nature letters to nature ... Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1 advance online publication

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1 vne online pulition... Mehnism for the lerning efiits in mouse moel of neurofiromtosis type 1 Rui M. Cost*, Nikoli B. Feerov*, Jeff H. Kogn*, Geoffrey G. Murphy*, Joel Stern*, Msuo Ohno*, Rju Kuherlpti, Tyler Jks & Alino J. Silv* * Deprtments of Neuroiology, Psyhitry n Psyhology, BRI, University of Cliforni t Los Angeles, Los Angeles, Cliforni , USA Deprtment of Moleulr Genetis, Alert Einstein College of Meiine, 13 Morris Prk Avenue, Bronx, New York, New York 1461, USA Deprtment of Biology, Msshusetts Institute of Tehnology, Cmrige, Msshusetts 2139, USA... Neurofiromtosis type I (NF1) is one of the most ommon single-gene isorers tht uses lerning efiits in humns 1. Mie rrying heterozygous null muttion of the Nf1 gene (Nf1 +/2 ) show importnt fetures of the lerning efiits ssoite with NF1 (ref. 2). Although neurofiromin hs severl known properties n funtions, inluing Rs GTPse-tivting protein tivity 3,4, enylyl ylse moultion 5,6 n mirotuule ining 7, it is unler whih of these re essentil for lerning in mie n humns. Here we show tht the lerning efiits of Nf1 +/2 mie n e resue y geneti n phrmologil mnipultions tht erese Rs funtion. We lso show tht the Nf1 +/2 mie hve inrese GABA (g-mino utyri i)-meite inhiition n speifi efiits in long-term potentition, oth of whih n e reverse y eresing Rs funtion. Our results inite tht the lerning efiits ssoite with NF1 my e use y exessive Rs tivity, whih les to impirments in long-term potentition use y inrese GABA-meite inhiition. Our finings hve implitions for the evelopment of tretments for lerning efiits ssoite with NF1. Visul sptil prolems re mong the most ommon ognitive efiits etete in iniviuls ffete with NF1 (refs 1, 8). Stuies hve shown tht Nf1 +/2 mie hve norml sptil lerning 2 when teste in the hien version of the wter mze tsk tht is sensitive to hippompl lesions 9. Stuies suggest tht n upregultion of Rs tivity my ount for the lerning efiits in NF1 oth in mie 1 n humns 11. To test this hypothesis, we rosse the Nf1 +/2 mie (C57B6/N kgroun) with mie heterozygous for null muttion in the K-rs gene 12 (K-rs +/2, 129T2/SvEmsJ), n teste the F 1 esennts (hyri isogeni kgroun) in the hien version of the wter mze tsk. Beuse K-rs 2/2 mie, like Nf1 2/2 (ref. 13), ie in utero 12, we use K-rs +/2 mie, whih re vile n show no pprent evelopmentl efets. Mie were trine with two trils per y. In ll the following experiments, no ifferenes etween genotypes n/or tretments were oserve in quisition (Fig. 1,, g), floting, thigmotxi ehviour or swimming spee (t not shown). Sptil lerning ws ssesse in proe tril 14 (trining y 7), in whih the pltform ws remove from the pool. The time spent serhing in the trining qurnt ws ifferent mong the ifferent genotypes (nlysis of vrine, ANOVA, F 3,61 = 7.27, P,.5). Wil-type mie spent Present ress: Memory Phrmeutils Corportion, Philips Prkwy, Montvle, New Jersey 7645, USA nture This vne online pulition (AOP) Nture pper shoul e ite s Author(s) Nture vne online pulition, 16 Jnury 22 (DOI 1.138/nture711). One the print version (ientil to the AOP) is pulishe, the ittion eomes Author(s) Nture volume, pge (yer); vne online pulition, 16 Jnury 22 (DOI 1.138/nture711). letters to nture signifintly more time serhing in the trining qurnt thn i Nf1 +/2 mie (Fisher s protete lest signifint ifferene, PLSD, P,.5; Fig. 1), onfirming tht the Nf1 +/2 mie hve impire sptil lerning. K-rs +/2 mie were lso impire (P,.5; Fig. 1); however, mie rrying heterozygous muttions in oth the Nf1 n the K-rs genes (Nf1 +/2 /K-rs +/2 ) spent s muh time s wiltype mie in the trining qurnt (P..5; Fig. 1), n signifintly more time thn Nf1 +/2 n K-rs +/2 mie (P,.5). This inites tht the lerning efiits in Nf1 +/2 mie n e resue y the K-rs +/2 muttion. Furthermore, oth wil-type (pire t-test, t 23 = 5.935, P,.5) n Nf1 +/2 /K-rs +/2 mie serhe seletively for the missing pltform; tht is, they serhe loser to the ext pltform position thn to the opposite position in the pool 15 (t 1 = 4.9, P,.5; Fig. 1), wheres Nf1 +/2 n K-rs +/2 mie i not (Nf1 +/2, t 14 = 1.83; K-rs +/2, t 14 =.149; P..5). Muttions tht erese Rs funtion o resue the efiits of the Nf1 +/2 mie euse null heterozygous muttion in N-rs (N-rs +/2 ), nother mmmlin rs gene with overlpping funtion n similr expression to tht of K-rs 12, lso reverses the sptil lerning efiits of the Nf1 +/2 mie (Fig. 1e, f; see Methos). During the proe tril, there ws signifint effet of genotype on serhing strtegy (F 3,32 = 2.83, P,.5; Fig. 1e). Nf1 +/2 /N-rs +/2 mie were inistinguishle from wil type (Fisher s PLSD, P..5), n serhe signifintly more in the trining qurnt thn i Nf1 +/2 mie (P,.5). Also, nlysis of proximity sores showe tht oth wil-type n Nf1 +/2 /N-rs +/2 mie serhe seletively (wil type, t 9 = 3.621; Nf1 +/2 /N-rs +/2, t 8 = 5.84; P,.5; Fig. 1f), wheres Nf1 +/2 mie i not (t 14 = 1.83, P..5). The N-rs +/2 muttion y itself i not proue ny etetle phenotype (P..5; Fig. 1e), onsistent with previous stuies initing tht Rs signlling is less impire in N-rs thn in K-rs mutnts 12. The rs muttions tht reverse the Nf1 +/2 sptil lerning efiits shoul result in erese in Rs funtion throughout the life of the mie. To etermine whether ereses in Rs funtion speifilly uring trining oul reverse the lerning efiits of the Nf1 +/2 mie, we use frnesyl-trnsferse inhiitor (FTI; BMS ; ref. 16). This gent reues Rs signlling y loking frnesyltion, post-trnsltionl moifition tht is essentil for Rs funtion 17. FTIs hve een shown to resue other NF1 phenotypes, suh s Shwnn ell prolifertion in humn n murine ells 18,19. Wil-type n Nf1 +/2 mie were injete intrperitonelly every y 6 min efore trining with either sline or 5 mg per kg (oy mss) BMS ; the trining onitions n the geneti kgroun of the nimls were the sme s in the Nf1 +/2 /K-rs +/2 experiment. Anlysis of the y 7 proe tril showe n effet of genotype tretment intertion on serhing strtegy (F 3,32 = 2.83, P,.5; Fig. 1h). Nf1 +/2 mie trete with BMS serhe signifintly longer in the trining qurnt thn Nf1 +/2 mie trete with sline (P,.5), n were inistinguishle from wil-type mie (P..5). Moreover, nlysis of proximity sores showe tht Nf1 +/2 mie trete with BMS serhe seletively (t 18 = 4.13, P,.5; Fig. 1i), s i wil-type mie (t 17 = 4.33, P,.5), wheres Nf1 +/2 mie trete with sline i not (t 19 =.266, P..5). Aministrtion of 5 mg per kg BMS i not versely ffet wil-type nimls, s these mie serhe equivlently to wil-type mie trete with sline (P..5; Fig. 1h). The fining tht the lerning efiits of Nf1 +/2 mie n e reverse with n FTI onfirms our geneti results, n shows tht these lerning efiits re reversile in ult mie. Lerning is thought to our through tivity-epenent synpti moifitions in neuronl networks 2. To etermine whether hnges in synpti plstiity might ount for the sptil (hippompl-epenent 9 ) lerning impirments of Nf1 +/2 mutnts, we exmine long-term potentition (LTP) t Shffer ollterl/ca1 NATURE 16 Jnury 22 DOI 1.138/nture Mmilln Mgzines Lt 1

2 letters to nture vne online pulition Aquisition 6 5 K-rs +/ 4 /K-rs +/ Lteny (s) Lteny (s) g Lteny (s) Dys N-rs +/ /N-rs +/ Dys sl sl FTl FTl Dys Proe tril qurnt 5 TQ Figure 1 Lerning efiits of Nf1 +/2 mie re Rs epenent., Hien version of the wter mze for the Nf1 +/2 /K-rs +/2 popultion (wil type (), n = 24; Nf1 +/2, n = 15; K-rs +/2, n = 15; Nf1 +/2 /K-rs +/2, n = 11)., Lteny to get to the pltform over ys., Per ent time spent in eh qurnt uring proe tril, Averge proximity to the ext position where the pltform ws uring trining, ompre with proximity to the opposite position in the pool. f, Aquisition, per ent time in qurnt, n proximity Time in qurnt (%) e Time in qurnt (%) h Time in qurnt (%) Proe tril proximity 7 TQ OP iproximity (m)proximity (m) Proximity (m) 3 K-rs +/ /K-rs +/ K-rs +/ /K-rs +/ 3 N-rs +/ /N-rs +/ N-rs +/ /N-rs +/ 3 sl FTl FTl sl FTl FTl f t for the Nf1 +/2 /N-rs +/2 popultion (, n = 1; Nf1 +/2, n = 1; N-rs +/2, n =7; Nf1 +/2 /N-rs +/2, n = 9). g i, Aquisition, per ent time in qurnt, n proximity t for the ifferent genotypes n tretments uring the FTI resue experiment ( FTI, n = 19; sline, n = 18; Nf1 +/2 FTI, n = 18; Nf1 +/2 sline, n = 18). Qurnts re trining qurnt (TQ), jent right, jent left n opposite qurnt (OP). synpses in hippompl slies from these mie. Shffer ollterl stimultion (6 ma) eliite similr seline exittory postsynpti potentil (EPSP) in Nf1 +/2 n wil-type mie (Nf1 +/2 = 1.2 ^.6 mv, wil type = 1.9 ^.5, F 1,54 =.58, P..5). We first inue LTP using thet-urst stimultion (TBS) protool, whih mimis the in vivo tivity of hippompl neurons uring explortory ehviour 21. This protool onsiste of two, five or ten ursts. The two-urst protool eliite persistent potentition in wil-type nimls (Fig. 2), whih ws signifintly lrger thn tht triggere in Nf1 +/2 mie (F 1,11 = 6.98, P,.5). This ifferene ws lso seen with five (F 1,13 = 8.27, P,.5; Fig. 2) n ten ursts (F 1,16 = 6.93, P,.5; Fig. 2). Thus, TBS stimultion shows tht there is n LTP efiit in Nf1 +/2 mie (Fig. 2e). We lso exmine LTP inue uner high-frequeny stimultion (HFS, Hz for 1 s; Fig. 2e). With this protool, the potentition inue ws equivlent in wil-type n Nf1 +/2 mie (F 1,15 =.6, P..5; see elow). To etermine whether the TBS-inue LTP efiits in Nf1 +/2 mie were use y inrese Rs funtion, we teste Nf1 +/2 / K-rs +/2 mie (of the sme geneti kgroun s those teste in the wter mze). The mgnitue of CA1 LTP inue with two-urst protool iffere ross genotypes (F 3,35 = 4.6, P,.5; Fig. 2). The mount of LTP inue in Nf1 +/2 /Krs +/2 ws signifintly higher thn tht inue in Nf1 +/2 mie (Fisher PLSD, P,.5), n equivlent to tht of wil-type mie (P..5; Fig. 2f). K-rs +/2 mie were lso signifintly impire reltive to oth Nf1 +/2 /Krs +/2 n wil-type mie (LTP t 4 min 114 ^ 4.47, P,.5; t not shown). These results show tht the K-rs +/2 muttion n resue the LTP efiits in Nf1 +/2 mie, just s it n lso resue their sptil lerning efiits. Nf1 +/2 mie lso showe erese in the input output funtion of extrellulrly reore EPSPs t the Shffer ollterl/ca1 synpse (F 1,16 = 4.11, P,.5, Fisher PLSD ( ma), P,.5; Fig. 3). Neither this impirment, nor the LTP efiit, seeme to e use y norml presynpti funtion, euse eletrophysiologil phenomen sensitive to presynpti hnges suh s pirepulse filittion (F 1,19 =.43, P..5; Fig. 3), ugmenttion, epletion n proility of glutmte relese 22 were norml in Nf1 +/2 mie (t not shown). The LTP efiits of Nf1 +/2 mie were revele y TBS, ut not HFS stimultion. LTP inue y TBS is more sensitive to hnges in GABA-meite inhiition thn is LTP inue y HFS 23. In ition, Rs n ffet hlorie urrents in renl gln ells 24. We therefore investigte whether GABAmeite inhiition ws ltere in Nf1 +/2 mie. We mesure evoke inhiitory postsynpti potentils (IPSPs) in CA1 pyrmil ells of hippompl slies from Nf1 +/2 mie. We foun tht Nf1 +/2 mie hve lrger IPSPs thn those of wil-type ontrols (ANOVA, F 1,88 = 5.31, P,.5; Fisher PLSD, P,.5; Fig. 3). We lso mesure monosynptilly evoke IPSPs in the presene of AP5 (2-mino-5-phosphonopentnoi i), n NMDA (N-methyl Mmilln Mgzines Lt NATURE 16 Jnury 22 DOI 1.138/nture711

3 vne online pulition letters to nture e /K-rs +/ 16 2-urst 5-urst 1-urst Hz D-sprtte) reeptor ntgonist, n CNQX (6-yno-7-nitroquinoxline-2,3-ione), n AMPA (-mino-3-hyroxy-5-methyl-isoxzole-4-propioni i) reeptor ntgonist (Fig. 3). One gin, IPSPs were lrger in Nf1 +/2 thn in wil-type mie (ANOVA, F 2,237 = 5.9, P,.5; Fisher PLSD, P,.5; Fig. 3). The inrese inhiition in Nf1 +/2 mie ws lso resue y mnipultions tht erese Rs funtion: IPSPs in Nf1 +/2 /K-rs +/2 mie re smller thn in Nf1 +/2 mie (P,.5; Fig. 3) n inistinguishle from wil-type ontrols (P..5). Results from severl of our experiments suggest tht this inrese in GABA-meite inhiition is the use for the LTP efiits in Nf1 +/2 mie. First, pirotoxin (1 mm), GABA A reeptor ntgonist, resues the LTP (two-urst) efiits of Nf1 +/2 mie (F 1,9 =.78, P..5; Fig. 4). Seon, LTP efiits re evient in Nf1 +/2 mie (Fig. 2) t synpti stimultion strengths (6 ma) tht use n inrese in GABA-meite inhiition, ut re sent t synpti stimultion strengths (35 ma) tht o not use inreses in inhiition (F 1,27 =.263, P..5; Figs 3 n 4). Note tht in wiltype mie the LTP inue t stimultion strength of 6 ma is lrger thn tht inue t 35 ma (F 1,16 = 18.1, P,.5), wheres in Nf1 +/2 mie it is not (F 1,22 =.18, P..5; Fig. 4). This is onsistent with the lrger inhiition in Nf1 +/2 mie t 6 ma (Fig. 4). Thir, LTP inue with HFS, whih is less sensitive to inhiition thn LTP inue with TBS, is norml in Nf1 +/2 mie (Fig. 2e). Our results show tht the sptil lerning impirments in mouse moel of NF1 n e resue y two ifferent geneti f /K-rs +/ Figure 2 Rs-epenent long-term potentition efiits in Nf1 +/2 nimls. Perentge of seline fiel EPSP (fepsp) is plotte over time., Two-urst inution protool (wil type (), n =5;Nf1 +/2, n = 8)., Five-urst inution protool (, n =7;Nf1 +/2, n = 8)., Ten-urst inution protool (, n =7;Nf1 +/2, n = 11)., Two-urst inution protool. LTP efiits in Nf1 +/2 mie re Rs epenent (, n = 13; Nf1 +/2, n = 13; Nf1 +/2 /K-rs +/2, n = 8). Representtive tres re shown from left to right for, Nf1 +/2 n Nf1 +/2 /K-rs +/2. Horizontl r, 1 ms; vertil r, 1 mv. e, LTP mesure 4 min fter inution uner ifferent stimultion protools. f, LTP mesure 4 min fter inution in the Nf1 +/2 /K-rs +/2 resue experiment. fepsp (slope) IPSP (mv) Nf1 +/ mnipultions tht erese Rs levels. Our t lso show tht the lerning efiits in Nf1 +/2 mie n e reverse phrmologilly in the ult n importnt fining for the evelopment of tretments for lerning efiits ssoite with NF1. Our results suggest tht normlly high or low Rs tivity n isrupt lerning, initing tht preise Rs moultion y neurofiromin is essentil for lerning n memory. Although the lerning efiits in Drosophil tht lk neurofiromin re epenent on enylyl ylse 6, Nf1 +/2 mie hve norml enylyl ylse tivity 25. These mie, however, hve upregulte Rs/mitogen-tivte protein kinse tivity 26. Our t inite tht inrese Rs tivity, use y prtil loss of neurofiromin, les to normlly high GABA-meite inhiition, n tht this enhnement in inhiition my e responsile for impirments in synpti plstiity tht might unerlie the lerning efiits ssoite with NF1. These finings suggest tht strtegies tht erese either Rs tivity or GABA-meite inhiition might e use to tret the lerning efiits ssoite with NF1. A Methos Stimultion (µa) Stimultion (µa) Interstimulus intervls (ms) Animls Genertion of the ifferent genetilly moifie mie hs een esrie 12,27,28. For the Nf1 +/2 /N-rs +/2 experiment, we generte nimls y rossing Nf1 +/2 mie, whih h een previously krosse five genertions to the C57BL/6N kgroun from 129T2/SvEmsJ (N5 into C57BL/6N), with N-rs +/2 lso krosse five genertions to the C57BL/6N kgroun. All the other nimls were 129T2/SvEmsJ C57BL/6N F 1 hyris generte y rossing Nf1 +/2 mie (krosse more thn 11 genertions to the C57BL/6N kgroun) with wil-type or K-rs +/2 mie on the 129T2/SvEmsJ kgroun. Thus, in every experiment the mie from eh genotype were littermtes n of isogeni geneti kgroun. All experiments were rrie out lin with respet to genotype or tretment. Wter mze The si protool for the wter mze experiments hs een esrie 1. Mie from the fepsp (% filittion) IPSP (mv) /K-rs +/ Stimultion (µa) Figure 3 Rs-epenent enhne inhiition in Nf1 +/2 mie., Input output funtion t the CA3 CA1 synpse (wil type (), n =9;Nf1 +/2, n = 9) with representtive tres (left, ; right, Nf1 +/2 ). Horizontl r, 5 ms; vertil r,.5 mv., Pire-pulse filittion (, n =9;Nf1 +/2, n = 12) t ifferent interstimulus intervls., Evoke IPSPs mesure intrellulrly in CA1 pyrmil ells using ifferent stimultion strengths (, 5 mie, 13 neurons; Nf1 +/2, 4 mie, 17 neurons)., Monosynptilly evoke IPSPs mesure in the presene of AP5 n CNQX (, 7 mie, 17 neurons; Nf1 +/2, 11 mie, 23 neurons; Nf1 +/2 /K-rs +/2, 6 mie, 13 neurons).,, Representtive tres re shown left to right for, Nf1 +/2 n Nf1 +/2 /K-rs +/2 mie. Horizontl r, ms; vertil r, 5 mv. NATURE 16 Jnury 22 DOI 1.138/nture Mmilln Mgzines Lt 3

4 letters to nture vne online pulition µa 6 µa T2/SvEmsJ C57B/6N F 1 kgroun were given two trils per y (3-s intertril intervls) with proe tril (6 s) t the en of trining y 7. Beuse mie from the C57B/6N geneti kgroun tke longer to lern the wter mze 29, mie from the Nf1 +/2 /N-rs +/2 popultion (N5 into C57BL/6N) were given three loks of two trils per y, with proe tril t trining y 5. We use ontextul fer onitioning in wil-type mie (129T2/SvEmsJ C57BL/6N F 1 ) to etermine the ose use in the BMS experiments. With one single injetion 6 h efore trining, ose of 1 mg per kg (oy mss) i not ffet lerning ut higher osges i (time freezing: sline, 63.3%; 1 mg per kg, 55.8%; 3 mg per kg, 34.4%; 45 mg per kg, 46.7%). But 1 mg per kg injete for 7 ys (s in the wter mze experiment) ffete lerning in wil-type mie (sline, 82.9%; 1 mg per kg, 52.2%; F 1,1 = 9.43, P,.5). As shown, 5 mg per kg injete for 7 ys i not ffet lerning in wil-type mie. We issolve BMS in sterile sline solution n injete it every y 1 h efore the experiment strte. Eletrophysiology For the fiel potentils, reorings were me from trnsverse hippompl slies (4 mm thik) in sumerge reoring hmer perfuse (2 ml min 21 ) with rtifiil ererospinl flui (ACSF) ontining (in mm): 12 NCl, 3.5 KCl, 2.5 CCl 2, 1.3 Mg 2 SO 4, 1.25 NH 2 PO 4, 26 NHCO 3 n 1 D-gluose t 3 8C (sturte with 95% O 2 n 5% CO 2 ). For the LTP experiments, EPSPs were evoke lterntively in seprte pthwys (ontrol n tetnize) in the CA1 Shffer ollterl/ommissurl fferents with -ms test pulses through two stimulting eletroes (,3 mm from the Pt/Ir reoring eletroe). Unless inite otherwise, the stimultion strength in oth stimulting eletroes ws set to 6 ma. After 1-min seline perio, LTP ws inue in one pthwy oring to HFS ( Hz for 1 s) or TBS protool (two, five or ten ursts, eh urst four pulses t Hz, 2-ms interurst intervls). We exlue from further nlysis slies in whih there ws signifint rift in the ontrol pthwy or in whih seizures evelope. The mount of potentition ws lulte s perentge of the seline EPSP slope. For the experiments with pirotoxin, the rug ws issolve (1 mm) in ACSF n ws present throughout the experiment. For the input output urves, we pplie ifferent stimultion strengths (from 1 to ma in steps of 1 ma). For eh slie, five EPSPs were verge per stimultion strength. Pire-pulse filittion ws ssesse y mesuring the per ent inrese in the slope of the seon pulse in reltion to the first (2-, 5-, -, 2-, 3-, 4-, 5-ms interpulse intervls). The stimultion strength ws juste to one-thir of the mximum EPSP mplitue. To ssess inhiition in Nf1 +/2 mie, we mesure IPSPs from CA1 pyrmil neurons using whole-ell (lin tehnique 3 ) rige moe reorings (Axolmp 2B, Axon Instruments). IPSPs were evoke through stimulting eletroe ple in the Shffer ollterl/ommissurl fferents (,75 mm from the reoring pipette when AP5 n CNQX were pplie) tht pplie ifferent stimultion strengths (from 1 to ma in steps of 1 ma). The IPSP mplitue ws mesure, with five IPSPs verge for eh neuron per stimultion strength. The intrellulr solution ontine (in mm): 135 potssium gluonte, 5 HEPES, 2 Mg 2+ -ATP, 5 MgCl 2,.3 GTP,.5 EGTA (pipette resistne 7 11 MQ). To evoke IPSPs monosynptilly, AP5 n CNQX (1 mm) were present in the ACSF. In ll experiments, t were verge n entere into nlysis s single sujet, n therefore reflet iniviul mie rther thn iniviul slies or neurons Figure 4 Long-term potentition efiits in Nf1 +/2 mie re use y inrese inhiition., LTP inution uner pirotoxin using two-urst inution (wil type (), n ¼ 6; Nf1 +/2, n ¼ 5)., LTP inue with two ursts using stimultion strength of 35 ma (evokes similr IPSPs in n Nf1 +/2 mie, see Fig. 3) is not ifferent in (n ¼ 13) n Nf1 +/2 (n ¼ 16) mie., In mie the LTP inue t stimultion strength of 6 ma is lrger thn LTP inue t 35 ma, wheres in Nf1 +/2 mie it is not (6 ma evokes lrger IPSPs in Nf1 +/2 mie). Sttistil nlysis We nlyse quisition t from the wter mze y repete-mesures ANOVA. Per ent time in trining qurnt for the ifferent genotypes ws nlyse using single-ftor ANOVA; post-ho omprisons (Fisher s PLSD) etween genotypes were rrie out when pproprite. Plnne omprisons using pire t-test were use to nlyse the proximity t. LTP ws nlyse using single-ftor ANOVA on the verge mount of LTP 3 4 min fter inution. We nlyse the inhiition n input output urves using ANOVA, n post-ho omprisons were performe when pproprite. Reeive 1 August 21; epte 21 Novemer 21. Pulishe online 16 Jnury 22, DOI 1.138/nture North, K. Neurofiromtosis type 1. Am. J. Me. Genet. 97, (2). 2. Silv, A. J. et l. A mouse moel for the lerning n memory efiits ssoite with neurofiromtosis type I. Nture Genet. 15, (1997). 3. Bllester, R. et l. The NF1 lous enoes protein funtionlly relte to mmmlin GAP n yest IRA proteins. Cell 63, (199). 4. Xu, G. F. et l. The tlyti omin of the neurofiromtosis type 1 gene prout stimultes rs GTPse n omplements ir mutnts of S. erevisie. Cell 63, (199). 5. Guo, H. F., The, I., Hnnn, F., Bernrs, A. & Zhong, Y. Requirement of Drosophil NF1 for tivtion of enylyl ylse y PACAP38-like neuropepties. Siene 276, (1997). 6. Guo, H. F., Tong, J., Hnnn, F., Luo, L. & Zhong, Y. A neurofiromtosis-1-regulte pthwy is require for lerning in Drosophil. Nture 43, (2). 7. Xu, H. & Gutmnn, D. H. Muttions in the GAP-relte omin impir the ility of neurofiromin to ssoite with mirotuules. Brin Res. 759, (1997). 8. Ozonoff, S. Cognitive impirment in neurofiromtosis type 1. Am. J. Me. Genet. 89, (1999). 9. Morris, R. G., Grru, P., Rwlins, J. N. & O Keefe, J. Ple nvigtion impire in rts with hippompl lesions. Nture 297, (1982). 1. Cost, R. M. et l. Lerning efiits, ut norml evelopment n tumour preisposition, in mie lking exon 23 of Nf1. Nture Genet. 27, (21). 11. Klose, A. et l. Seletive istivtion of neurofiromin GAP tivity in neurofiromtosis type 1. Hum. Mol. Genet. 7, (1998). 12. Johnson, L. K-r. et l. rs is n essentil gene in the mouse with prtil funtionl overlp with N-rs. Genes Dev. 11, (1997). 13. Brnnn, C. I. et l. Trgete isruption of the neurofiromtosis type-1 gene les to evelopmentl normlities in hert n vrious neurl rest-erive tissues. Genes Dev. 8, (1994). 14. Brneis, R., Brnys, Y. & Yehu, S. The use of the Morris wter mze in the stuy of memory n lerning. Int. J. Neurosi. 48, (1989). 15. Gllgher, M., Burwell, R. & Burhinl, M. Severity of sptil lerning impirment in ging: Development of lerning inex for performne in the Morris wter mze. Behv. Neurosi. 17, (1993). 16. Muthlif, M. M. et l. Contriution of Rs GTPse/MAP kinse n ytohrome P45 metolites to eoxyortiosterone-slt-inue hypertension. Hypertension 35, (2). 17. Gis, J. B. et l. Frnesyltrnsferse inhiitors versus Rs inhiitors. Curr. Opin. Chem. Biol. 1, (1997). 18. Yn, N. et l. Frnesyltrnsferse inhiitors lok the neurofiromtosis type I (NF1) mlignnt phenotype. Cner Res. 55, (1995). 19. Kim, H. A., Ling, B. & Rtner, N. Nf1-efiient mouse Shwnn ells re ngiogeni n invsive n n e inue to hyperproliferte: reversion of some phenotypes y n inhiitor of frnesyl protein trnsferse. Mol. Cell. Biol. 17, (1997). 2. Aott, L. F. & Nelson, S. B. Synpti plstiity: tming the est. Nture Neurosi. 3, (2). 21. Lrson, J., Wong, D. & Lynh, G. Ptterne stimultion t the thet frequeny is optiml for the inution of hippompl long-term potentition. Brin Res. 368, (1986). 22. Hessler, N. A., Shirke, A. M. & Mlinow, R. The proility of trnsmitter relese t mmmlin entrl synpse. Nture 366, (1993). 23. Chpmn, C. A., Perez, Y. & Lille, J. C. Effets of GABAA inhiition on the expression of long-term potentition in CA1 pyrmil ells re epenent on tetniztion prmeters. Hippompus 8, (1998). 24. Chorvtov, A., Genron, L., Biloeu, L., Gllo-Pyet, N. & Pyet, M. D. A Rs-epenent hlorie urrent tivte y renoortiotropin in rt renl zon glomerulos ells. Enorinology 141, (2). 25. Tong, J. et l. NF1-regulte enylyl ylse pthwy. So. Neurosi. Astr. strt no (Soiety for Neurosiene, New Orlens, 2). 26. Ingrm, D. A. et l. Hypertivtion of p21(rs) n the hemtopoieti-speifi Rho GTPse, R2, ooperte to lter the prolifertion of neurofiromin-efiient mst ells in vivo n in vitro. J. Exp. Me. 194, (21). 27. Jks, T. et l. Tumour preisposition in mie heterozygous for trgete muttion in Nf1. Nture Genet. 7, (1994). 28. Umnoff, H., Eelmnn, W., Pellier, A. & Kuherlpti, R. The murine N-rs gene is not essentil for growth n evelopment. Pro. Ntl A. Si. USA 92, (1995). 29. Voikr, V., Koks, S., Vsr, E. & Ruvl, H. Strin n gener ifferenes in the ehviour of mouse lines ommonly use in trnsgeni stuies. Physiol. Behv. 72, (21). 3. Blnton, M. G., Lo Turo, J. J. & Kriegstein, A. R. Whole ell reoring from neurons in slies of reptilin n mmmlin ererl ortex. J. Neurosi. Methos 3, (1989). Aknowlegements We thnk V. Mnne for the BMS191563, n E. Friemn for tehnil ssistne in erlier experiments. We re grteful to M. Br, D. Buonomno, T. Cnnon, J. Colielli, P. Frnkln, L. Kzmrek, A. Mtyni, M. Sners n D. Smith for isussions, n to C. Brnnn n S. Shlussel for enourgement. R.M.C. reeive support from the Grute Progrm in Bsi n Applie Biology (GABBA) of the University of Oporto, the Portuguese Fountion for Siene n Tehnology (FCT) n the Ntionl Neurofiromtosis Fountion (NNF). This work ws lso supporte y generous 4 22 Mmilln Mgzines Lt NATURE 16 Jnury 22 DOI 1.138/nture711

5 vne online pulition ontion from K. M. Spivk, n y grnts from the NIH, Neurofiromtosis In. (Ntionl, Illinois, Mss By Are, Minnesot, Arizon, Knss n Centrl Plins, Mi-Atlnti, n Texs hpters), the Merk n the NNF fountions to A.J.S. Competing interests sttement The uthors elre tht they hve no ompeting finnil interests. letters to nture Corresponene n requests for mterils shoul e resse to A.J.S. (e-mil: Silv@menet.ul.eu) NATURE 16 Jnury 22 DOI 1.138/nture Mmilln Mgzines Lt 5