Olfactory behavior and physiology are disrupted in prion protein knockout mice

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1 Olftory ehvior nd physiology re disrupted in prion protein knokout mie Clire E Le Pihon 1, Mtthew T Vlley 1, Mgdlini Polymenidou 2,3, Alexnder T Chesler 1, Botir T Sgdullev 1,3, Adrino Aguzzi 2 & Sturt Firestein 1 29 Nture Ameri, In. All rights reserved. The prion protein PrP C is infmous for its role in disese, ut its norml physiologil funtion remins unknown. Here we found previously unknown ehviorl phenotype of Prnp / mie in n odor-guided tsk. This phenotype ws mnifest in three Prnp knokout lines on different geneti kgrounds, whih provides strong evidene tht the phenotype is used y lk of PrP C rther thn y other geneti ftors. Prnp / mie lso showed ltered ehvior in seond olftory tsk, suggesting tht the phenotype is olftory speifi. Furthermore, PrP C defiieny ffeted osilltory tivity in the deep lyers of the min olftory ul, s well s dendrodendriti synpti trnsmission etween olftory ul grnule nd mitrl ells. Notly, oth the ehviorl nd eletrophysiologil ltertions found in Prnp / mie were resued y trnsgeni neuronl-speifi expression of PrP C. These dt suggest tht PrP C is importnt in the norml proessing of sensory informtion y the olftory system. Despite two dedes of reserh, the funtion of the ellulr prion protein PrP C is still unknown. It hd een hoped the Prnp / mouse would provide evidene for the funtion of this protein tht is so widely expressed in ll vertertes, t ll stges nd in lmost ll tissues, espeilly in the rin. Suh uiquity suggests tht PrP C might perform some essentil ellulr funtion. However, the first Prnp / mouse showed no overt phenotype, implying tht the protein ws dispensle 1. Insted, the mjor finding in Prnp / mie ws their resistne to prion disese 2. Nevertheless, it is unlikely tht the PrP protein would hve evolved simply to enle rre ftl disese. Indeed, sine the initil knokout mouse study, host of sutle phenotypes hve een desried, rnging from ehviorl hnges to eletrophysiologil nd iohemil ltertions 3. The reported ehviorl phenotypes re of disprte nture, s might e expeted from the widespred expression pttern of PrP C in the rin. They inlude ltered irdin rhythm 4, modified sleep ptterns 5, impired sptil lerning ehvior in the Brnes irulr mze 6 nd inresed sensitivity to seizure 7,8. Despite the wide gmut of ehviors tht hve een tested in PrP knokouts, lmost ll hve relied on sptiovisul or virissottile ues, wheres olftory-ued tsks hve, to the est of our knowledge, een overlooked. Beuse we nd others hd deteted widespred PrP C expression throughout the olftory system 9,1,we resoned tht olftory-medited ehviors might e ffeted in Prnp / mie. The sense of smell is ritil to the survivl of mny nimls, mediting essentil ehviors suh s feeding nd mting. The si iruit of the olftory system in mie nd other mmmls, from sensory epithelium to ortex, onsists of only two projetion synpses (peripherl sensory neuron to mitrl ell in the olftory ul nd mitrl ell to pyrmidl ell in the ortex) nd two lyers of inhiitory lterl proessing (periglomerulr nd grnule ells) in the olftory ul. In prtiulr, mitrl nd grnule ells mke unique dendrodendriti synpse in whih mitrl ells exite grnule ells tht reiprolly inhiit the mitrl ell. This inhiitory iruit is thought to e involved in synhronizing mitrl ell firing nd enling lterl inhiition 11,12. Here we unovered previously unknown phenotype of Prnp / mie in the olftory system y using omintion of geneti, ehviorl nd physiologil tehniques in systems pproh. We employed the so-lled ookie-finding tsk, test of rod olftory uity, to nlyze ttery of mie, inluding PrP C knokouts on multiple geneti kgrounds nd trnsgeni mie in whih Prnp expression ws driven y ell type speifi promoters. In this test, PrP-defiient mie showed impired ehvior tht ws resued in trnsgeni mie expressing PrP C speifilly in neurons ut not in mie expressing only extr-neuronl PrP C. Prnp / mie hd ltered ehvior in n dditionl olftory test (hitution-dishitution), whih ws lso resued y trnsgeni neuronl PrP expression, suggesting the phenotype ws olftory speifi. With this evidene tht the underlying ltertion resided eyond the periphery, we investigted the odor-evoked eletrophysiologil properties of the olftory ul of PrP knokouts. In these mie, we deteted ltertions in the ptterns of osilltory tivity in the olftory ul nd in the plstiity of dendrodendriti synpti trnsmission etween grnule ells nd mitrl ells. We propose tht eletrophysiologil 1 Deprtment of Biologil Sienes, Columi University, 1212 Amsterdm Avenue, New York, New York 127, USA. 2 Institute of Neuropthology, University Hospitl Zürih, Shmelzergstrsse 12, 891 Zürih, Switzerlnd. 3 Present ddresses: Ludwig Institute for Cner Reserh, University of Cliforni t Sn Diego, 95 Gilmn Drive, L Joll, Cliforni 9293, USA (M.P.) nd Weill Medil College of Cornell University, 785 Mmronek Avenue, White Plins, New York 165, USA (B.T.S.). Correspondene should e ddressed to S.F. (sjf24@olumi.edu). Reeived 24 July; epted 6 Novemer; pulished online 21 Deemer 28; doi:1.138/nn VOLUME 12 [ NUMBER 1 [ JANUARY 29 NATURE NEUROSCIENCE

2 29 Nture Ameri, In. All rights reserved. Figure 1 Impired ehvior of Zürih I Prnp / mie in the ookie-finding test. () Tril 1 of the ookie-finding test for (filled dots) nd Zürih I line (ZI) Prnp / (open dots) mie. Eh dot represents single individul. Individuls tht filed the tril were ssigned the onservtive sore of 6 s, whih orresponded to the totl test time. Blk lines represent medins. () Tril 2. Note tht tril times were redued to 5 min. Individuls tht filed tril 2 were given the onservtive sore of 3 s. () Shemti digrm of the geneti mkeup of the wild-type nd Zürih I line Prnp / mie on mixed B6 nd 129 kground. The red ross represents the knokout llele of Prnp. The lk nd yn setions represent lleles of B6 nd 129 origin, respetively. (d) Individul progression of eh mouse from tril 1 to tril 2. (e) Individul progression from tril 1 to tril 2 of eh Zürih I line Prnp / mouse, exluding nimls tht filed tril 1 or tht found the ookie in tril 1 with lteny greter thn 3 s nd lso filed tril 2. n is indited for points tht overlp extly. (f) Averge degree of improvement for wild-type (filled dots) nd knokout (open dots) mie, lulted s P (T1/ T2)/n, exluding nimls tht filed to find the ookie in tril 1 or tht found the ookie in tril 1 with lteny greter thn 3 s nd lso filed tril 2. Error rs represent ± s.e.m. P o.5 (one-tiled unpired t test), P o.1 (two-tiled Mnn-Whitney test). ltertions t the dendrodendriti synpse in the olftory ul ould underlie the ehviorl phenotype tht we found. RESULTS Prnp / mie show ltered ehvior in n olftory tsk We used test tht mesures olftory detetion (ookie-finding test; Fig. 1) 13. Mie tht re fster t retrieving the ookie re thought to hve etter sense of smell. The first of two suessive trils refleted nive olftory-medited finding nd the seond indited the mouse s ility to improve on the sis of positive reinforement reeived in the first tril. In tril 1, wild-type mie retrieved the ookie in medin lteny time of 73 s, wheres Prnp / mie (Zürih I line; Fig. 1) were signifintly slower t 233 s (P o.1, Mnn-Whitney test; Fig. 1). Furthermore, lose to third of the Prnp / mie (6 out of 2) filed to find the ookie in the 1-min test time, wheres no wildtype mouse filed the test. In tril 2, Prnp / mie were gin signifintly slower thn wild types t retrieving the ookie (wild-type medin, 2 s; Prnp / medin, s; P o.1, Mnn-Whitney test; Fig. 1). Even if those Prnp / mie tht hd filed Tril 1 were exluded from the nlysis on the sis of their filure to hve reeived positive reinforement, the differenes were still signifint (wild-type medin, 2 s; Prnp / medin, 83.5 s; P o.1 Mnn-Whitney test). The slower ltenies of Prnp / mie in oth trils were not result of lk of explortion (s ssessed y the numer of rossings from one ge qudrnt to nother), lk of ppetite (s these mie redily onsumed the ookie on finding it) or metoli ltertion (ll tested mie showed similr weight nd dily food onsumption regrdless of genotype) (Supplementry Fig. 1 online nd dt not shown). Furthermore, the differene etween Prnp / nd wild-type mie did not result from loomotor defiieny in the knokouts, s oth performed similrly in ontrol version of this experiment in whih the ookie ws presented on the surfe of the edding insted of eing uried underneth it (Supplementry Fig. 1). Tril 1 Tril Zl Prnp / Zl Prnp / d e f Zl Prnp / Zl Prnp / (n = 9) 6 n = 6 Zl Prnp / (n = 13) n = Tril 1 Tril 2 Tril 1 Tril 2 Tril 1 Tril 2 Compring trils 1 nd 2, we oserved tht wild-type mie improved from medin of 73 s to 2 s, wheres Prnp / mie improved, t est, from 233 s to 83.5 s. Eight out of nine wild-type mie improved etween trils 1 nd 2 (Fig. 1d), ompred with only 8 out of 13 knokouts (nd exluding those tht hd filed tril 1 or 2; Fig. 1e). We lulted n improvement ftor orresponding to the verge rtio of the lteny in tril 1 versus tril 2. Overll, wild-type mie improved threefold (3.12 ±.53 s.e.m.), wheres Prnp / mie only improved twofold (1.97 ±.29; P o.5, one-tiled t test). The differene in the degree of improvement ws even greter onsidering the floor effet on wild-type ltenies resulting from the initil rpidity in tril 1. Thus, Prnp / mie showed impired ehvior in this odor-guided tsk. Prnp / mouse ehvior resemles tht of nosmi mie For omprison with negtive extreme of possile ehviors in this tsk, we tested known nosmi mouse, the denylyl ylse type 3 (AC3) knokout (Ady3 / ). AC3 is omponent of the olftory trnsdution sde tht is neessry for generting tion potentils in response to odornt inding t n odornt reeptor. Ady3 / mie hve een shown to e lrgely nosmi 14, lthough they retin residul olftory pity vi their vomeronsl orgn 15. To ontrol for the mixed geneti kground of oth the Prnp / nd Ady3 / mie, we lso tested oth pure prentl strins C57BL/6J (B6) nd 129/SvEv (129). Beuse of niml vilility, this experiment ws onduted in different fility, neessitting the retesting of the wildtype nd Zürih I line Prnp / mie for omprison with our previous results. The ltered environmentl onditions my explin the rw dt differenes for the nd Prnp / mie etween the two experiments (Fig. 2 versus Fig. 1). Despite these differenes, the sme trend ws pprent under oth experimentl onditions. All wild-type mie, regrdless of strin, hieved muh fster ltenies to retrieve the ookie thn either the Prnp / or Ady3 / mie, sustntil proportion of whih filed oth trils (Fig. 2,). In tril 1, Prnp / mie, similr to Ady3 / mie, trended towrd slower ltenies thn wild types (wild-type medins: Averge improvement ftor NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 1 [ JANUARY 29 61

3 29 Nture Ameri, In. All rights reserved B6 Tril 1 Tril 2 Prnp / Ady3 / s (129), 278 s (B6) nd 119 s (); Prnp / medin, 518 s; Ady3 / medin, 6 s; Fig. 2). In tril 2, Prnp / mie ontinued to resemle Ady3 / mie, filing to improve nd ontrsting signifintly with wild types (wild-type medins: 56 s (129), 79 s (B6) nd 73 s ( F1); Prnp / nd Ady3 / mie, oth 6 s, P o.1 nd P o.5, respetively, Dunn s multiple omprison test; Fig. 2). Prnp / phenotype extends to other geneti kgrounds Beuse the phenotypi impirment hd een deteted in Prnp / mouse on mixed geneti kground nd lking wild-type littermtes, it is possile tht the phenotype tht we deteted ws the result of geneti ftor other thn the sene of PrP C.Wethustestedtwo dditionl Prnp / lines (Fig. 3), one ongeni with B6 (Ngski; Fig. 3) nd one isogeni with 129 (Edinurgh; Fig. 3i), resoning tht if the phenotype were lso oservle on these kgrounds, it might indeed e ttriutle to PrP defiieny rther thn to nother geneti ftor. The Ngski Prnp / mouse line is not usully line of hoie for phenotypi nlysis of PrP defiieny, s the mie develop lte-onset txi s result of spurious upregultion of the downstrem gene B6 Prnp / Ady3 / Figure 2 Zürih I Prnp / mouse ehvior resemles tht of known nosmi mouse (Ady3 / ). () Stter plot showing performne of wild-type strins 129, B6 nd (lk dots), Zürih I line Prnp / (open), nd Ady3 / (gry) in tril 1 of the ookie-finding test. Individuls tht filed to find the ookie in the test time were ssigned the onservtive sore of 6 s, orresponding to the totl test time. Blk lines represent medin vlues. () Tril 2 performnes for the sme mie. P o.5, P o.1, Dunn s multiple omprison test. Note tht vlues for wild-type nd Zürih I line Prnp / mie differ from those in Figure 1 euse this test ws performed under lternte experimentl onditions. Figure 3 The ookie-finding phenotype is mnifest in Prnp knokouts on other geneti kgrounds. () Tril 1 of the ookie-finding test for B6 (filled) nd Ngski (Ng) Prnp / (open). () Tril 2 for mie shown in. Notethe redued timesle of 5 min. () Shemti digrm of geneti kground of the B6 wild-type nd ongeni Ngski Prnp / mie. Blk nd yn setions represent lleles of B6 nd 129 origin, respetively. The red ross represents the knokout llele. (d) Individul progression from tril 1 to tril 2 of eh B6 mouse. (e) Individul progression from tril 1 to tril 2 of eh Ngski Prnp / mouse. (f) Averge degree of improvement for B6 (filled) nd Ngski Prnp / (open). Error rs ± s.e.m. (g) Tril 1 of the ookiefinding test for Edinurgh (Ed) Prnp +/+ (filled) nd Edinurgh Prnp / (open) mie. (h) Tril 2 for mie shown in g. (i) Shemti digrm of geneti kground of the Edinurgh Prnp / nd isogeni wild-type littermtes (129/Ol kground). Cyn setions represent lleles of 129/Ol origin. The red ross represents the knokout llele. (j) Individul progression from tril 1 to tril 2 of eh 129/Ol wild-type mouse. (k) Individul progression from tril 1 to tril 2 of eh Edinurgh Prnp / mouse. (l) Averge degrees of improvement for Edinurgh Prnp / (open) nd Prnp +/+ littermtes (filled) were not signifintly different euse of the low n. Error rs ± s.e.m. Blk lines represent medin vlues. P o.5 (one-tiled unpired t test), P o.1 (two-tiled Mnn Whitney test). Prnd 16. However, these mie show no symptoms efore 1 yer of ge, nd we tested them t the presymptomti ge of 7 1 weeks, whih is well efore their deline (7 weeks). We notied n effet of predominntly B6 geneti kground on ookie-finding ehvior; Ngski Prnp / mie sored fster ltenies thn the Zürih I line (Ngski medin, 155 s; Zürih I line medin, 223s). In tril 1, not single Ngski Prnp / mouse filed to find the ookie, wheres 6 out of 2 Zürih I line Prnp / mie filed (Fig. 3 versus Fig. 1). Nevertheless, the Ngski knokouts were signifintly slower thn their wild-type ounterprts (wild-type medin, 76.5 s; Prnp /, medin 155 s; P o.5, Mnn-Whitney test; Fig. 3), thus reveling phenotype similr to the one tht we deteted in the Zürih I Prnp / line. In tril 2, Ngski knokouts were signifintly slower thn their wild-type ounterprts (wild-type medin, 27.5 s; Prnp / medin, 89.5 s; P o.1, Mnn-Whitney test; Fig. 3). The fstest knokout ltenies in tril 2 lustered round 62 s, lose to doule the medin wild-type lteny (Fig. 3). Although the knokouts tended to improve in tril 2 (Fig. 3e), they filed to improve s muh s wild types (Fig. 3d). Overll, the Prnp / mouse improvement ws lmost twofold less thn wild types (wild-type improvement ftor, 3.84 ±.68 s.e.m.; Prnp / improvement ftor, 1.96 ±.32; P o.5, one-tiled unpired t test; Fig. 3f). The phenotype exhiited y Zürih IlinePrnp knokouts ws thus onfirmed y nother knokout line. However, euse of residul 129 lleles tht re tightly linked to the knokout llele in the Ngski Prnp / mouse line, whih is otherwise ongeni with B6, we still ould not fully ttriute the phenotype to the sene of PrP. We thus tested third Prnp / mouse line, the d g Tril Ed Prnp +/+ Ed Prnp / h Tril Ed Prnp +/+ Ed Prnp / j k l Tril 1 B6 Ng Prnp / B6 Tril 1 Tril 2 e Ed Prnp +/+ Ed Prnp / Ed Prnp +/+ (n = 6) Ed Prnp / (n = 5) 6 n = n = n = 2 n = 4.5 Tril 1 Tril 2 Tril 1 Tril 2 Tril 1 Tril Tril 2 B6 Ng Prnp / Ng Prnp / f Averge improvement ftor i Averge improvement ftor B6 Ng Prnp / Tril 1 Tril 2 Tril 1 Tril 2 Ed Prnp +/+ (129/OI) Ed Prnp / B6 (n = 18) Ng Prnp / (n = 1) 62 VOLUME 12 [ NUMBER 1 [ JANUARY 29 NATURE NEUROSCIENCE

4 Tle 1 Mouse strins tested for ookie finding ehvior Strin nme Arevition Type Desription Referene numer Geneti kground PrP C expression 29 Nture Ameri, In. All rights reserved. C57BL/6J 129/Sv Wild type Wild-type hyrid Methods 5% C57BL/6J, Wild type F1 hyrid 5% 129S/Sv Zürih I Prnp / Knokout Prnp knokout 1 C57BL/6J 129/Sv None mixed kground Ngski Prnp / Knokout Prnp knokout with lte-onset 42 Congeni C57BL/6J None txi s result of upregultion of Prnd Edinurgh Prnp / Knokout Prnp knokout 38 Isogeni 129/Ol None Tg2 Tg2 Trnsgeni Prnp driven y endogenous Prnp promoter ( hlf-genomi onstrut ) 43 Zürih I Prnp / kground Overexpression in wild-type lotions Trnsgeni Prnp driven y Eno2 promoter 44 Zürih I Prnp / kground Neurons only (entrl nd peripherl nervous system) MBP-PrP MBP-PrP Trnsgeni Prnp driven y Mp promoter 45 Zürih I Prnp / kground Oligodendroytes nd Shwnn ells only Tg36 CD19-PrP Trnsgeni Prnp driven y CD19 promoter 46 Zürih I Prnp / kground B ells only Tg33 Lk-PrP Trnsgeni Prnp driven y Lk promoter 47 Zürih I Prnp / kground T ells nd some neurons Prn knokout Prn / Knokout Doule knokout of Prnp nd homologous downstrem gene Prnd 48 C57BL/6J 129/Sv mixed kground None All knokout nd trnsgeni nimls presented in this tle re on the Zürih I mixed B6 nd 129 geneti kground. Notly, the trnsgeni lines were generted y miroinjetion of Prnp trnsgenes into homozygous Zürih I Prnp / zygotes, thus enling diret omprisons etween the different lines. Edinurgh line, on pure 129/Ol kground. These mie re isogeni with their wild-type ounterprts, thus irumventing the prolem of mixed kground (Fig. 3i). On this kground too the phenotype ws pprent. Although Prnp +/+ mie only trended towrd fster ltenies in tril 1 (wild-type medin, s; Prnp / medin, 227 s; Fig. 3g), they were signifintly fster in tril 2 (wild-type medin, 26 s; Prnp / medin, 6 s; P o.5, Mnn-Whitney test; Fig. 3h). In tril 2, four out of six wildtype mie improved to very fst ltenies (Fig. 3j), wheres Prnp / mie showed no ler trend towrd improvement, with four out of six filing the tril (Fig. 3k). The verge improvement ftors were not signifintly different s result of the smll smple size (wild type, 5.16 ± 2.7; Prnp /, 2.44 ± 2.; Fig. 3l). Thus, lthough the severity of the phenotype vried with the geneti kground, we found tht Prnp / mie showed impired ehvior in the ookie-finding test on mixed B6 129, ongeni B6 nd n isogeni 129/Ol kground. The Prnp / phenotype is resued y neuronl PrP expression We next sked whether neuronl-speifi PrP C expression ould seletively resue the phenotype. We tested ttery of knokout nd trnsgeni mie tht were ll on the Zürih I mixed kground (Tle 1 nd Fig. 4). We pooled nimls ording to whether or not they expressed Prnp in neurons, whih we onfirmed y in situ hyridiztion, nd exmined whether neuronl PrP C improved ookie-finding performne. The differene etween the two groups ws notle. In oth trils, the mie lking neuronl PrP C were twie s slow s mie tht expressed PrP C in neurons (P o.1, Mnn- Whitney test; Fig. 4,). This differene ws not result of the effet of ny prtiulr strin, s shown y seprting the groups into individul dtsets (Fig. 4,d). Overexpression of PrP C on Prnp / kground (Tg2 mouse line) exerted resuing effet, s did NSE-driven expression of PrP C (tht is, neuronl-speifi expression, mouse line). Both these lines losely resemled the wild types (Fig. 4,d) with tril 1 medins ll eing elow 1 s nd tril 2 medins eing elow 4 s. In ontrst, when PrP ws expressed in non-neuronl ells suh s myelinting gli (MBP-PrP mouse line) or B ells (CD19-PrP mouse line), the mie were not resued nd phenotypilly resemled the Zürih I line Prnp / mie (Fig. 4,d). In ddition, Prnp nd Prnd ( downstrem gene of Prnp) doule-knokout mie (Prn / ) were lso impired. All mie lking neuronl PrP C showed medin ltenies ove 16 s in tril 1 (Fig. 4) nd ove 125 s intril 2(Fig. 4d). The se of the Lk- PrP mouse line (Fig. 4,d) will e disussed elow. Lk-PrP mie express neuronl Prnp in the olftory ul Notly, the Lk-PrP trnsgeni line (Fig. 4,d) ppered to e t lest prtilly resued y its prtiulr pttern of PrP expression. Lk enodes lymphoyte protein tyrosine kinse nd is highly expressed in T ells. By in situ hyridiztion, however, we found tht the Lk promoter drove Prnp expression in severl rin res (Fig. 5), inluding the olftory ul (in juxtglomerulr ells, mitrl/tufted ells nd grnule ells; Fig. 5) nd the ereellum (Fig. 5). In ontrst, CD19-PrP mie (B ell speifi expressers) showed no suh stining (Fig. 5d). Other reports hve lso deteted n tive Lk promoter in neurons of the rin, inluding in olftory res 17 (Allen Brin Atls, We exluded the Lk-PrP line from the groups listed ove (Fig. 4,), s PrP C ws expressed in some, ut not ll, neurons in these mie. However, the sustntil resue medited y the prtiulr pttern of PrP expression in Lk-PrP mie ould, in ft, point to neuroehviorl regions of importne. In prtiulr, PrP C ws not expressed in the olftory epithelium of these mie (Fig. 5), suggesting tht the sis for the impirment ws not peripherl. In ddition, we oserved norml odor-evoked eletro-olftogrm responses from Prnp / olftory epithelium (Supplementry Fig. 2 online). The physiologil orreltes underlying the impired ehvior thus ppered to reside in entrl strutures. To stremline our investigtion, we restrited our susequent experiments to the use of the wild type, the Zürih I Prnp / line nd the trnsgeni line (Fig. 4e f). Altered ehvior of Prnp / mie in nother olftory tsk To help sertin whether the phenotype of the Prnp / mie in the ookie-finding test ws indeed olftory speifi, we rried out n NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 1 [ JANUARY 29 63

5 29 Nture Ameri, In. All rights reserved. Tril 1 Tril Neuronl PrP C Neuronl PrP C +Neuronl PrPC Neuronl PrP C d 3 e 6 f Tg2 Lk-PrP ZI Prnp / MBP-PrP CD19-PrP Prn / dditionl olftory ehvior test, the hitution-dishitution ssy 18. In this test, suessive presenttions of the sme stimulus odor result in derese of investigtory ehvior (hitution). An inrese in the mouse s interest when novel odor is presented (dishitution) is interpreted s n ility to disriminte the differene etween the two odornts. We used penut utter odor s the hitution odor, mixture of penut utter nd vnill s the first novel odor nd myl ette s seond novel odor. Zürih I line Prnp / mie hituted to the first odor similr to ontrols ( nd ). However, lthough the ontrols showed inresed interest in the novel odors, Prnp / mie did not, inditing ltered olftory ehvior (Fig. 4g). Together with the results of the ookie-finding test, this result strongly suggests tht the phenotype ws indeed olftory speifi ZI Prnp / Tg2 Lk-PrP ZI Prnp / ZI Prnp / MBP-PrP CD19-PrP Prn / Altered responses to odor input in Prnp / olftory ul We foused on the eletrophysiologil properties of the olftory ul iruitry euse the olftory ul is the first rin re to proess olftory informtion nd the ehviorlly resued Lk-PrP mie expressed PrP C in neurons of the olftory ul. We reorded lol field potentils (LFPs, Fig. 6) from this re euse they reflet the verge urrent flow from synpti nd spiking tivity round the g Investigtion durtion (s) ZI Prnp / Penut utter 1 Penut utter 2 Penut utter 3 Penut utter 4 Penut utter + vnill Amyl ette Figure 4 Neuronl PrP expression resues the ookie-finding phenotype. (). Tril 1 for ll lines, neuronl PrP C expressers (+Neuronl PrP C :, Tg2 nd ; filled) nd neuronl PrP C -defiient mie ( Neuronl PrP C :Zürih I Prnp /, MBP-PrP, CD19-PrP nd Prnp / ; open). For +Neuronl PrP C : minimum ¼ 19 s, lower qurtile ¼ 53 s, medin ¼ 7.5 s, upper qurtile ¼ 133 s, mximum ¼ 569 s. For Neuronl PrP C : minimum ¼ 37 s, lower qurtile ¼ 16.5 s, medin ¼ 187 s, upper qurtile ¼ s, mx ¼ 6 s. () Tril 2. Note redued timesle of 5 min. For +Neuronl PrP C : minimum ¼ 6s, lower qurtile ¼ 19.5 s, medin ¼ 3 s, upper qurtile ¼ 49 s, mximum ¼ 3 s. For Neuronl PrP C :min¼ 23 s, lower qurtile ¼ 64 s, medin ¼ 14 s, upper qurtile ¼ s, mx ¼ 3 s. Individuls tht filed the tril were given the onservtive sore of the totl tril length. P o.1, two-tiled Mnn-Whitney test. (,d) Brekdown y strin of dt in nd, respetively. Filled dots represent strins expressing PrP C in neurons nd open dots represent those tht do not. Beuse it expressed PrP C only in some neurons, Lk-PrP (gry) ws not inluded in either group in nd. Lines represent medins. (e,f) Results for the, Zürih I line Prnp / nd mouse lines, our three representtive strins. (g) Altered phenotype of Zürih I line Prnp / mie in the hitution-dishitution test. All mie hituted to the first odor (penut utter). (lk) nd (gry) mie showed strong renewed interest in the novel odors (penut utter + vnill mix nd myl ette), wheres Zürih I line Prnp / mie (open dots) filed to respond to them. Error rs ± s.e.m. P o.5, P o.1, P o.1, two-wy ANOVA, Bonferroni post test. Figure 5 Lk-PrP trnsgeni mie express some neuronl PrP C. ( d) Fluoresent in situ hyridiztion for detetion of Prnp trnsripts in the olftory ul nd ereellum of trnsgeni Lk-PrP mie ( ) nd CD19- PrP mie (d). Left, signl from ntisense Prnp proe. Right, negtive ontrol sense proes. The Prnp proe used here ws suh tht it only reognized wild-type Prnp trnsripts nd not the trunted Prnp trnsript tht is produed from the Zürih I Prnp knokout llele. All slides were deteted over n equl mount of time. Prnp ws expressed in ells of the olftory ul in Lk-PrP mie (). From left to right, rrows point to exmples of Prnp-positive ell in the externl plexiform lyer, mitrl ell nd grnule ells. Prnp expression in the ereellum of Lk-PrP mie (). Arrow points to n exmple of Prnp-positive ell in the moleulr lyer. Some Purkinje ells nd grnule ells re lso leled. Lk-PrP mie do not express Prnp in the olftory epithelium (). Prnp ws not expressed in the olftory ul of CD19-PrP mie (d). The low signl tht n e oserved is result of the kground stining. Sle r represents 1 mm. Olftory ul Lk-PrP Cereellum Olftory epithelium CD19-PrP Olftory ul d Antisense Sense 64 VOLUME 12 [ NUMBER 1 [ JANUARY 29 NATURE NEUROSCIENCE

6 29 Nture Ameri, In. All rights reserved. LFP Brething Prnp / LFP Brething Olftory epithelium Puff odor Amyl ette Olftory ul Eletrode Inhle trnsition Exhle Lterl olftory trt Gmm Power (mv 2 Hz 1 ) e f 1 Gmm power High-gmm power Non-odor reth 6 8 Frequeny Odor Gmm (4 7 Hz) High gmm (7 1 Hz) 1 4 Odor reth Breth Odor d Breth 6 8 Frequeny 1 12 Breths Breths (n = 11) Prnp / (n = 1) (n = 5) % dey Figure 6 Power of LFPs nd durtion of the odor response in Prnp / mie. () LFPs were mesured in vivo from the grnule ell lyer in the min olftory ul of nesthetized mie. () Exmple LFP tres from mouse (top) nd Prnp / mouse (ottom) with orresponding rething tres. Odor presenttion ws triggered y the first inhltion/exhltion trnsition (, irle in left inset), ut the odor ws not deteted until the following inhltion. Right inset, exmple of gmm-rnge osilltions. () Averge power spetr of non-odor reth from wild-type (, n ¼ 11), PrP knokout (Prnp /, n ¼ 1) nd neuronl PrP expressing trnsgeni (, n ¼ 5) mie. The power of high-gmm osilltions ws signifintly lower in PrP knokouts ompred with oth ontrol strins. (d) In n odor-ontining reth, the power of ll frequenies inresed in ll groups. (e) The power of gmm osilltions in eh reth is plotted s fold hnge from seline for 3 reths round 2-s pulse of myl ette. Prnp / mie hd n extended osilltory response to odor in the gmm frequeny nd, s indited y the time (in numer of reths) for the response to dey to 9% of its pek. (f) High-gmm osilltions in the PrP knokout lso showed signifintly longer dey ompred with oth ontrol strins. P o.5 using one-wy ANOVA with post ho PLSD. Sle rs represent 2 ms in nd 5 ms in inset. Prnp / Prnp / reording site (Fig. 6). Furthermore, s vrious frequenies of LFP osilltions speifilly reflet different proesses, n LFP signl simultneously ssys different types of physiologil events. For exmple, gmm osilltions in nesthetized mie (4 1 Hz) reflet tivity originting from speifi synpse etween output neurons (mitrl ells) nd interneurons (grnule ells) lled the dendrodendriti synpse 19,2. In the olftory ul of nesthetized mie, LFP osilltions re oupled to the rething yle, llowing us to use reths s mesure of time (Fig. 6). We mesured the power of LFP osilltions t frequenies rnging from 2 12 Hz over sequene of reths surrounding odor stimultion. We did not find ltertions t et (1 4 Hz) nd delt frequenies (rething rte, 2 3 Hz; dt not shown), nd so we foused our nlysis on gmm (4 7 Hz) nd high-gmm (7 1 Hz) osilltions. During norml (odorless) respirtion, Prnp / nimls showed signifintly lower power thn nd ontrol mie t 88 Hz (men power in 1 3 mv 2 Hz 1 ; Prnp /, 9.1 ± 2.11;, 18.8 ± 2.96;, 22.1 ± 5.57; ANOVA, P o.5; Fig. 6), 93 Hz (Prnp /, 5.81 ± 1.17;, 17. ± 3.5;, 18. ± 4.56; ANOVA, P o.5) nd 98 Hz (Prnp /, 3.99 ±.73;, 12.8 ± 2.32;, 1.5 ± 1.92; ANOVA, P o.5). Similr nlysis of the first reth of odor showed n inrese in the power of osilltions in gmm nd high-gmm ompred with odorless respirtion ut without ny signifint differenes etween the groups (Fig. 6d). Anlysis with finer temporl resolution ws neessry to resolve ny differenes (Fig. 7). Plotting the verge nd power for every reth llowed us to oserve hnges in the kinetis of the odor response. Odor stimultion eliited strong response in oth gmm (Fig. 6e) nd high-gmm (Fig. 6f) nds, visile s shrp inrese in power followed y slow dey. In Prnp / mie, this dey ourred over signifintly lrger numer of reths thn in the ontrol group for oth gmm-nd osilltions (dey time in men numer of reths: Prnp /, 12. ± 1.8;, 5.5 ± 1.8;, 5. ± 1.7; ANOVA, P o.5; Fig. 6e) nd high-gmm osilltions (Prnp /, 1. ± 1.9;, 2.2 ±.3; NSE- PrP, 2.6 ±.8; ANOVA, P o.1; Fig. 6f). Together, the lower power nd sustined durtion of high-frequeny osilltions in the Prnp / mie suggested tht the temporl struture of osilltions in single reth might lso e ltered. LFP osilltions poorly timed to rething in Prnp / mie To etter understnd the osilltory phenotype, we further nlyzed our LFP dt to mesure the emergene nd extintion of LFP osilltions in rething yle. Gmm osilltions in the grnule ell lyer of the olftory ul emerged during exhltion nd were NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 1 [ JANUARY 29 65

7 29 Nture Ameri, In. All rights reserved. 1 Hz 7 Hz 4 Hz 1 Hz Prnp / LFP Brething 12 db db 12 db LFP Brething extinguished shortly fter (Fig. 7 ). To our surprise, the totl rnge of osilltory power during reth ws smller in the Prnp / mie (Fig. 7) ompred with nd mie (Fig. 7,). Furthermore, the distriution of osilltory power in the Prnp / mie ws temporlly diffuse ross n odor reth (Fig. 7), n ltertion tht ws sustined in series of reths following odor exposure (Supplementry Fig. 3 online). We quntified the rnge of osilltory power in non-odor nd n odor-ontining reth (Fig. 7 nd Supplementry Fig. 3) y tking the differene etween the pek of power nd the following trough. In non-odor reth, the hnge in power of high-gmm osilltions in 1 Hz 7 Hz 4 Hz 1 Hz Reord A D No odor 25 Stim 8 ms ISI MC Glu GC MC LOT GC Odor Amyl ette 25 GABA fepsp (GC) fipsp (MC) 2 mv 1 ms 25 LOT fipsp2/fipsp1 fepsp2/fepsp Hz 7 Hz 4 Hz 1 Hz d Power (db) No odor 25 No odor Gmm High gmm LFP Brething Power (db) Prnp / ISI (ms) Prnp / ISI (ms) Figure 8 Pired-pulse synpti plstiity of field potentils in the GCL fter LOT stimultion. () Top, digrm illustrting the stimultion proedure. GC, grnule ell; Glu, glutmte; LOT, lterl olftory trt; MC, mitrl ell. Middle, exmple tre following LOT pired-pulse stimultion (8 ms interstimulus intervl, ISI). () Pired-pulse rtio of the evoked negtive potentil orresponding to mitrl ell fipsp. indites signifint differenes etween oth ontrol groups nd Prnp / (one-wy ANOVA, P o.5); indites signifine from (one-wy ANOVA with post ho PLSD, P o.5). () Pired-pulse rtio of the evoked positive potentil, orresponding to grnule ell fepsp for (filled lk dots, n ¼ 7), Prnp / (open dots, n ¼ 9) nd (filled gry dots, n ¼ 1) mie. 1 1, 1, Odor Amyl ette Odor Gmm High gmm Figure 7 High-frequeny osilltions in PrP knokouts re dmpened in the ourse of single reth. Exmple wveforms from single mie demonstrting how nd power in high-gmm (G, 7 1 Hz), gmm (g, 4 7 Hz) nd et (, 1 4 Hz) frequenies hnged round the point t whih mouse egn to exhle ( trnsition, the midline of the spetrogrms s mrked y vertil lines, units in ms). The left exmple is reth without odor stimultion nd the right ontins the first inhltion of n odor pulse. Below eh LFP nd rething wveform is the verged spetrogrm from the entire group, orresponding to reth 1 nd reth 5 (dshed oxes in Supplementry Fig. 3). ( ), () Prnp / () nd () mieehshowed similrly strutured osilltory ptterns round non-odor reth nd n odor reth. (d) However, the differene etween the nd-verged pek nd susequent trough of spetrl power indite tht Prnp / mie hve less hnge in the highgmm nd nd less hnge during odor presenttion in the gmm nd. P o.5 using one-wy ANOVA with post ho PLSD. the Prnp / mie ws redued ompred with nd mie (men hnge in power (db): Prnp /, 6.5 ±.9;, 11.5 ±.8;, 11.6 ± 1.5; Fig. 7d). Similrly, in n odor-ontining reth, oth gmm nd high-gmm osilltions showed less hnge in power in Prnp / mie (Prnp /, 5.9 ±.9;, 9.7 ± 1.1;, 9.5 ±.6; Fig. 7d). Osilltions t these high-frequeny nds (gmm nd high gmm) re elieved to result from tivity t the dendrodendriti synpse 19,2. The oserved ltertions in oth power nd timing suggested tht the properties of the dendrodendriti synpse might e ffeted in the Prnp / mie. Altered pired-pulse plstiity of dendrodendriti synpse We next exmined the Prnp / dendrodendriti synpse for hnges tht ould underlie the oserved ehviorl phenotypes. We foused on the short-term plstiity of this synpse, s our LFP results suggested tht Prnp / mie might hve disrupted synhroniztion etween rething nd osilltions, perhps refleting ltered filittion or depression of this synpse. We therefore rried out pired-pulse stimultion of the synpse y ntidromilly exiting mitrl ells from their xon undle in the lterl olftory trt (LOT) (Fig. 8). This stimultion proedure produes distint field potentils orresponding to grnule ell exittion (field exittory postsynpti potentils, fepsps) followed y mitrl ell inhiition 21,22 (field inhiitory postsynpti potentils, fipsps; Fig. 8). In Prnp / mie, reiprol inhiition of mitrl ells (fipsp) showed unusul filittion over rnge of interstimulus intervls (Fig. 8). nd mie hd signifintly filitted pired-pulse rtio from the Prnp / mie t intervls etween 8 nd 1 ms, nd mie lso showed signifintly different rtio t 5 ms (ANOVA with Fisher s PLSD, P o.5). Notly, filittion of the fipsp in the Prnp / mie ws not ompnied y ny differenes in the plstiity of the grnule ell fepsps (Fig. 8). DISCUSSION Here we desrie previously unknown olftory ehviorl phenotype of Prnp / mie nd physiologil ltertions in their olftory ul. The ookie-finding phenotype ws mnifest in three Prnp / lines on lternte geneti kgrounds, whih is strong evidene of its 66 VOLUME 12 [ NUMBER 1 [ JANUARY 29 NATURE NEUROSCIENCE

8 29 Nture Ameri, In. All rights reserved. dependene on PrP C rther thn on other geneti ftors. PrP knokouts lso showed ltered ehvior in the hitution-dishitution tsk, suggesting tht the phenotype ws proly olftory speifi. Prnp / mie hd widespred ltertions of osilltory tivity in the olftory ul nd ltered pired-pulse plstiity t the dendrodendriti synpse. Notly, oth the ehviorl nd eletrophysiologil phenotypes ould e resued y neuronl PrP C expression. These dt suggest ritil role for PrP C in the norml proessing of sensory informtion y the olftory system. Prnp / mouse ookie-finding ehvior mrkedly resemled tht of the nosmi Ady3 / mie; however, Prnp / mie re lerly not nosmi. Indeed, no spet of Prnp / mouse survivl suggested tht they might hror defiit in n odor-guided tsk. Anosmi pups hve n 8% neontl ftlity rte s result of diffiulty in sukling t irth nd indequte mternl re, nd those tht survive hve low ody weight during their first 3 months 14.Inontrst,Prnp / mie hve helthy litters of verge size (B6-9 pups per litter) tht grow to norml ody weights. The lk of outwrd signs of nosmi is proly reson why olftory tsks hve een overlooked in previous ehviorl hrteriztions of Prnp / mie. The ehviorl impirment tht we deteted in PrP knokouts did not originte in the periphery. This is supported y the norml pperne of odor-evoked eletro-olftogrm responses from Prnp / olftory epithelium nd y the resued ehvior of Lk-PrP mie tht do not express PrP C in their olftory sensory neurons ut do in susets of entrl neurons, inluding in the olftory ul. Therefore, the Prnp / ehvior defiit proly rises from ltertions in entrl proessing events in the olftory ul nd/or higher enters. One initil onern regrding the ehviorl phenotype ws the mixed kground of the Zürih I Prnp / mie. Any phenotype of Zürih I knokout mie ould e the result of genes with 129 origin tht re linked to Prnp rther thn to the knokout llele itself. The mrked impirments tht we hd deteted thus neessitted utious interprettion. We onfirmed the phenotype of the Zürih I knokouts through two strtegies: testing lternte Prnp / mouse lines with different geneti kgrounds (B6 ongeni (Ngski) nd 129 isogeni (Edinurgh)) nd using trnsgeni lines on the Zürih I kground tht express PrP C in speifi ell sutypes. The use of multiple knokout lines illustrted how geneti kground n modulte phenotypi severity. For exmple, lthough the Ngski knokouts sored onsistently slower ltenies thn their wild-type B6 ounterprts, they were, on verge, fster thn the Zürih I line knokouts. Furthermore, no Ngski individul filed the test wheres third of Zürih I line knokouts filed eh tril. A predominntly B6 kground thus ppered to ttenute the phenotype, lthough no differene ws pprent etween wild-type B6 nd 129 strins, perhps s result of floor effet, with ll wild types hieving n unsurpssle threshold of rpidity. The trnsgeni pproh demonstrted tht the phenotype ws neuronl speifi. nd Lk-PrP mie were resued, wheres MBP-PrP nd CD19-PrP mie were not. Additionl tested lines ll segregted in similr fshion, ording to whether or not they expressed neuronl PrP C. Perhps most notly, the resued ehvior of the mie indites tht the Prnp / phenotype ws indeed the result of the sene of PrP nd not of genes in the viinity of Prnp, s the introdution of the PrP trnsgene lone suffied to medite the resue. Thus, lthough the phenotype ws ttriutle to lk of PrP, its olftory speifiity remined unertin euse of the ehviorl omplexity of the ookie-finding test. Food onsumption nd ody weight of Prnp / mie ppered to e no different from ontrols, llowing us to rule out ny possile ltertions in metolism or ppetite. Knokouts performed similrly to wild types in ontrol version of the experiment, in whih the food stimulus ws no longer oneled eneth the edding. Knokouts were therefore fully ple of nvigting the test ge nd loting the visile ookie, suggesting the defiit in the ookie-finding test ws thus neither loomotor nor explortory. Furthermore, Prnp / mie hve een doumented to perform normlly in tests using extensive loomotor skills, suh s the Morris wter mze 1. Notly, in n dditionl olftory ssy, Prnp / mie lso showed ltered ehvior, filing to ret to novel odor tht ws disriminle y nd mie. Together, the phenotypes in the ookie-finding nd the hitution-dishitution tests pointed to n olftory-speifi phenotype. We therefore foused our follow-up investigtion on the olftory ul, s it ontins the initil synpse of the olftory system nd the first iruit to integrte sensory nd higher ortil informtion. We oserved disruptions in LFP osilltions nd in the plstiity of the dendrodendriti synpse, either or oth of whih ould ontriute to the Prnp / ehviorl phenotype. Osilltory LFPs my t to orgnize informtion flow in the olftory system 23,24 y onstrining the timing of mitrl ell tion potentils 25. In ddition, gmm osilltions re speifilly implited in ehviorl performne in olftory tsks Therefore, ltertions in osilltory timing during odor exposure my pertur olftory ul output to higher enters y disrupting how informtion is pkged in rething yle. Altering the dendrodendriti synpse my hve multiple funtionl onsequenes. This synpse my medite lterl inhiition etween ensemles of mitrl ells nd my e ritil for olftory disrimintion 29,3. In ddition, euse grnule ells reeive onvergent informtion onto their proximl dendriti ror from multiple higher rin res 31, disruption of the dendrodendriti synpse my lter the trnsmission of entrifugl modultion to olftory ul mitrl ells. High-frequeny osilltions in the olftory ul (gmm nd high gmm) hve een shown in vitro to result from the rpid nd reiprol intertions etween grnule nd mitrl ells ross the dendrodendriti synpse 19,2. Therefore, our dt ould imply tht inresed filittion of the mitrl ell IPSP following repetitive spiking dereses the dynmi rnge nd inreses the durtion of gmm osilltions ross the oundries of reth. Unfortuntely, not enough is urrently known out how hnges in si prmeters of synpti physiology mnifest themselves on the sle of lol field potentils in vivo. Thus, lthough oth osilltory nd synpti effets ould e reversed y neuronl PrP C expression, we nnot lim usl link etween these findings. Other physiologil ltertions reported in Prnp / mie inlude ltered GABA-medited synpti urrents in CA1 neurons of the hippompus 32 (ut see ref. 33), ltered long-term nd post-tetni potentition 34, nd ltered pired-pulse plstiity in the dentte gyrus 6. Given tht PrP C is memrne ssoited, synptilly enrihed 35 nd present in the externl plexiform lyer of the olftory ul 1,PrP C my funtion s memer of the synpti mhinery in the olftory ul nd the hippompus. Puttive moleulr prtners of PrP C inlude synpti moleules suh s synpsin I 36. We oserved tht mitrl ells reeived filitted inhiition in Prnp / mie. This filittion ould result from either pre- nd/or postsynpti hnges to the dendrodendriti synpse. Future work should determine the preise synpti loliztion of the PrP C protein nd its iohemil intertions with synpti mhinery. It lso remins to e seen whether higher enters involved in olftory proessing nd NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 1 [ JANUARY 29 67

9 29 Nture Ameri, In. All rights reserved. memory re similrly ffeted y lk of PrP C or whether nlogous synpti ltertions n e deteted in other rin regions. Furthermore, the trnsgeni resue strtegy tht we used nnot indite whether the oserved phenotypes re the result of developmentl hnges in olftory iruitry. Future use of onditionl strtegies using tissue-speifi promoters my llow more preise dissetion of the physiologil nd ehviorl importne of PrP C for olftory proessing. Although the physiologil funtion of PrP C is unknown, its role in the pthogenesis of prion diseses hs een estlished eyond resonle dout 2. The srity of ny mrked pthologil phenotypes, prtiulrly in the nervous system, of Prnp-lted mie ws originlly tken s evidene tht loss-of-funtion phenomen re not importnt in prion diseses 37. Our findings suggest tht more nuned view my e pproprite nd tht t lest some omponents of the neurologil phenotype of prion infetions my e ssigned to the mlfuntion of PrP C -dependent neuronl events. METHODS Animls. All of the PrP-relted knokout nd trnsgeni nimls shown in Tle 1 were provided y A. Aguzzi (University Hospitl of Zürih). Beuse the Zürih I Prnp / mie 1 re on mixed C57BL6/J (B6) nd 129/SvEv (129) geneti kground nd lk wild-type littermtes, we used the F1 hyrid strin of B6 nd 129 () s the wild-type ontrol. Ady3 / mie, lso on mixed B6 nd 129 kground, were otined from D. Storm (University of Wshington) 14. Use of the Edinurgh Prnp / mie nd wild-type littermtes 38 ws kindly permitted y the Institute for Animl Helth nd J. Mnson (University of Edinurgh). All nimls were housed t either Columi University or the University Hospitl of Zürih in ordne with institutionl requirements for niml re. The Columi University Institutionl Animl Cre nd Use Committee nd the Cntonl Animl Experimenttion Committee (Zürih) reviewed nd pproved these experiments. Cookie finding ehvior test. In this test, ookie ws uried under the ge edding so s to offer purely olftory ue, nd the time tken y mouse to retrieve the ookie ws reorded. Hitution-dishitution test. The initil interest in n odor presented severl times in suession is expeted to derese with eh presenttion s the niml hitutes to the odor. On the fifth presenttion, novel odor is presented. The novelty of the odor should indue n inrese in the niml s investigtion time nd this is interpreted s n ility to disriminte etween odors 1 nd 2. Odor delivery. A ustom-mde olftometer ws dpted from previous design 39. Compressed ir ws humidified nd pssed y the mouse s nose. Odor puffs (2 s) were diverted into the rrier strem. For every mouse, odor ws delivered t lest seven times, sped prt with pulses of solvent hedspe. Eletrophysiology reordings. The nesthetized mouse s nose ws inserted into n ir-tight gs msk through whih humidified ir from the olftometer ws pssed. Two rniotomies were performed for insertion of ustom-mde tungsten reording eletrode 4 into the grnule ell lyer of the min olftory ul, nd ustom-mde ipolr tungsten stimulting eletrode into the LOT. Brething ws monitored with piezoeletri fore-trnsduer (Stoelting); this signl ws used to relily trigger odor delivery t the trnsition of inhltion to exhltion. LFP signl proessing nd nlysis. All signl proessing ws done off-line using ustom-written sripts in Spike2, nd in Mtl (Mthworks) using omintion of ustom-written sripts nd eegl Sttistis. For our ehvior experiments, sttistil nlysis ws performed using Prism softwre (GrphPd). Cookie-finding dt were nlyzed using nonprmetri sttistis, s the ltenies to retrieve the ookie did not follow norml distriution. The Mnn-Whitney test ws used for omprison etween two groups. For omprison etween more thn two groups, we used the Kruskl-Wllis one-wy ANOVA followed y Dunn s post ho nlysis when signifint overll min effet ws found (P o.5). Hitutiondishitution dt were nlyzed using one-wy ANOVA followed y the Bonferroni test when signifint min effet ws found (P o.5). For our physiology experiments, sttistil nlysis ws rried out using SttView 5. (SAS Institute). Dt from three experimentl groups ws ompred using one-wy ANOVA test followed y post ho nlysis using Fisher s PLSD when signifint overll min effet ws found (P o.5). For full detils, see Supplementry Methods online. Note: Supplementry informtion is ville on the Nture Neurosiene wesite. ACKNOWLEDGMENTS The uthors thnk memers of the Aguzzi lortory for their ssistne, espeilly G. Miele nd P. Shwrtz. We lso thnk J. Mnson for the use of the Edinurgh Prnp / line, D.-J. Zou nd D. Kelley for helpful omments on the ehviorl experiments, nd J. Gordon for vlule disussions on the eletrophysiology dt. This work ws supported y grnts from the Ntionl Institute on Defness nd Other Communition Disorders (S.F., C.E.L.P., M.T.V., B.T.S. nd A.T.C.). C.E.L.P. lso reeived Short-Term Fellowship from the Europen Moleulr Biology Orgniztion. A.A. nd M.P. were supported y grnts from the Europen Community nd the Swiss Ntionl Siene Foundtion. AUTHOR CONTRIBUTIONS C.E.L.P., A.T.C., M.P., A.A. nd S.F. designed the ehvior experiments. M.T.V., B.T.S. nd S.F. oneived the eletrophysiology experiments. C.E.L.P., A.T.C. nd M.P. rried out the ookie-finding ehvior experiments nd C.E.L.P. nd M.T.V. performed the hitution-dishitution test. C.E.L.P. nlyzed ll of the ehvior experiments nd rried out the ehvior ontrol experiments. B.T.S. designed the eletrophysiology setup. M.T.V. performed the eletrophysiology experiments nd their nlysis. C.E.L.P., M.T.V. nd S.F. wrote the pper. All the uthors disussed the results nd ommented on the mnusript. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Bueler, H. et l. Norml development nd ehviour of mie lking the neuronl ellsurfe PrP protein. Nture 356, (1992). 2. 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