andB). Immunofluorescent stainingwas performed to extend these findings. Sections of hippocam-pus were immunostained for phosphorylated Akt to visualizeactivation of PI3K. As shown in Fig. 1C, stressed rats exhibitedincreased numbers of immunopositive cells and labeling inten-sity for phosphorylated Akt in the CA1 pyramidal layer com-paredwith unstressed rats. In addition, stress protocol-inducedphosphorylated Akt increases colocalized with the neuronalmarker NeuN and the pyramidal cell makers MAP2. Together,these results indicate that PI3K in the hippocampal CA1 regionis activated following the stress protocol application.Previous findings from our laboratory demonstrated a paral-
lel in time course of the increased ERK1/2 activation as well asthe effects of stress protocol on LTP and LTD; additionally, apharmacological blockade of the ERK1/2 signaling pathwaycompletely prevented the stress protocol effects, suggesting acritical role of sustained ERK1/2 activation in mediating theeffects of the stress protocol (7, 13). In addition, it has beenshown that PI3K is essential for NMDA receptor-mediatedERK1/2 activation in cultured neurons (29, 45). Therefore, weexamined the possible contribution of PI3K to stress protocol-induced increase of ERK1/2 activation. As expected, we foundthat the stress protocol induced a marked increase of ERK1/2phosphorylation in the hippocampal CA1 region. This increasewas severely impaired by prior intrahippocampal injection ofLY294002 (Fig. 1A) or wortmannin (Fig. 1B), demonstratingthat this event is mostly PI3K-dependent. There was no signif-icant variation in total ERK1/2 in the hippocampal CA1 regionof stressed rats when compared with unstressed rats (supple-mental Fig. S1A).A well established pathway for activation of ERK1/2 is via
stimulation of Ras and formation of a Ras-Raf-1-MEK1/2 com-plex (46). Previous studies have shown that the cross-talk
between the PI3K and ERK1/2 signaling pathwaysmay occur atthe level of Raf kinase (47, 48). We therefore tested whetherPI3K inhibitors blocked the stress protocol-induced increasesin the activation of ERK1/2 by suppressing Raf-1 and MEK1/2activity. In agreement with our previous observations on theeffects of the stress protocol (13), area CA1 excised from thehippocampal slices of stressed rats exhibited a significantincrease in both Raf-1 and MEK1/2 phosphorylation in com-parison with slices from unstressed control rats (supplementalFig. S2,A andC).We found that bilateral injection of LY294002or wortmannin into area CA1 of the hippocampus before stress
A
P-Akt(Ser473)
Akt
P-ERK1/2
ERK1/2
C-Veh S-Veh C-LY2 S-LY2
Rel
ativ
e de
nsito
met
ry (%
)
0
100
200
300 P-Akt (Ser473)P-ERK1/2
(n = 5)
* **
C-Veh S-Veh C-LY2 S-LY2
(P < 0.05)
*
BC-Veh S-Veh C-Wort S-Wort
Akt
P-ERK1/2
ERK1/2
(n = 5)
Rel
ativ
e de
nsito
met
ry (%
)
0
100
200
300
C-Veh S-Veh C-Wort S-Wort
* *
*
(P < 0.05)
(n = 5)
**
P-Akt(Ser473)
C
Control
Stress
P-Akt
P-Akt
NeuN
NeuN
Merge
Merge
Control
Stress
P-Akt
P-Akt
MAP2 Merge
MergeMAP2 Merge
FIGURE 1. Stress protocol induces PI3K activation in hippocampal CA1neurons. A, representative immunoblots and corresponding densitometricanalysis showing changes in phosphorylation of Akt (Ser473) and ERK1/2(Thr202 and Tyr204) in the hippocampal CA1 region after a stress protocol.Administration of PI3K inhibitor LY294002 (LY2) 30 min before the stress pro-tocol inhibited the stress protocol effects on both Akt and ERK1/2. B, repre-sentative immunoblots and corresponding densitometric analysis showingthat stress protocol-induced increased Akt and ERK1/2 phosphorylation issignificantly blocked by wortmannin (Wort) administration before stress pro-tocol application. C, representative photomicrographs showing fluorescentstaining of anti-phospho-Akt (Ser473)/anti-rabbit Alexa 488 (green), anti-NeuN/anti-mouse Alexa 568 (red), or anti-MAP2/anti-mouse Alexa 568 (red) inthe CA1 region immediately after the stress protocol. The merged imageshows overlapping localization (yellow) (arrow). Phospho-Akt labeling wasobserved in cell bodies and dendrites immunoreactive to MAP2. The stressprotocol increased the percentage of CA1 pyramidal neurons stained forphospho-Akt. The number of experiments per group is indicated by n. Theasterisk denotes a significant difference compared with the control-vehicleslices (p � 0.05).
PI3K in Stress Protocol Effects
2634 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 5 • FEBRUARY 1, 2008
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
protocol exposure significantly suppressed the increases inRaf-1 andMEK1/2 phosphorylation by the stress protocol. Thestress protocol showed no effect on total levels of Raf-1 orMEK1/2 (supplementary Fig. S2, B and D). These results indi-cate that activation of PI3K is required, at least in part, for theactivation of Raf-1,MEK1/2, and ERK1/2 triggered by the stressprotocol in the hippocampal CA1 region.We next test whether PI3K activity is necessary formaintain-
ing the effects of stress protocol on subsequent LTP and LTDinduction in the hippocampal CA1 region. In accordance withprevious results (11–13), the hippocampal slices from stressedrats exhibited impaired LTP (6.8 � 5.7%, n � 6; p � 0.05) butfacilitated LTD (33.5 � 6.8% of base line, n � 6; p � 0.05)comparedwith slices from unstressed control rats (LTP, 53.5�8.2%, n � 6; LTD, 5.5 � 5.7%, n � 6) (Fig. 2, A and B). Bilateralintrahippocampal injection of LY294002 almost completelyprevented the blockade of LTP (48.7 � 7.3%, n � 6) and thefacilitation of LTD (6.3� 4.7%, n� 6) by stress protocol. Injec-tion of LY294002 in unstressed rats had no effect on the induc-tion of both LTP (51.9� 6.5%, n� 6) and LTD (4.4� 3.5%, n�6). In contrast to LY294002, the inactive analog LY303511 (10mM, 0.5 �l/side) did not significantly affect the effects of stressprotocol (LTP, 11.3 � 5.3%, n � 5; LTD, 32.8 � 6.2%, n � 5).These results strongly support the view that the activation ofPI3K signaling pathways is an obligatory component of bio-
chemical bases that serves theeffects of stress protocol on subse-quent LTP and LTD induction.Glucocorticoid Receptors Mediate
Stress Protocol-induced PI3KActivation—Having confirmed therole of PI3K in mediating the effectsof stress protocol on hippocampalplasticity, we next investigated pos-sible signaling events that underliethe stress protocol-induced PI3Kactivation. We have previouslydemonstrated that this acuterestraint-tail shock stress protocolcan modulate the subsequent hip-pocampal CA1 LTP and LTDthrough the corticosterone releaseto activate glucocorticoid receptors(13). To test the possible role of glu-cocorticoid receptor activation inmediating the stimulatory effect ofstress protocol on PI3K activity, thespecific glucocorticoid receptorantagonist RU38486 (40mg/kg) wasintraperitoneally injected 30 minbefore stress protocol application.As expected, RU38486 almost com-pletely prevented the stress protocoleffects, as indicated by normal LTP(48.6� 6.8%, n� 5) and LTD (5.7�4.1%, n � 5) in slices from stressedrats (Fig. 3,A andB). In line with theelectrophysiological observations,
RU38486 also completely blocked the stress protocol-inducedPI3K activation. We found no difference between vehicle-treated unstressed and RU38486-treated stressed rats in thelevels of phosphorylated Akt at both Thr308 and Ser473 in thehippocampal area CA1 (Fig. 3C). In contrast, prior administra-tion of specificmineralocorticoid receptor antagonist RU28318(10 mg/kg) did not affect the effects of stress protocol on LTP(9.7 � 4.6%, n � 5) and LTD (26.3 � 6.1%; n � 5) (Fig. 3, A andB). Likewise, the ability of stress protocol to increase the Aktphosphorylation on either the Thr308 or Ser473 site was notsignificantly affected by RU28318 pretreatment (Fig. 3C). Injec-tion of either RU28318 or RU38486 in unstressed rats had noeffects on the induction of LTP or LTD or the levels of Aktphosphorylation. In addition, neither RU28318 nor RU38486treatment showed an effect on total levels of Akt (supplementalFig. S1B). These results support the hypothesis that stress pro-tocol modulates the PI3K activity through the release of corti-costerone to activate glucocorticoid receptors.Blockade of NMDA Receptors Prevents Stress Protocol-in-
duced PI3K Activation—It has previously been shown thatstressmodifies hippocampal plasticity through the activation ofNMDA receptors (11). It has been further evident that pharma-cological blockade of NMDA receptors during stress prevents astress-induced facilitation of the trace eye blink conditioning inrats (49). We therefore investigated whether the stress proto-
Time (min)-10 0 10 20 30 40 50 60
fE
PSP
slop
e (%
of b
asel
ine)
0
50
100
150
200
250
300C-VehS-VehC-LY2S-LY2
1 HFS 2
10 ms0.5 mV
1 1 1 1
2 2 2 2
C-Veh S-Veh C-LY2 S-LY2
LT
P (%
)
0
20
40
60
80
100
C-Veh
S-Veh
C-LY2
S-LY2
C-LY3
S-LY3
(6)
(6)
(6) (6)(5)
(5)
*
(P < 0.05)
(P < 0.05)
*
A
Time (min)-10 0 10 20 30 40 50 60 70 80
fE
PSP
slop
e (%
of b
asel
ine)
020406080100120140
1
1 Hz
2
10 ms0.5 mV1 1 1 1
2 2 2 2
C-Veh S-Veh C-LY2 S-LY2
LT
D (%
)
0
20
40
60
80
C-Veh
S-Veh
C-LY2
S-LY2
C-LY3
S-LY3
(6)
(6)
(6) (6) (5)
(5)*
(P < 0.05)
(P < 0.05)
*
B
FIGURE 2. Activation of PI3K mediates the effects of stress protocol on LTP and LTD. A, stressed rat slicesdisplayed a deficit in HFS-induced LTP, whereas HFS induced a robust LTP in slices from stressed rats adminis-tered with LY294002 (LY2). Administration of the inactive analog LY303511 (LY3) did not significantly affect theeffect of stress protocol. The bar graphs show a comparison of the magnitude of LTP 50 min after HFS. B, LFSinduced a reliable LTD in slices from stressed rats but not in slices from stressed rats administered withLY294002. The bar graphs show a comparison of the magnitude of LTD 50 min after LFS (1 Hz for 15 min). Thenumber of experiments per group is indicated by n. *, significant difference compared with the control-vehicleslices (p � 0.05).
PI3K in Stress Protocol Effects
FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2635
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
col-induced PI3K activation also depends on the activation ofNMDA receptors by using bilateral injection of a competitiveNMDA receptor antagonist, APV, into the CA1 region of thehippocampus. The advantages of using APV as aNMDA recep-tor antagonist include the reversibility of its effect and its abilityto selectively inhibitNMDAreceptors. Confirming and extend-ing previous findings (11), when administered 30 min beforethe start of the stress protocol, APV (25mM, 0.5�l/side) almostcompletely prevented the effects of stress protocol on LTP(58.7 � 6.9%, n � 5) and LTD (6.2 � 3.5%, n � 5) (Fig. 4, A andB). A similar blockade of the enhancing effect of stress protocolon PI3K activation was also observed in APV-treated stressedrats (Fig. 4C). When APV was applied to a group of unstressedrats, it showed no effects on the induction of LTP or LTD or thelevels of Akt phosphorylation. APV treatment showed no effect
on total levels of Akt (supplementalFig. S1C). These results suggest thatstress protocol activates PI3Kthrough aNMDAreceptor-depend-ent mechanism.In subsequent experiments, we
investigated the downstream sig-naling components activated byNMDA receptors to increase PI3Kactivity. Because BDNF was shownto play an important role as a medi-ator of stress response in the hip-pocampus (50) and the activation ofPI3K signaling is one of key signal-ing responsible for binding of BDNFto its receptor TrkB (tyrosine kinaseB) (51, 52), the rapid increase inBDNF expression during stress pro-tocol exposure could result in theactivation of PI3K signaling path-ways by NMDA receptors. To eval-uate this possibility, we examinedthe activation of PI3K in slices fromrats that received intracerebroven-tricular injection of BDNF anti-sense, sense, or scrambled oligonu-cleotide (50 pmol twice per day,bilateral injection for 5 consecutivedays). As shown in Fig. 5A, treat-ment with BDNF antisense oligonu-cleotide completely prevented PI3Kactivation by the stress protocol. Incontrast, BDNF sense or scrambledoligonucleotide treatment did notshow any significant influence onthe effects of the stress protocol onPI3K activity. Furthermore, BDNFantisense, sense, or scrambled oli-gonucleotide treatment showed noeffect on total levels of Akt (supple-mental Fig. S1D). To confirm theeffect of intracerebroventricularinjection of BDNF antisense oligo-
nucleotide on BDNF synthesis, the BDNF protein levels ofstressed rats were also determined. Fig. 5B shows that theBDNFprotein levels in the hippocampi of antisense oligonucle-otide-treated rats were significantly lower than those in vehicle,sense, or scrambled oligonucleotide-treated rats. Remarkably,microinjection of APV into the bilateral hippocampus areaCA1 before stress protocol application also completely pre-vented the stress protocol-induced increase in BDNF synthesis,further supporting that these effects were specifically attribut-able to the activation of NMDA receptors.Stress Protocol Stimulates PSD-95 Protein Translation
through the PI3K-Akt-mTOR Signaling Pathway—How doesactivation of PI3K lead to the effects of stress protocol on hip-pocampal synaptic plasticity? Protein synthesis is required foreffects of stress protocol on LTP and LTD (53). In addition, it
Time (min)-10 0 10 20 30 40 50 60 70 80
fE
PSP
slop
e (%
of b
asel
ine)
020406080100120140
1 Hz
LT
D (%
)
0
20
40
60
80
C-Veh
S-Veh
C-RU28
318
S-RU28
318
(6)
(6)
(5)
(5)
*
(P < 0.05)
*
(5) (5)
C-RU38
486
S-RU38
486
B
Time (min)-10 0 10 20 30 40 50 60
fE
PSP
slop
e (%
of b
asel
ine)
0
50
100
150
200
250
300C-RU28318S-RU28318C-RU38486S-RU38486
HFS
LT
P (%
)
0
20
40
60
80
100
C-Veh
S-Veh
C-RU28
318
S-RU28
318
(6)(5)
(6)
(5)
*
(P < 0.05)
(5)
(5)
C-RU38
486
S-RU38
486
*
A
Phos
pho-
Akt
(%)
0
100
200
300 P-Akt (Thr308)P-Akt (Ser473)(n = 4)
** (P < 0.05)
C-Veh
S-Veh
C-RU28
318
S-RU28
318
C-RU38
486
S-RU38
486
**P-Akt (Thr308)
P-Akt (Ser473)Akt
C-V
eh
S-V
eh
C-R
U28
318
S-R
U28
318
C-R
U38
486
S-R
U38
486
DC
FIGURE 3. Activation of glucocorticoid receptors mediates the effects of the stress protocol. A, HFSinduced robust LTP in slices from stressed rats administered RU38486 but not RU28318. The bar graphs show acomparison of the magnitude of LTP 50 min after HFS. B, LFS failed to induce LTD in slices from stressed ratsadministered RU38486. The bar graphs show a comparison of the magnitude of LTD 50 min after LFS. C and D,representative immunoblot and corresponding densitometric analysis showing that RU38486 administrationspecifically prevented the stress protocol-induced increase in Akt phosphorylation at both Thr308 and Ser473
sites. The number of experiments per group is indicated by n. *, significant difference compared with thecontrol-vehicle slices (p � 0.05).
PI3K in Stress Protocol Effects
2636 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 5 • FEBRUARY 1, 2008
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
has been shown that BDNF can increase protein synthesis byactivating initiation and elongation steps in mRNA translationthrough a TrkB-PI3K-mTOR-dependent mechanism (54). Wetherefore hypothesized that a PI3K-regulated translational acti-vation is involved inmediating the effects of the stress protocol.To further ascertain that the stress protocol can rapidly activatethe PI3K-Akt-mTOR signaling pathway, we conducted a seriesof experiments tomeasure the PDK1, Akt, andmTOR activitiesin hippocampal slices from stressed rats. For PDK1, we used anantibody specific for phosphorylated Ser241, which is on theactivation loop of PDK1 and is essential for kinase activity (55).For mTOR, we used an antibody specific for phosphorylatedSer2448, which has been shown to be important in the control ofmTOR activity (56). As shown above, the levels of phosphoryl-atedAkt on Ser473 were strongly up-regulated in slices obtainedfrom stressed rats. A significant increase of Akt phosphoryla-tionwas observed 0–6 h after stress protocol (Fig. 6A); the timecourse correlated well with the effects of stress and LTP andLTD induction (13, 14). In addition, a significant increase in thePDK1 phosphorylation was observed immediately after the
stress protocol and 0.5 h later (Fig.6B). Furthermore, we found a signif-icant increase of mTOR phospho-rylation at 0–12 h after the stressprotocol (Fig. 6C). The stress proto-col showed no effect on total levelsof Akt, PDK1, or mTOR (supple-mental Fig. S3, A–C).
Because mTOR activation mayregulate translation by direct orindirect phosphorylation of riboso-mal protein S6K (57), we used anantibody that recognizes S6K duallyphosphorylated on Thr229 andThr389 sites after stress protocol.Phosphorylation of S6K on Thr229and Thr389 is crucial for kinaseactivities (58). As shown in Fig. 6B,the levels of phosphorylation of S6Kwere strongly elevated within 0–24h after the stress protocol. The totalS6K levels did not exhibit anychange after stress protocol (sup-plemental Fig. S3D).eIF4B is an RNA-binding protein
that has the ability to stimulatetranslation and to promote ribo-some binding to mRNA (59). eIF4Bis a key downstream target of S6Kandmaymediate some of the effectsof the S6K on translation (60). Inaddition, eIF4B hyperphosphoryla-tion on Ser422 stimulated by serumor mitogen correlates well withincreased translation rates (61). Wethen asked whether stress protocol-induced S6K activation was accom-panied by enhanced eIF4B phos-
phorylation on Ser422. Consistent with the time course of S6Kactivation after stress protocol, we found a strong increase ofeIF4B phosphorylation within 0–24 h after the stress protocol(Fig. 6E). Total levels of eIF4B were unaffected after stress pro-tocol, supporting a specific effect on the activation of eIF4B(supplemental Fig. S3E). These data strongly suggest that stressprotocol can trigger a translation initiation in the hippocampalCA1 region through the activation of PI3K-Akt-mTOR signal-ing pathways.To further explore a role of mTOR-coupled mechanism
mediating stress effects, we performed parallel electrophysi-ological studies to examine the inducibility of LTP and LTD inslices from rats treated with the mTOR inhibitor, rapamycin (1ng/�l; bilateral intrahippocampal injection 30 min beforestress). These effects of the stress protocol were inhibited by theprior administration of rapamycin. Reliable LTP was elicited(56.3 � 7.6%, n � 5), and LTD was no longer induced (5.3 �3.6%, n � 6) in slices from stressed rats (Fig. 7, A and B). Injec-tion of rapamycin in unstressed rats had no effect on the induc-tion of both LTP (53.2 � 6.7%, n � 5) and LTD (5.1 � 3.2%,
P-ERK1/2
Rel
ativ
e de
nsito
met
ry (%
)
0
100
200
300 P-Akt (Ser473)P-ERK1/2(n = 4)
* *
C-Veh S-Veh C-APV S-APV
(P < 0.05)(P < 0.05)
Time (min)-10 0 10 20 30 40 50 60
fE
PSP
slop
e (%
of b
asel
ine)
0
50
100
150
200
250
300C-VehS-VehC-APVS-APV
HFS
LT
P (%
)
0
20
40
60
80
100
C-Veh S-Veh C-APV S-APV
(6)
(5)
(5) (5)
*
(P < 0.05)
Time (min)-10 0 10 20 30 40 50 60 70 80
fE
PSP
slop
e (%
of b
asel
ine)
020406080100120140
1 Hz
LT
D (%
)
0
20
40
60
80
C-Veh S-Veh C-APV S-APV
(6)
(5)
(5)(5)
*
(P < 0.05)
C-Veh S-Veh C-APV S-APVP-Akt
(Ser473)Akt
ERK1/2
A B
DC
FIGURE 4. Activation of NMDA receptors mediates the effects of stress protocol. A, HFS induced robust LTPin slices from stressed rats administered NMDA receptor antagonist APV. The bar graphs show a comparison ofthe magnitude of LTP 50 min after HFS. B, LFS failed to induce LTD in slices from stressed rats administered APV.The bar graphs show the comparison of the magnitude of LTD 50 min after LFS. C and D, representativeimmunoblot and corresponding densitometric analysis showing that APV administration specifically pre-vented the stress protocol-induced increase in Akt phosphorylation at Ser473. The number of experiments pergroup is indicated by n. *, significant difference compared with the control-vehicle slices (p � 0.05).
PI3K in Stress Protocol Effects
FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2637
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
n� 5).However, the inactive analogue ascomycin (1 ng/�l) (62)infused bilaterally into the CA1 before stress did not producechanges in the effects of stress protocol on the induction of bothLTP (10.5 � 6.3%, n � 5) and LTD (32.2 � 5.8%, n � 4).In subsequent experiments, we investigated which plastic-
ity-related protein synthesis is possibly involved in mediat-ing the effects of the stress protocol on hippocampal synap-tic plasticity. PSD-95 (postsynaptic density-95) is suggestedas a scaffolding protein containing multiple PSD-95/Discslarge/Zona occluens-1 domains to anchor and associate glu-tamate receptors with other functional proteins in the PSD
(63). The activation of PI3K-Akt-mTOR signaling pathwayby estrogen or insulin has been shown to rapidly stimulatedendritic PSD-95 synthesis in NG108-15 neuroblastomacells (64) and the hippocampal CA1 region (36). Further-more, like stress protocol does, it has been reported recentlythat overexpression of PSD-95 impairs LTP but enhancesLTD induction in the hippocampal slice cultures (65). Wetherefore examined whether stress protocol may stimulatenew PSD-95 protein synthesis. Consistent with this notion,we found a profound increase in the levels of PSD-95 proteinin hippocampal CA1 homogenates within 0–24 h after stressprotocol (Fig. 6F). The highest level was found at 0.5 h afterstress. Given that PSD-95 is more centrally located in thepostsynaptic density compartment, it was important toestablish that the stress protocol also increases the expres-sion of PSD-95 in the postsynaptic component. In order totest this, we generated synaptoneurosome, a biological prep-aration that enriched for postsynaptic fractions (66) andcontained untranslated PSD-95 mRNA (36). Consistent withresults obtained with total homogenates, an increase in thePSD-95 protein levels was observed within 0–24 h afterstress protocol in our hippocampal CA1 synaptoneurosomepreparations (supplemental Fig. S4A). Noticeably, the stressprotocol-induced up-regulation of PSD-95 protein in totalhomogenates was higher than in the synaptoneurosomes. Inaddition, in the synaptoneurosomes, this effect of the stressprotocol was specifically inhibited by APV, LY294002,K252a, and rapamycin but not by inactive analogs,LY303511, K252b, and ascomycin pretreatment (supple-mental Fig. 4, B–D). However, infusion of the permeableMEK1/2 inhibitor U0126 (2 �g/�l, 0.5 �l/side) bilaterallyinto the hippocampal area CA1 region 30 min before thestress protocol application did not significantly affect theup-regulation of PSD-95 protein expression by stress. Thesefindings strongly suggest that PI3K-Akt-mTOR signalingpathway is a major pathway that couples TrkB receptorsto enhance PSD-95 translation during stress protocolexposure.Finally, we investigated the plausible mechanisms by which
stress protocol-induced up-regulation of PSD-95 protein mod-ulates the induction of hippocampal synaptic plasticity. PSD-95is known to inhibit NMDA receptor function through suppres-sion of the tyrosine kinase activity of Src by interacting directlywith the Src SH2 domain (67). Because theNR2B subunit of theNMDA receptors is themajor tyrosine-phosphorylated proteinin the brain (68) and the level of Src-mediated tyrosine phos-phorylation of the NR2B subunit has been found to increase inLTP in CA1 (69) and dentate gyrus (70) of the hippocampus, itis therefore possible that the changes in the level of NR2B tyro-sine phosphorylation by a stress protocol may contribute to itseffects on LTP and LTD. To test this prediction, we thereforeassessed the effect of a stress protocol on tyrosine phosphoryl-ation of the NR2B subunit in the CA1 region by immunopre-cipitation with NR2B-specific antibody followed by immuno-blotting with antiphosphotyrosine antibody.We found that thebasal tyrosine phosphorylation level of the NR2B subunit wasconsistently reduced in slices from stressed rats compared withslices from unstressed rats (Fig. 8A). There is a clear inverse
APh
osph
o-A
kt (%
)
0
50
100
150
200
250
300
C-Veh
S-Veh
C-Anti-
sense
S-Anti-
sense
C-Sense
S-Sense
C-Scramble
S-Scramble
* *
(4)
(4)
(4) (4) (4) (4)
(4)(4)*
P-Akt(Ser473)
Akt
BD
NF
prot
ein
(pg/
g tis
sue
wei
ght)
0
1000
2000
3000
4000
StressAnti-sense
SenseScramble
D-APV
- + - - - -+ + + +- - + + - - - - - -- - + +- - - - - -- - + +- --- - -- - ++- - - -- -
(4)
(4)
* * *
(4) (4)(4)
(4)
(4)
(4)
(4) (4)
B
FIGURE 5. BDNF signaling pathway mediates the effects of stress proto-col. A, representative immunoblot and corresponding densitometric analysisshowing that administration of BDNF antisense oligonucleotide specificallyprevented the stress protocol-induced increased Akt phosphorylation atSer473. B, administration of BDNF antisense oligonucleotide or APV specifi-cally suppressed the stress protocol-induced increase in BDNF protein levelsin the whole rat hippocampi. The number of experiments per group is indi-cated by n. *, significant difference compared with the control-vehicle slices(p � 0.05).
PI3K in Stress Protocol Effects
2638 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 5 • FEBRUARY 1, 2008
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
correlation between the levels of PSD-95 and NR2B tyrosinephosphorylation. In accordancewith previous findings (71), thetyrosine phosphorylation level of the NR2B subunit was ele-vated after LTP induction. The amount of LTP-inducedincrease of tyrosine phosphorylation of NR2B was found to besignificantly lower in slices from stressed rats than in slicesfrom unstressed rats (Fig. 8B). Furthermore, prior administra-tion of LY294002 prevented the stress-induced up-regulation
of PSD-95 and was effective toreverse the inhibitory effect of stresson tyrosine phosphorylation ofNR2B. Total protein levels of NR2Bwere unaffected after stress proto-col with or without LY294002.
DISCUSSION
In this study, we have identified acritical role of PI3K in the stressprotocol-induced alterations in theinducibility of LTP and LTD in theCA1 region of the hippocampus.We have found that an acuterestraint-tail shock stress protocolincreases the circulating corticos-terone levels, leading to the activa-tion of glucocorticoid receptors,which in turn facilitates the activa-tion of NMDA receptors of hip-pocampal CA1 neurons. This thenresults in provoking the synthesisand release of BDNF acting on theTrkB receptors, which leads to acti-vation of the PI3K-Akt-mTOR sig-naling pathway. mTOR, through itsdownstream translation regulatorymolecules, p70 S6K and eIF4B, acti-vates translational machinery andstimulates the synthesis of dendriticscaffolding protein PSD-95 (Fig. 9).Furthermore, we have shown thatstress protocol-induced up-regu-lation of PSD-95 inhibits theenhanced tyrosine phosphorylationof the NR2B subunit of NMDAreceptors in LTP.PI3K is an unusual lipid signal-
ing kinase that has been impli-cated in a wide range of biologicalfunctions most commonly associ-ated with cell growth and survival(16–18). Beyond its role in neuro-nal growth and survival (19), thePI3K also directly regulates theneuritic outgrowth (71) and syn-aptic plasticity (20–22). Using thespecific inhibitors of PI3K, recentstudies have shown that activationof PI3K is necessary for the induc-
tion (72) or, in contrast, the expression (21, 22) of LTP inhippocampal area CA1. In addition, PI3K has also beenshown to be implicated in the induction of a metabotropicglutamate receptor-dependent form of LTD (73). In thepresent study, the fact that the parallel time course of theincreased PI3K activation and the effects of the stress proto-col on LTP and LTD and pharmacological blockade of PI3Kcompletely prevented the stress effects strongly suggests
F
US S0 S0.5 S1 S2 S6 S12 S24 P-p70S6K
p70S6K
D
Phos
pho-
p70S
6K (%
)
0
50
100
150
200
250
300
**
* **
(5)
(5)
(5)(5)
(5)
(5) (5)(5)
* *
P-eIF4BeIF4B
Phos
pho-
eIF4
B (%
)
050100150200250300350
(4)
(4)
**(4)
(4) (4)(4) (4)
(4)* * * * *
PSD-95
β-actin
PSD
-95/β-
actin
(%)
050100150200250300350
US S0 S0.5 S1 S2 S6 S12 S24
(5)
(5)
*
*(5)
(5) (5)(5)
(5)(5)
* * * * *
Phos
pho-
PDK
-1 (%
)
0
50
100
150
200
(5)
(5)* *
(5)
(5) (5) (4) (4) (4)
P-PDK-1
PDK-1
Phos
pho-
Akt
(%)
0
50
100
150
200
250
300
* * ** *(6)
(6)(5) (5)
(5)
(4) (4)
(4)
US S0 S0.5 S1 S2 S6 S12 S24 P-Akt(Ser473)
Akt
A
B
Phos
pho-
mT
OR
(%)
0
50
100
150
200
US S0 S0.5 S1 S2 S6 S12 S24
(6)
(6)* *
(6) (6)
(6)(5)
(5)
(5)
**
**
CP-mTOR
mTOR
E
FIGURE 6. Stress protocol induces a sustained activation of PI3K signaling cascade. A, representativeimmunoblot and corresponding densitometric analysis showing time course of Akt phosphorylation at Ser473
after the stress protocol. B, representative immunoblot and corresponding densitometric analysis showing thetime course of PDK-1 phosphorylation after the stress protocol. C, representative immunoblot and correspond-ing densitometric analysis showing the time course of mTOR phosphorylation after the stress protocol.D, representative immunoblot and corresponding densitometric analysis showing the time course of p70 S6Kphosphorylation after the stress protocol. E, representative immunoblot and corresponding densitometricanalysis showing time course of eIF4B phosphorylation after the stress protocol. F, representative immunoblotand corresponding densitometric analysis showing the time course of PSD-95 expression in the hippocampalCA1 whole tissue homogenates after the stress protocol. The sample at S0 was obtained immediately after thecompletion of a 1-h stress protocol. The number of experiments per group is indicated by n. *, significantdifference compared with the slices from unstressed control rats (p � 0.05).
PI3K in Stress Protocol Effects
FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2639
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
that the PI3K pathway is an important cascade implicated inmediating the blockade of LTP and the facilitation of LTDinduced by stress protocol.
To explore the functional conse-quences of activation of PI3K andto identify intracellular signalingevents implicated in mediating theeffects of the stress protocol on syn-aptic plasticity, we investigated theregulation of different upstreamactivators and downstream effec-tors of the PI3K signaling pathway.Consistent with a role of glucocorti-coid receptor in mediating theeffects of stress (11, 13, 74, 75), theglucocorticoid receptor antagonistRU38486, when administeredbefore stress protocol application,completely prevented the effects ofthe stress protocol on both PI3Kactivation and hippocampal synap-tic plasticity. Moreover, we havedemonstrated that the competitiveNMDA receptor antagonist APV,when administered before stress,completely prevented the stressprotocol-induced PI3K activation.Thus, the activation of NMDAreceptors can initiate the molecularprocess that contributes to activatePI3K signaling. This is in accord-
ance with an early report (11) indicating that pharmacologicalblockade of NMDA receptors during stress prevented theeffects of stress protocol on subsequent LTP and LTD induc-tion.Moreover, previous studies reported thatNMDA receptorstimulation can activate PI3K-Akt signaling pathways in cul-tured cortical (76) and striatal (29) neurons. How glucocorti-coid receptor enhances the activation of NMDA receptorsremains an important question which needs to be addressed. Incultured rat hippocampal neurons, previous work has shownthat corticosterone can induce a rapid and nongenomic prolon-gation of NMDA receptor-mediated Ca2� elevation via puta-tive membrane surface glucocorticoid receptors (77).How might NMDA receptor activation lead to the sustained
PI3K activation? The NMDA receptor could regulate PI3Kthrough a direct activation process or indirectly through stim-ulating the release of an endogenous mediator. A number ofstudies have pointed toward an important role for BDNF in avariety of NMDA receptor-mediated biological activities. Forexample, NMDAhas been shown to promote BDNF expressionin cerebellar granule cells, and the neuroprotective activity ofNMDA in cerebellar granule cells has been associated withincreased BDNF release and TrkB receptor activation (78–80).Our results also suggest this possibility, since stress protocolapplication induced a significant increase in BDNF proteinexpression in the whole rat hippocampi in a NMDA receptor-dependent manner, and BDNF antisense oligonucleotideintracerebroventricular injection completely prevented thisincrease and the effects of stress protocol on PI3K activation.Moreover, we also found that the stress protocol-induced PI3Kactivationwas suppressed by theTrkB receptor inhibitorK252a
Time (min)-10 0 10 20 30 40 50 60
fE
PSP
slop
e (%
of b
asel
ine)
0
50
100
150
200
250
300
HFS
LT
P (%
)
0
20
40
60
80
100
C-Veh
S-Veh
C-Rap
amyc
in
S-Rap
amyc
in
(5)(5)
(5) (5)
*
(P < 0.05)
*
(5)(5)
C-Asco
mycin
S-Asco
mycin
Time (min)-10 0 10 20 30 40 50 60 70 80
fE
PSP
slop
e (%
of b
asel
ine)
020406080100120140
1 Hz
LT
D (%
)
0
20
40
60
80
C-Veh
S-Veh
C-Rap
amyc
in
S-Rap
amyc
in
(5)
(5)
(5) (6)
*
(P < 0.05)
*
(3)
(4)
C-Asco
mycin
S-Asco
mycin
A BC-RapamycinS-RapamycinC-AscomycinS-Ascomycin
FIGURE 7. The protein kinase mTOR signaling mediates the effects of the stress protocol on LTP and LTD.A, HFS induced robust LTP in slices from stressed rats administered the mTOR inhibitor rapamycin but notascomycin. The bar graphs show a comparison of the magnitude of LTP 50 min after HFS. B, LFS failed to induceLTD in slices from stressed rats administered rapamycin. The bar graphs show a comparison of the magnitudeof LTD 50 min after LFS.
P-NR2BNR2B
PSD-95
Control Stress
A
P-NR2BNR2B
PSD-95
Base LTP Base LTP Base LTPControl Stress + Stress
BLY2
Phos
pho-
NR
2B (%
)
0
50
100
150
Control Stress
(6)
*(6)
Phos
pho-
NR
2B (%
)
0
50
100
150
200 BaselineLTP
(6)
*
Control Stress LY2+Stress
*(6)
(6)(6)
(5)
(5)
FIGURE 8. The stress protocol suppresses the increase in tyrosine phospho-rylation of NR2B subunits after LTP. A, representative immunoblot and corre-sponding densitometric analysis showing the basal levels of tyrosine phospho-rylation of NR2B subunits in slices from control and stressed rats. The NR2Bsubunit was immunoprecipitated with anti-NR2B antibody, and phosphorylationof NR2B was detected with anti-phosphotyrosine antibody. Stress decreased thephosphorylation state of NR2B subunits. B, representative immunoblot and cor-responding densitometric analysis showing stress protocol application inhibitedthe increased tyrosine phosphorylation of NR2B subunits 20 min after LTP induc-tion. Administration of LY294002 blocked the effects of the stress protocol onLTP-induced increase in tyrosine phosphorylation of NR2B subunits. The numberof experiments per group is indicated by n. *, significant difference in comparisonwith slices from unstressed control rats (p � 0.05).
PI3K in Stress Protocol Effects
2640 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 5 • FEBRUARY 1, 2008
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
(data not shown). These observations, therefore, suggest thatthe increased BDNF synthesis then acting on the TrkB receptormay lie downstream of NMDA receptors tomediate the activa-tion of the PI3K signaling pathway by a stress protocol,although other signaling processesmay also be involved. In thisregard, our previous study also demonstrated that the increasedBDNF release and resultant TrkB receptor-ERK1/2 MAPKactivation is an important series of initial events to underlie theeffects of the stress protocol on hippocampal CA1 LTP andLTD (13).
PI3K can stimulate multiple signaling pathways via PDK1and Akt in both neuronal and nonneuronal cells (17, 81). Onemajor downstream target of this signaling pathway is proteinkinase mTOR, which can stimulate translational initiation andelongation. Using a specific mTOR inhibitor, rapamycin, weconfirm a significant role of mTOR-coupled signaling in medi-ating the stress protocol effects. Furthermore, our results alsoreveal that stress protocol increases the phosphorylation of S6Kand eIF4B, which can result in an enhanced translational rate of5�-oligopyrimidine tract-containing mRNAs that encodenumerous components of the translational machinery (57, 60).Our results suggest that a stress protocol can promote transla-tion of specific target proteins in the hippocampal CA1 regionthrough the activation of PI3K-Akt-mTOR-S6K-eIF4B signal-ing pathway. This is in line with the previous finding of a role ofnew protein synthesis in mediating the effects of stress on hip-pocampal synaptic plasticity (53). In search of likely proteinsynthesis for the alterations of LTP and LTD by a stress proto-col, our data suggest that one such candidate is dendritic spinescaffolding protein PSD-95. The increase was observed imme-diately after the stress protocol, in agreement with previousevidence showing that mRNA encoding PSD-95 is present atthe synapses and can be rapidly stimulated following the acti-vation of the PI3K-Akt-mTOR signaling pathway (36, 64). It isnoteworthy that a significantly higher stress protocol-inducedPSD-95 expression was found in the hippocampal CA1 homo-genates than in the synaptoneurosomes within 0.5–24 h afterstress (Fig. 6F and supplemental Fig. S4A). A possible explana-tion for this difference is that stress protocol-induced rapidPSD-95 protein synthesis in the synaptoneurosomes is primar-ily dependent on the existing population of PSD-95 mRNAalready transcribed, whereas some stress protocol-induced newPSD-95mRNA transcription in the neuronal cell body also par-ticipates in subsequent new protein synthesis observed in thehomogenates. In support of this assertion, we have found thatstress protocol-induced PSD-95 expression in the homoge-nates was significantly inhibited by the administration of tran-scriptional inhibitor actinomycin-D.3 However, we cannot ruleout the increases in PSD-95 trafficking to the synapses involvedin this difference. Indeed, it has been shown that both synapticNMDA receptor stimulation and BDNF applied to culturedcortical neurons can increase the trafficking of PSD-95 to den-drites and synapses through the activation of PI3K-Akt signal-ing pathways (82).The precisemechanism by which PSD-95 interacts with syn-
aptic plasticity remained to be elucidated.We found that stressprotocol-induced up-regulation of PSD-95 is accompanied by areduction in the amount of basal and LTP-induced increase intyrosine phosphorylation of NR2B subunits of NMDA recep-tors. Furthermore, pharmacological blockade of stress proto-col-induced up-regulation of PSD-95 also effectively reversedthe effects of the stress protocol on NR2B subunit tyrosinephosphorylation and the induction of long term synaptic plas-ticity. Given that tyrosine phosphorylation of NR2B subunits isessential for the induction and maintenance of hippocampal
3 P. C. Yang and K. S. Hsu, unpublished observation.
Environmentalstress protocol
HPA responseCRHACTHCorticosterone
Glucocorticoid R
p70S6K
PDK1
Akt
mTOR
LTP LTD
NMDA R
BDNF
PI3K
PSD-95
Raf-1
MEK1/2
ERK1/2
Ion channels
Ras
FIGURE 9. Schematic model illustrating the proposed mechanisms under-lying the effects of the stress protocol on the subsequent induction ofhippocampal CA1 LTP and LTD. Autonomic signals conveying acute stress-ful stimuli reach the hypothalamus releasing corticotropin-releasing hor-mone (CRH) from paraventricular nucleus to influence rapid secretion ofACTH. ACTH induces secretion of corticosterone from adrenal glands, andthese interact with glucocorticoid receptors in the hippocampus, thus initiat-ing signal transduction, leading to provocation of the synthesis and release ofBDNF. The increased BDNF subsequently acts on specific TrkB receptors onthe hippocampal neurons and, hence, results in a sustained activation ofERK1/2 MAPK signaling cascade through a mechanism involving the sequen-tial PKC, Ras-Raf-1, and MEK1/2 activation. BDNF also acts on TrkB receptors toactivate the PI3K-PDK-1-Akt-mTOR signaling pathway. mTOR, through itsdownstream translation regulatory molecules, p70 S6K and eIF4B, activatestranslational machinery and stimulates the synthesis of dendritic scaffoldingprotein PSD-95. The activated ERK1/2 MAPK would activate some ion chan-nels, and the up-regulation of PSD-95 may inhibit the enhanced tyrosinephosphorylation of the NR2B subunit of NMDA receptors, which are impor-tant components of the machinery required to support LTP, thus then occlud-ing LTP and facilitating LTD induction. Activation of the PI3K-Akt signalingpathway also leads to some activation of the Ras-ERK1/2 signaling pathway.
PI3K in Stress Protocol Effects
FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2641
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
CA1 LTP (69, 70), it seems possible that the increased PSD-95induced by the stress protocol may negatively regulate NMDAreceptor activity and thereby synaptic plasticity, but additionalstudies will be necessary to examine this issue. Related to thisidea, recent work demonstrates that PSD-95 may suppress, viaits binding to the SH2 domain of Src, Src catalytic activity withthe NMDA receptor complex and suppresses the induction ofLTP (67).We have recently shown an obligatory role of the Ras-
ERK1/2 signaling pathway in the stress protocol-induced alter-ations in the inducibility of LTP and LTD in the CA1 region ofthe hippocampus (13, 83). In the present study, we haveextended these findings by showing that PI3K-Akt signalingpathways can cross-talkwithRas-ERK1/2 signaling pathways inthe hippocampal CA1 neurons to mediate the stress protocoleffects. Our results indicate that stress protocol-induced acti-vation of Raf-1 in the hippocampal CA1 neurons appears to bepartially dependent on PI3K activation, since inhibition of PI3Kprevented activation of Raf-1 and its downstream MEK1/2-ERK1/2. In fact, there is some evidence indicating that PI3Kcan, through its protein kinase activity, regulate Raf kinaseactivity (47, 48). Whether or not PI3K may lie upstream ordownstream of Ras in our observed stress protocol-inducedERK1/2 activation is currently unknown.In conclusion, this study has underscored the importance of
activation of PI3K signaling pathways not only directly mediat-ing synaptic plasticity but also serving to determine the polarityof subsequent synaptic plasticity via a metaplastic function.Our results also support the hypothesis that stress may affectsubsequent hippocampal plasticity by sharing the same molec-ular machinery required to support LTP. These findings pro-vide new insights into the molecular mechanisms underlyingstress-related memory disorders, which, in turn, might providea new avenue for development of more selective medicationthat targets these pathways and prevents their malfunctioning.
REFERENCES1. Eichenbaum, H. (2000) Nat. Rev. Neurosci. 1, 41–502. Rolls, E. T., Stringer, S. M., and Trappenberg, T. P. (2002) Proc. Biol. Sci.
269, 1087–10933. McEwen, B. S. (1999) Annu. Rev. Neurosci. 22, 105–1224. Kim, J. J., and Yoon, K. S. (1998) Trends Neurosci. 21, 505–5095. Garcia, R. (2001) Synapse 40, 180–1836. Kim, J. J., and Diamond, D. M. (2002) Nat. Rev. Neurosci. 3, 453–4627. Huang, C. C., Yang, C. H., and Hsu, K. S. (2005) Mol. Neurobiol. 32,
223–2358. Foy, M. R., Stanton, M. E., Levine, S., and Thompson, R. F. (1987) Behav.
Neural Biol. 48, 138–1499. Shors, T. J., Seib, T. B., Levine, S., and Thompson, R. F. (1989) Science 244,
224–22610. Diamond, D. M., Bennett, M. C., Stevens, K. E., Wilson, E. L., and Rose,
G. M. (1990) Psychobiology 18, 273–28111. Kim, J. J., Foy, M. R., and Thompson, R. F. (1996) Proc. Natl. Acad. Sci.
U. S. A. 93, 4750–475312. Xu, L., Anwyl, R., and Rowan, M. J. (1997) Nature 387, 497–50013. Yang, C. H., Huang, C. C., and Hsu, K. S. (2004) J. Neurosci. 24,
11029–1103414. Yang, C. H., Huang, C. C., and Hsu, K. S. (2006) J. Physiol. 577, 601–61515. Whitman, M., Downes, C. P., Keeler, M., Keller, T., and Cantley, L. (1988)
Nature 332, 644–64616. Leevers, S. J., Vanhaesebroeck, B., and Waterfield, M. D. (1999) Curr.
Opin. Cell Biol. 11, 219–225
17. Brunet, A., Datta, S. R., andGreenberg,M. E. (2001)Curr.Opin.Neurobiol.11, 297–305
18. Hawkins, P. T., Anderson, K. E., Davidson, K., and Stephens, L. R. (2006)Biochem. Soc. Trans. 34, 647–662
19. Frebel, K., and Wiese, S. (2006) Biochem. Soc. Trans. 34, 1287–129020. Kelly, A., and Lynch, M. A. (2000) Neuropharmacology 39, 643–65121. Raymond, C. R., Redman, S. J., andCrouch,M. F. (2002)Neuroscience 109,
531–53622. Sanna, P. P., Cammalleri, M., Berton, F., Simpson, C., Lutjens, R., Bloom,
F. E., and Francesconi, W. (2002) J. Neurosci. 22, 3359–336523. Barros, D. M., Mello e Souza, T., de Souza, M. M., Choi, H., DeDavid e
Silva, T., Lenz, G., Medina, J. H., and Izquierdo, I. (2001) Behav. Pharma-col. 12, 629–634
24. Mizuno, M., Yamada, K., Takei, N., Tran, M. H., He, J., Nakajima, A.,Nawa, H., and Nabeshima, T. (2003)Mol. Psychiatry 8, 217–224
25. Horwood, J. M., Dufour, F., Laroche, S., and Davis, S. (2006) Eur. J. Neu-rosci. 23, 3375–3384
26. Lin, C. H., Yeh, S. H., Lin, C. H., Lu, K. T., Leu, T. H., Chang, W. C., andGean, P. W. (2001) Neuron 31, 841–851
27. Chen, X., Garelick, M. G., Wang, H., Lil, V., Athos, J., and Storm, D. R.(2005) Nat. Neurosci. 8, 925–931
28. Man, H. Y.,Wang, Q., Lu,W. Y., Ju,W., Ahmadian, G., Liu, L., D’Souza, S.,Wong, T. P., Taghibiglou, C., Lu, J., Becker, L. E., Pei, L., Liu, F., Wymann,M. P., MacDonald, J. F., and Wang, Y. T. (2003) Neuron 38, 611–624
29. Perkinton, M. S., Ip, J. K., Wood, G. L., Crossthwaite, A. J., and Williams,R. J. (2002) J. Neurochem. 80, 239–254
30. Cammalleri, M., Lutjens, R., Berton, F., King, A. R., Simpson, C.,Francesconi,W., and Sanna, P. P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100,14368–14373
31. Diamond, D. M., Park, C. R., and Woodson, J. (2004) Hippocampus 14,281–291
32. Shors, T. J., and Thompson, R. F. (1992) Synapse 11, 262–26533. Paxinos, G., and Watson, C. (1998) The Rat Brain in Stereotaxic Coordi-
nates, Academic Press, Inc., San Diego34. Seligman, M. E., and Maier, S. F. (1967) J. Exp. Psychol. 74, 1–935. Wells, D. G., Dong, X., Quinlan, E. M., Huang, Y. S., Bear, M. F., Richter,
J. D., and Fallon, J. R. (2001) J. Neurosci. 21, 9541–954836. Lee, C. C., Huang, C. C., Wu, M. Y., and Hsu, K. S. (2004) J. Biol. Chem.
280, 18543–1855037. Huang, C. C., and Hsu, K. S. (2006) J. Neurochem. 96, 179–19438. Okragly, A. J., and Haak-Frendscho, M. (1997) Exp. Neurol. 145, 592–59639. Acheson, A., Conover, J. C., Fandl, J. P., DeChiara, T. M., Russell, M.,
Thadani, A., Squinto, S. P., Yancopoulos, G. D., and Lindsay, R. M. (1993)Nature 374, 450–453
40. Baker-Herman, T. L., Fuller, D. D., Bavis, R.W., Zabka, A. G., Golder, F. J.,Doperalski, N. J., Johnson, R. A., Watters, J. J., and Mitchell, G. S. (2004)Nat. Neurosci. 7, 48–55
41. Dash, P. K.,Orsi, S. A., andMoore,A.N. (2006) J. Neurosci.26, 8048–805642. Izquierdo, I., Schroder, N., Netto, C. A., and Medina, J. H. (1999) Eur.
J. Neurosci. 11, 3323–332843. Saal, D., Dong, Y., Bonci, A., and Malenka, R. C. (2003) Neuron 37,
577–58244. Franke, T. F., Yang, S. I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison,
D. K., Kaplan, D. R., and Tsichlis, P. N. (1995) Cell 81, 727–73645. Chandler, L. J., Sutton, G., Dorairaj, N. R., and Norwood, D. (2001) J. Biol.
Chem. 276, 2627–263646. Hagemann, C., and Blank, J. L. (2001) Cell. Signal. 13, 863–87547. Bondeva, T., Pirola, L., Bulgarelli-Leva, G., Rubio, I., Wetzker, R., and
Wymann, M. P. (1998) Science 282, 293–29648. Moelling, K., Schad, K., Bosse, M., Zimmermann, S., and Schweneker, M.
(2002) J. Biol. Chem. 277, 31099–3110649. Shors, T. J., and Servatius, R. J. (1995) Neuroreport 6, 677–68050. Marmigere, F., Givalois, L., Rage, F., Arancibia, S., and Tapia-Arancibia, L.
(2003) Hippocampus 13, 646–65551. Segal, R. A., and Greenberg, M. E. (1996) Annu. Rev. Neurosci. 19,
463–48952. Taoka, T., Tokuda, M., Tasaka, T., Hatase, O., Irino, S., and Norman,
A. W. (1990) Biochem. Biophys. Res. Commun. 170, 1151–1156
PI3K in Stress Protocol Effects
2642 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 5 • FEBRUARY 1, 2008
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
53. Xu, L., Holscher, C., Anwyl, R., and Rowan, M. J. (1998) Proc. Natl. Acad.Sci. U. S. A. 95, 3204–3208
54. Takei, N., Kawamura, M., Hara, K., Yonezawa, K., and Nawa, H. (2001)J. Biol. Chem. 276, 42818–42825
55. Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342,287–292
56. Brunn, G. J., Fadden, P., Haystead, T. A., and Lawrence, J. C., Jr. (1997)J. Biol. Chem. 272, 32547–32550
57. Hay, N., and Sonenberg, N. (2004) Genes Dev. 18, 1926–194558. Dufner, A., and Thomas, G. (1999) Exp. Cell Res. 253, 100–10959. Trachsel, H., Erni, B., Schreier, M. H., and Staehelin, T. (1977) J. Mol. Biol.
116, 755–76760. Raught, B., Peiretti, F., Gingras, A. C., Livingstone, M., Shahbazian, D.,
Mayeur, G. L., Polakiewicz, R. D., Sonenberg, N., andHershey, J.W. (2004)EMBO J. 23, 1761–1769
61. Duncan, R., and Hershey, J. W. (1985) J. Biol. Chem. 260, 5486–549262. Petros, A. M., Neri, P., and Fesik, S. W. (1992) J. Biomol. NMR 2, 11–1863. Hering, H., and Sheng, M. (2001) Nat. Rev. Neurosci. 2, 880–88864. Akama, K. T., and McEwen, B. S. (2003) J. Neurosci. 23, 2333–233965. Stein, V., House, D. R., Bredt, D. S., and Nicoll, R. A. (2003) J. Neurosci. 23,
5503–550666. Hollingsworth, E. B., McNeal, E. T., Burton, J. L., Williams, R. J., Daly,
J. W., and Creveling, C. R. (1985) J. Neurosci. 5, 2240–225367. Kalia, L. V., Pitcher, G. M., Pelkey, K. A., and Salter, M. W. (2006) EMBO
J. 25, 4971–498268. Moon, I. S., Apperson, M. L., and Kennedy, M. B. (1994) Proc. Natl. Acad.
Sci. U. S. A. 91, 3954–3958
69. Nakazawa, T., Komai, S., Tezuka, T., Hisatsune, C., Umemori, H., Semba,K., Mishina, M., Manabe, T., and Yamamoto, T. (2001) J. Biol. Chem. 276,693–699
70. Rostas, J. A., Brent, V. A., Voss, K., Errington, M. L., Bliss, T. V., and Gurd,J. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10452–10456
71. Leemhuis, J., Mayer, U., Barth, H., Schmidt, G., and Meyer, D. K. (2004)Naunyn Schmiedeberg’s Arch. Pharmacol. 370, 211–222
72. Opazo, P., Watabe, A. M., Grant, S. G., and O’Dell, T. J. (2003) J. Neurosci.23, 3679–3688
73. Hou, L., and Klann, E. (2004) J. Neurosci. 24, 6352–636174. Pavlides, C., Watanabe, Y., and McEwen, B. S. (1993) Hippocampus 3,
183–19275. Kerr, D. S., Huggett, A. M., and Abraham, W. C. (1994) Psychobiology 22,
123–13376. Sutton, G., and Chandler, L. J. (2002) J. Neurochem. 82, 1097–110577. Takahashi, T., Kimoto, T., Tanabe, N., Hattori, T., Yasumatsu, N., and
Kawato, S. (2002) J. Neurochem. 83, 1441–145178. Favaron, M., Manev, R. M., Rimland, J. M., Candeo, P., Beccaro, M., and
Manev, H. (1993) Neuroreport 4, 1171–117479. Marini, A. M., Rabin, S. J., Lipsky, R. H., and Mocchetti, I. (1998) J. Biol.
Chem. 273, 29394–2939980. Bhave, S. V., Ghoda, L., and Hoffman, P. L. (1999) J. Neurosci. 19,
3277–328681. Wymann,M. P., Zvelebil,M., and Laffargue,M. (2003)Trends Pharmacol.
Sci. 24, 366–37682. Yoshii, A., and Constantine-Paton, M. (2007) Nat. Neurosci. 10, 702–71183. Yang, C.H.,Huang, C. C., andHsu, K. S. (2005) J. Neurosci. 25, 4288–4293
PI3K in Stress Protocol Effects
FEBRUARY 1, 2008 • VOLUME 283 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2643
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
Ping-Chun Yang, Chih-Hao Yang, Chiung-Chun Huang and Kuei-Sen HsuModification of Hippocampal Synaptic Plasticity
Phosphatidylinositol 3-Kinase Activation Is Required for Stress Protocol-induced
doi: 10.1074/jbc.M706954200 originally published online December 5, 20072008, 283:2631-2643.J. Biol. Chem.
10.1074/jbc.M706954200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
Supplemental material:
http://www.jbc.org/content/suppl/2007/12/06/M706954200.DC1
http://www.jbc.org/content/283/5/2631.full.html#ref-list-1
This article cites 82 references, 28 of which can be accessed free at
by guest on Decem
ber 19, 2020http://w
ww
.jbc.org/D
ownloaded from
