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Biphasic activation of the mTOR pathway in the gustatory cortex is correlated with and necessary for taste learning

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Biphasic activation of the mTOR pathway in the gustatory cortex is correlated with and necessary for taste learning
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/26282104 Biphasic Activation of the mTOR Pathway inthe Gustatory Cortex Is Correlated with andNecessary for Taste...  Article   in  The Journal of Neuroscience : The Official Journal of the Society for Neuroscience · July 2009 DOI: 10.1523/JNEUROSCI.3809-08.2009 · Source: PubMed CITATIONS 46 READS 33 4 authors , including:Katya BelelovskyBar Ilan University 14   PUBLICATIONS   408   CITATIONS   SEE PROFILE Kobi RosenblumUniversity of Haifa 83   PUBLICATIONS   3,497   CITATIONS   SEE PROFILE All content following this page was uploaded by Hanoch Kaphzan on 07 January 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Behavioral/Systems/Cognitive Biphasic Activation of the mTOR Pathway in the Gustatory Cortex Is Correlated with and Necessary for Taste Learning KatyaBelelovsky,HanochKaphzan,AlinaElkobi,andKobiRosenblum Department of Neurobiology and Ethology, Faculty for Science, University of Haifa, Haifa 30905, Israel Differentformsofmemoriesandsynapticplasticityrequiresynthesisofnewproteinsatthetimeofacquisitionorimmediatelyafter.Weareinterestedintheroleoftranslationregulationinthecortex,thebrainstructureassumedtostorelong-termmemories.Themamma-lian target of rapamycin, mTOR (also known as FRAP and RAFT-1), is part of a key signal transduction mechanism known to regulatetranslationofspecificsubsetofmRNAsandtoaffectlearningandsynapticplasticity.WereportherethatnoveltastelearninginducestwowavesofmTORactivationinthegustatorycortex.Interestingly,thefirstwavecanbeidentifiedbothinsynaptoneurosomalandcellularfractions,whereasthesecondwaveisdetectedinthecellularfractionbutnotinthesynapticone.InhibitionofmTOR,specificallyinthegustatory cortex, has two effects. First, biochemically, it modulates several known downstream proteins that control translation andreducestheexpressionofpostsynapticdensity-95 invivo .Second,behaviorally,itattenuateslong-termtastememory.Theresultssuggestthat the mTOR pathway in the cortex modulates both translation factor activity and protein expression, to enable normal taste memory consolidation. Introduction Long-term taste memory, in a similar way to other memories, isassumed to be stored at least partially in the cortex (Bures et al.,1998; Rosenblum, 2008). Different forms of learning and synap-tic plasticity, including taste memory consolidation in the gusta-tory cortex (GC), are sensitive to general protein synthesis inhi-bition during or immediately after learning (Rosenblum et al.,1993; Meiri and Rosenblum, 1998). However, the molecularmechanismthatregulatesneuronaltranslationandmemorycon-solidation processes in the cortex is not clear. Correlative studiesidentifydifferentpost-translationalmodificationsintheGCafternovel taste learning (Berman et al., 1998; Belelovsky et al., 2005;Merhav et al., 2006; Yefet et al., 2006). Specifically, some of thesecorrelative events between protein phosphorylation in the GCand taste learning are known to affect and modulate the functionofthetranslationmachinery(Belelovskyetal.,2005).Inaddition,theinvolvementofdifferenttranslationinitiationfactors,includ-ing eIF2  , 4E-BP2, and S6K, in taste learning was reported intransgenic mice studies (Banko et al., 2007; Costa-Mattioli et al.,2007; Antion et al., 2008).Some of the correlative modifications, observed after noveltaste or conditioned taste aversion (CTA) learning, are detectedin proteins that are either direct or indirect targets of the mam-maliantargetofrapamycin(mTOR).ThemTORpathwaycanbeactivated by a number of growth factors and nutrients, resultingin increased phosphorylation of mTOR on residue Ser2448 (Hay andSonenberg,2004).TwostructurallyandfunctionallydistinctTOR complexes, TORC1 and TORC2, exist. TORC1 mediatesrapamycin-sensitive, TOR-shared signaling to the translationmachinery, the transcription apparatus, and other targets.TORC2mediatesrapamycin-insensitive,TOR2-uniquesignalingto the actin cytoskeleton (Loewith et al., 2002). The inhibition of mTOR by rapamycin interferes with the translation of specificsubpopulations of mRNAs, an action which significantly distin-guishes the action of rapamycin from other general protein syn-thesisinhibitors,suchasanisomycinorcycloheximide(Raughtetal., 2001). The downstream targets of mTOR are all componentsof translation machinery, including ribosomal protein kinase(S6K1)andelongationfactors1Aand2(eEF1AandeEF2).Theseproteins are mostly involved in ribosome recruitment to mRNA,and they regulate both the initiation and elongation phases of translation (Hay and Sonenberg, 2004).The significant role of the mTOR pathway in different formsof synaptic plasticity has been pointed out in several studies (Ca-sadio et al., 1999; Tang et al., 2002; Cammalleri et al., 2003; Hor- wood et al., 2006; Tsokas et al., 2007). Rapamycin applicationprevented long-term facilitation in  Aplysia  (Casadio et al., 1999)and blocked high-frequency and BDNF-induced long-term po-tentiation in the rat hippocampus (Tang et al., 2002). Further-more,mTOR-dependentactivationofdendriticS6K1wasshownto be necessary for the induction phase of protein synthesis-dependent synaptic plasticity (Cammalleri et al., 2003). How-ever, only few studies have investigated the role of mTOR signal-ing in learning and memory formation, focusing mainly onhippocampus and amygdala-dependent forms of learning (Tis-chmeyeretal.,2003;Dashetal.,2006;Parsonsetal.,2006;Bekin- schtein et al., 2007).In the current study, we sought to determine the role of mTORsignalingintheGCafternoveltastelearningintherat.To Received July 2, 2008; revised Nov. 12, 2008; accepted Jan. 9, 2009.ThisworkwassupportedbyanIsraelScienceFoundationgranttoK.R.WethankDr.PaulSkehelforconstructivecomments on this manuscript.CorrespondenceshouldbeaddressedtoDr.KobiRosenblum,DepartmentofNeurobiologyandEthology,Univer-sity of Haifa, Haifa 31905, Israel. E-mail: kobir@psy.haifa.ac.il.DOI:10.1523/JNEUROSCI.3809-08.2009Copyright © 2009 Society for Neuroscience 0270-6474/09/297424-08$15.00/0 7424  •  The Journal of Neuroscience, June 10, 2009  •  29(23):7424–7431  establish this, we examined the effect of rapamycin microinfu-sions to the GC before and after taste learning. Next, we assessedthetemporalpatternofmTORpathwayactivationatseveraltimepoints after taste learning. Moreover, we characterized the effectof rapamycin application on total and phospho-levels of severaltranslation regulators, affecting both the initiation and elonga-tion phases. Finally, we addressed the relationship betweenmTOR-dependent translation control and the expression levelsof postsynaptic protein postsynaptic density-95 (PSD-95) that isknown to stabilize synaptic plasticity (Ehrlich et al., 2007) andwhich induced expression in the GC is necessary for taste mem-ory consolidation (Elkobi et al., 2008). MaterialsandMethods Subjects Adult male Wistar rats, weighing 200–250 g (Harlan), were maintainedon a 12 h light/dark cycle. The procedures were performed in strict ac-cordance with the University of Haifa regulations and the US NationalInstitutes of Health (NIH) guidelines (NIH publication number 8023). Behavioral procedures Conditioned taste aversion and latent inhibition.  CTA was performed asdescribed previously (Rosenblum et al., 1993). Saccharin (0.1% w/v, so-dium salt) or NaCl (0.3%) were used as the unfamiliar taste in training[i.e., the conditioned stimulus (CS)] and injection of LiCl (0.15 m, 2%body weight, i.p.) as the malaise-inducing agent [unconditioned stimu-lus (UCS)]. At the beginning of the behavioral experiment, the rats weretrained for 3 d to get their daily water ratio once a day for 15 min fromtwo pipettes, each containing 10 ml of water. On the conditioning day,they were allowed to drink the saccharin solution instead of water fromsimilarpipettesfor15min,and50minlatertheywereinjectedwithLiCl.Under these conditions, 2 d after training the conditioned rats preferredwater to saccharin in a multiple choice test situation (three pipettes with5 ml of saccharin each and three with 5 ml of water each), whereasnonconditioned rats preferred saccharin to water. The behavioral dataare presented in terms of aversion index, defined as [ml water/(ml waterplus ml saccharin)] consumed in the test; 0.5 is a chance level, and thehigher the aversion index, the more the rats prefer water to the condi-tioned taste.In some experiments, a latent inhibition (LI) procedure (Lubow,1989) was combined with CTA to further isolate the effect of taste learn-ing from the potential confounding effects of the UCS and the CS–UCSassociation. Latent inhibition is a process by which pre-exposure to asensory stimulus diminishes the ability of that same stimulus to serve asan associated stimulus in subsequent learning. Thus, exposure of rats toan unfamiliar taste several days before this same taste serves as the CS inCTA training and significantly reduces the acquired aversion (Rosen-blum et al., 1993). Under such conditions, the degree of aversion afterCTAtrainingisameasureofthememoryforsaccharinacquiredinciden-tally (Hebb, 1949) in the pre-CTA trial. In LI experiments, the rats wereexposed to two 10 ml pipettes of saccharin for 15 min, 2 d before CTAtraining, as described above, in which saccharin was used as the CS.Testing was also as described above for the usual CTA procedure. Incidental taste learning.  Two groups of rats were water deprived for24 h and then pretrained for 3 d to get their daily water ration once a day for 15 min from two pipettes each containing 10 ml of water. On thefourthday,theexperimentalgroupwasexposedfor15mintounfamiliartaste (saccharin 0.1% or NaCl 0.3%), whereas the control group wasexposed for the same time period to water (Rosenblum et al., 1993;Berman et al., 1998).  Microsurgery and microinfusion Microinfusions into the gustatory cortex were performed via chronically implanted cannulae. Rats were anesthetized with equithesine (0.45 ml/100 g; 2.12% w/v MgSO 4 , 10% v/v ethanol, 39.1% v/v 1,2,-propranolol,0.98% w/v sodium pentobarbital, and 4.2% w/v chloral hydrate), re-strained in a stereotactic apparatus (Stoelting), and implanted bilaterally with a 10-mm-guide stainless cannula (23 gauge) aimed at the rat gusta-tory cortex (anteroposterior,  1.2 mm relative to bregma; lateral,  5.5mm;ventral,  5.5mm)(PaxinosandWatson,1986).Thecannulaewerepositioned in place with acrylic dental cement and secured by two skullscrews.Astyluswasplacedintheguidecannulatopreventclogging.Afterthe microsurgery, animals were injected intramuscularly with antibioticand were allowed to recuperate for 1 week.For microinfusion, the stylus was removed from the guide cannula,and a 28 gauge injection cannula, extending 1.0 mm from the tip of theguide cannula, was inserted. The injection cannula was connected viaPE20 tubing to a Hamilton microsyringe driven by a microinfusionpump (CMA/100; Carnegie Medicin). Microinfusion was performed bi-laterally in a 1.0   l volume per hemisphere delivered over 1 min. Theinjection cannula was left in position before withdrawal for an additional 1mintominimizedraggingoftheinjectedliquidalongtheinjectiontract.For the behavioral set of experiments, the rats were injected bilaterally either with rapamycin (10   M ; Sigma) or with vehicle (0.2% DMSO insaline), 25 min before or 100 min after the pre-exposure in the LI para-digm. For the biochemical part, to provide a within-subject control,rapamycinandvehicleorU-0126andvehiclewereinjectedtoeachGCof the same animal. To avoid lateral bias, animals were injected inter-changeably between both sides.The rats were decapitated 15 min or 45 min after the microinjectionwith rapamycin and 20 min after the microinjection with U-0126, andthe gustatory cortices were subjected to Western blot analysis. Hippocampal slice preparation After decapitation, the brain was immediately immersed in cold (4°C)carboxygenated (95% O 2 , 5% CO 2 ) artificial CSF [aCSF (in m M ): 124NaCl, 5 KCl, 1.2 MgSO 4  1.2 NaH 2 PO 4 , 26 NaHCO 3 , 10  D -glucose, 2.4CaCl], and after  120 s, both hippocampi were dissected out in a platefilled with cold (4°C) aCSF on ice. The hippocampi were put on a cooledstand of a McIlwain tissue chopper TC752 (Campden Instruments), cutinto400  mslices,andthenputbackintoachamberfilledwithcarboxy-genated cold (4°C) aCSF.The slices were transferred to a holding chamber for  20–30 min, toreach room temperature, and were then transferred to a six-chamber phar-macologicalinstrument,designedtoourspecificationsbyScientificSystemsDesignCompany.Alloftheslicestestedinanyoneexperiment(i.e.,inallsix chambers)wereproducedbythesameprocedurefromthesamerat.The hippocampal slices were heated to 32°C and were kept in thechamberfor5hbeforeanypharmacologicalintervention.Eachchambercontained four slices. The slices were perfused with heated and carboxy-genated aCSF via a model MP3 peristaltic pump (Gilson), at a rate of   2ml/min. The chamber space was carboxygenated and humidified. Thechamberwasaninterfacetype,andthesliceswereplacedonalenspaper.Thesliceswereanalyzedoccasionallyfortheirviabilityusingextracellularrecordings of field EPSP in the CA1 region.Afterinsulinapplication(10min;1.5  M )/controlcondition,thesliceswere removed from the pharmacological chamber and snap frozen ondry ice. After freezing, slices were homogenized in SDS sample buffer asdescribed previously (Rosenblum et al., 1997). Four slices from eachchamber were combined as two pairs, and the two slices of each pair werehomogenizedasasinglesample,sothateachchamberyieldedtwosamples. Sample preparation and Western blotting  Preparation of total sample.  At different time points after the incidentaltaste learning (0,15, 30, and 180 min) and after the microinfusion in thepharmacologicalexperiments(15and45min),theratsweredecapitated,the brain was removed, and the gustatory cortices were dissected out.EachGCwashomogenizedin300  lice-coldlysisbufferinaglass-Teflonhomogenizer. The lysis buffer was composed of the following (in m M ,unless indicated otherwise): 10 HEPES, 2 EDTA, 2 EGTA, 5 Na fluoride,0.5 DTT, 0.1 Na orthovanadate, 0.1 phenylmethylsulfonyl fluoride, 10  g/ml leupeptin, 10  g/ml aprotin, and 1% phosphatase inhibitor mix-ture 1 (Sigma). Protein content was determined by Bradford assay (Bio-Rad). Appropriate volumes of 2  SDS sample buffer were added to thehomogenates, and samples were boiled for 5 min and stored at  20°C. Preparation of synaptoneurosomal fraction.  The protocol was adoptedfrom Quinlan et al. (1999) and is similar to the protocol used in Kelleher Belelovsky et al. • mTOR in Cortical-Dependent Learning J. Neurosci., June 10, 2009  •  29(23):7424–7431  • 7425  et al. (2004). In brief, fresh cortex was homogenized in a glass/glass 5 mltissue grinder in 1 ml of lysis buffer (as detailed above). An aliquot of thehomogenized tissue (100   l) was retained, and after determination of protein amount, it was mixed with appropriate volume of 2  SDS sam-ple buffer (total fraction). The remaining material was passed oncethrough a 100   m filter and once through a 5   m filter (Millipore Bio-scienceResearchReagents),attachedtoa5mlsyringe.Thehomogenizedtissuewascentrifugedat1000   g  for10minat4°C.Thepelletcontainedthe synaptoneurosome fraction, whereas nonspecific material remainedin the supernatant. Lysis buffer (100   l) was added to the pellet, theprotein amount of both the pellet and supernatant fractions was deter-mined, and 2  SDS sample buffer was added at appropriate volume toboth; the samples were boiled for 5 min and stored at  20°C. Westernblotting. Samples(8–15  gofproteinperwell)wereloadedon7.5% or 10% SDS-PAGE gels and resolved by standard electrophoresis.The gels were transferred electrophoretically onto nitrocellulose mem-branes(poresize,0.45  m;Invitrogen).Membraneswereblockedfor1hat room temperature with blocking buffer [3% BSA in TBS containing0.1% Tween 20 (TBS-T)] and probed overnight at 4°C using primary antibodiesforp44/42mitogen-activatedprotein(MAP)kinase[extracel-lular signal-regulated protein kinase (ERK)], 1:1000; phospho- p44/42MAP kinase (p-ERK), 1:1000; p70 S6 kinase (S6K1), 1:500; phospho-(Thr389)p70 S6 kinase (p-S6K1), 1:750; mTOR, 1:500 and phospho-mTOR (p-mTOR), 1:1000; eEF2, 1:1000 and phospho-eEF2 (p-eEF2),1:1000, all rabbit polyclonal and all from Cell Signaling Technology.eEF1A,1:1000,mousemonoclonalwasfromUpstateBiotechnology,and  -actin, 1:3000, goat polyclonal, was purchased from Santa Cruz. Afterwashing in TBS-T (three washes, 10 min each), the membranes wereincubatedwithgoatanti-rabbit(IgG),goatanti-mouse(IgG),ordonkey anti-goat (IgG) horseradish peroxidase-conjugated (all from JacksonImmunoResearch), and proteins were visualized using chemilumines-cence (ECL and ECL plus Western blotting analysis system; GEHealthcare).Quantification was performed with a charge-coupled device camera(XRS; Bio-Rad). Each sample was measured relative to the background,and phosphorylation levels were calculated as the ratios between theresults from the antibody directed against the phospho-proteins andthose from the antibody directed against the phosphorylation state-independent forms of the proteins; a ratio of unity would indicate thatthere was no difference in protein phosphorylation. The results are ex-pressedasmeans  SEM.Forstatisticalanalysis,weusedthepaired t  test, t   test assuming equal variances, the univariate ANOVA test, andrepeated-measures ANOVA test. For  post hoc   analysis, we used Tukey honestly significant differences test. Results Novel taste learning induces the activation of mTOR pathway in the GC To examine the effect of novel taste (saccharin) drinking on theexpression of mTOR pathway, we collected the GC of rats atseveral time points after the taste learning. Our results indicatethatincidentaltastelearningresultsintwowavesofmTORphos-phorylation on Ser2448, the site known to be critical for mTOR activation (Raught et al., 2001). No change in mTOR phosphor- ylation was observed immediately after the exposure to noveltaste; however, within 15 min, it increased by 21% ( t   test,  t  (14)  2.43;  p  0.03). Thirty minutes after the learning, the levels of phospho-mTOR returned to the baseline and remained un-changed at the time point of 90 min after learning. With thepassage of 180 min, we detected a second wave of mTOR activa-tion of 27% ( t   test,  t  (15)  2.93;  p  0.01) (Fig. 1  A ).Next, we analyzed the phosphorylation levels of S6K1 at sametime points studied for mTOR. Again, we observed a two-wavepattern of S6K1 phosphorylation, increasing 15 min (29%) and180 min (22%) after the taste learning ( t   test,  t  (8)  2.55;  p  0.05 and  t  (15)  2.45;  p  0.03, respectively) (Fig. 1 B ).Because S6K1 is a known substrate of mTOR, we examinedthe correlation between mTOR and S6K1 activation. Indeed, wefound that a significant positive correlation exists between thetwo phosphorylation events in the GC after novel taste learning(Spearman’s  r   0.341;  p  0.02; data not presented). S6K1 phosphorylation is increased in synaptoneurosomalfraction 15 min but not 180 min after novel taste After the observation that S6K1 phosphorylation is correlatedwith taste learning in the gustatory cortex, we analyzed S6K1phosphorylation in a synaptoneurosomal fraction made fromgustatory cortex. First, we confirmed the enrichment of the syn-aptic protein PSD-95 compared with total fraction ( t   test,  t  (8)  7.21;  p  0.001) (Fig. 2 B ). The levels of    -actin were compa-rable in both fractions [not significant (n.s.)]. Next, we analyzedthe total and phospho-levels of S6K1 in the synaptic and totalfractions.Surprisingly,thetotalexpressionofS6K1,calculatedasaratiobetweenS6K1and  -actinlevels,wassignificantlyreducedin synaptoneurosomes, compared with total fraction ( t   test,  t  (11)  4.5;  p  0.001) (Fig. 2  A , B ), proposing a decreased S6K1 ex-pression in the synapse. The analysis of S6K1 phosphorylationrevealed no difference between the fractions (Fig. 2 B ). However,the ratio between phospho- and total S6K1, which indicates acti-vatedS6K1levels,wassignificantlyincreased( t  test, t  (11)  2.22;  p  0.05) (Fig. 2  A ).We next evaluated S6K1 total and phospho-levels in synapto-neurosomesafternoveltaste.Wefocusedonthetwotime-points,wherephospho-S6K1wasincreasedinthetotallysate:15and180min. Here, we obtained a differential effect of activation: at thefirst point, p-S6K1 was increased ( t   test,  t  (7)  2.7;  p  0.03), Figure1.  mTORandS6K1arephosphorylatedinthegustatorycortexinabiphasicmanner.A protein phosphorylation is expressed as the ratio between saccharin and water values.  A ,mTORphosphorylationisincreased15and180minafternoveltaste( n  8,  p  0.03; n  9,  p  0.01, respectively). Here and in other figures, error bars are SEM, whereas asterisk repre-sents a significant change. The top panel depicts representative immunoblots of anti-proteinantibodyandanti-phospho-specificantibody(Ser2448)fromthegustatorycortex0,15,30,90,and180minafternoveltastesaccharin(sac)orfamiliartaste(water). B ,S6K1phosphorylationis also increased within 15 and 180 min of novel taste drinking ( n  5,  p  0.05;  n  8,  p  0.03,respectively).Thetoppaneldepictsrepresentativeimmunoblotsofanti-proteinantibodyand phospho-specific antibody (Thr389) from the gustatory cortex for all time points. 7426  •  J. Neurosci., June 10, 2009  •  29(23):7424–7431 Belelovsky et al. • mTOR in Cortical-Dependent Learning  whereas at the second one, no difference was observed ( t  (18)  0.8, n.s.) (Fig. 2 C  ). Rapamycin affects the phosphorylation and protein levels of different mTOR substrates in the GC  in vivo We examined the abundance and phosphorylation levels of sev-eral translation regulators, known to be substrates of mTOR.Specifically,weanalyzedproteinlevelsofeEF2,eEF1A,S6K1,andERK2, and the phosphorylation levels of eEF2, S6K1, and ERK2,at two time points (15 and 45 min) after the microinfusion of rapamycin (10  M ) to the GC of naive rats.At 15 min, no modulation was observed. However, 45 minafter the injection, the phosphorylation and total levels of severalmTOR substrates were modulated.ThephosphorylationlevelofS6K1wassignificantlydecreased(paired  t   test,  t  (4)    4.7;  p    0.01) (Fig. 3  A ), indicating a de-creased initiation rate. As for the elongation phase, increasedlevels of eEF2 phosphorylation together with decreased levels of totaleEF1Awereobserved(paired t  test, t  (4)  2.9;  p  0.05and t  (4)  2.9;  p  0.05, respectively) (Fig. 3 B , C  ), both suggestingthat rapamycin inhibits the elongation rate of protein synthesis.Because it was previously shown that protein synthesis mightbe regulated in ERK-dependent manner (Kelleher et al., 2004)and that mTOR and ERK do crosstalk (Tsokas et al., 2007), wealso analyzed the effect of mTOR inhibition on ERK2 phosphor- ylation and vice versa: the effect of ERK2 inhibition on mTOR and S6K1 phosphorylation. No effect was observed: the phos-phorylationlevelsofERK2didnotchangeafterrapamycinappli-cation (paired  t   test,  t  (4)  1.5, n.s.) (Fig. 3 D ). Similarly, MEKinhibitor U-0126, injected into the gustatory cortex, did not af-fect the phosphorylation of either S6K1 (paired  t   test,  t  (4)  0.1,n.s.)(Fig.3 E  )ormTOR(paired t  test, t  (4)  0.3,n.s.)(Fig.3 F  ). Figure2.  DifferentialtemporalactivationofS6K1inthesynaptoneurosomesmadefromGC.  A ,  B , The levels of PSD-95 and total and phospho-S6K1 were examined by immunoblotting inthe synaptoneurosomal fraction (syn) and compared with the level in the initial lysate (total).The levels of postsynaptic marker PSD-95 are significantly increased in the synaptic fraction( n  5;  p  0.001).S6K1levelinthesynaptoneurosomalfractionislower( n  7;  p  0.001),butthelevelsofactivatedS6K1arehigher( n  7;  p  0.05)comparedwiththetotalfraction. C  ,S6K1activationisincreasedinthesynaptoneurosomalfractionoftheinsularcortexwithin15minbutnot180minofnoveltastelearning( n  5,  p  0.03; n  10,n.s.).Cumulativedataareshown in the graph and representative immunoblots of total and phospho-S6K1 are shown inthe inset. Figure3.  Biochemical effects 45 min after rapamycin microinfusion into GC of naive rats. Aproteinphosphorylation/expressionisexpressedastheratiobetweenrapamycinorU-0126andvehicle values.  A , The phosphorylation levels of S6K1 in brains of rapamycin-treated rats aresignificantly decreased ( n  5;  p  0.01).  B , Increased levels of eEF2 phosphorylation areobservedafterrapamycininjection( n  5;  p  0.05). C  ,ThetotallevelsofeEF1Aarereducedin rapamycin-injected rats ( n  5;  p  0.05).  D  , ERK2 phosphorylation is not modified afterrapamycinapplication( n  5;n.s.). E  ,mTORphosphorylationisnotaffectedbyU-0126appli-cation( n  5;n.s.). F  ,U-0126injectiondidnotchangethephosphorylationlevelsofS6K1( n  5;n.s.).Toppanelsateachpointdepictrepresentativeimmunoblotsfromthegustatorycortex. Belelovsky et al. • mTOR in Cortical-Dependent Learning J. Neurosci., June 10, 2009  •  29(23):7424–7431  • 7427
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