25 Nture Pulishing Group http://www.nture.om/ntureneurosiene Bistility of ereellr Purkinje ells moulte y sensory stimultion Yontn Loewenstein 1 3,6, Séverine Mhon 4,6, Pul Cherton 4, Kzuo Kitmur 4, Him Sompolinsky 2,5, Yosef Yrom 1,2 & Mihel Häusser 4 A persistent hnge in neuronl tivity fter rief stimuli is ommon feture of mny neuronl miroiruits. This persistent tivity n e sustine y ongoing reverernt network tivity or y the intrinsi iophysil properties of iniviul ells. Here we emonstrte tht rt n guine pig ereellr Purkinje ells in vivo show istility of memrne potentil n spike output on the time sle of seons. The trnsition etween memrne potentil sttes n e iiretionlly triggere y the sme rief urrent pulses. We lso show tht sensory tivtion of the liming fier input n swith Purkinje ells etween the two sttes. The intrinsi nture of Purkinje ell istility n its ontrol y sensory input n e expline y simple iophysil moel. Purkinje ell istility my hve key role in the short-term proessing n storge of sensory informtion in the ereellr ortex. Persistent firing of neurons fter trnsient stimulus is ommon feture of sensory proessing in neurl iruits tht hs een shown to e ssoite with short-term memory tsks 1. He propose tht suh persistent firing sttes re generte y ell ssemlies with mutully reinforing exittory feek onnetions. Inee, reent theoretil n experimentl work 2 4 hs shown tht lol reurrent networks re ple of generting suh sustine firing ptterns. An lterntive possiility is tht persistent firing is sustine y the intrinsi properties of the neuron 5. Severl neuronl types show istle ehvior: rief exittion proues prolonge inrese in firing rte through tivtion of persistent inwr urrents tht mintin epolriztion 5 8. Some neurons show multiple persistent firing sttes tht reflet the history of their synpti inputs 9. Theoretilly, single ell istle or multistle sttes n funtion s short-term memory uffers or enhne the short-term memory pilities of exittory reurrent networks 4,5,1 12. In ontrst to the ererl ortex, the ereellum lks prominent reurrent exittory synpti network. Purkinje ells provie the sole output of the ereellr ortex. They reeive exittory input from two istint soures, the prllel fiers n the liming fiers, n exert inhiitory ontrol on their trgets in the eep ereellr nulei. Eh Purkinje ell is innervte y over 1 5 prllel fiers, whih synpse on its elorte lef-like enriti tree. Eh Purkinje ell lso reeives input from single liming fier xon, originting from neuron in the inferior olive. The liming fier wrps roun the proximl enrites of the Purkinje ell, mking hunres of synpti ontts. Purkinje ells n fire spontneous soium tion potentils (known s simple spikes ) in the sene of synpti exittion 13. In response to liming fier tivtion, they generte stereotypi ishrge pttern lle omplex spike, whih n e istinguishe from ongoing simple spikes oth intrellulrly n extrellulrly 14. Previous intrellulr in vitro stuies hve shown tht Purkinje ells n show oth spontneous n urrent-evoke istle ehvior tht is orrelte with intermittent perios of simple spike ishrge 6,15. However, istility in Purkinje ells in vivo n its potentil implitions for ereellr informtion proessing hve not yet een investigte. Here we show tht Purkinje ells in vivo show prominent ongoing istility of memrne potentil n spike output. Notly, the stte of the neuron n e ontrolle y sensoryevoke liming fier input tht n inue stte trnsitions in oth iretions, triggering prolonge inreses or ereses in the simple spike firing rte. RESULTS Purkinje ell memrne potentil is istle in vivo To explore the ynmi interply etween intrinsi properties n physiologil synpti inputs in Purkinje ells, we otine in vivo whole-ell reorings from rt n guine pig Purkinje ells lote in the ereellr vermis n hemispheres. Purkinje ells, ientifie from the spontneous ourrene of omplex spikes t their hrteristi frequeny ( 1 Hz) 14, showe trnsitions in memrne potentil etween hyperpolrize stte ( own stte ) n epolrize stte ( up stte ). The hyperpolrize stte ws quiesent (tht is, evoi of ny simple spike tivity), wheres the epolrize stte ws usully ssoite with simple spike ishrge (Fig. 1,, left). Trnsitions etween the own stte n the up stte were oserve t either perisomti (Fig. 1) or enriti (Fig. 1) lotions, n the existene of these two istint sttes ws pprent in the imol istriution of the memrne 1 Deprtment of Neuroiology, 2 The Interisiplinry Center for Neurl Computtion, n 5 Rh Institute of Physis, The Herew University of Jeruslem, Jeruslem 9194, Isrel. 3 Deprtment of Brin n Cognitive Sienes, The Msshusetts Institute of Tehnology, Cmrige, Msshusetts 2139, USA. 4 Wolfson Institute for Biomeil Reserh n Deprtment of Physiology, University College Lonon, Gower Street, Lonon WC1E 6BT, UK. 6 These uthors ontriute eqully to this work. Corresponene shoul e resse to Y.L. (yontnl@mit.eu) or S.M. (s.mhon@ul..uk). Pulishe online 23 Jnury 25; oi:1.138/nn1393 22 VOLUME 8 NUMBER 2 FEBRUARY 25 NATURE NEUROSCIENCE
25 Nture Pulishing Group http://www.nture.om/ntureneurosiene 42 mv 57 mv 45 mv 6 mv 2 mv potentil (Fig. 1,, right). A ip test 16, whih gives the likelihoo of rwing smple from unimol istriution, ws use to ssess the sttistil signifine of this imolity. In ll reore Purkinje ells (n = 31), the istriution of the memrne potentil evite signifintly from unimol istriution (P <.2, n = 3 ells; P <.5, n = 1 ell). Purkinje ells showe the hrteristi eletrophysiologil properties reporte for this ell type. As previously esrie 15, Purkinje ells respone to long hyperpolrizing urrent pulses with slow epolrizing sg towrs the prestimulus memrne potentil, initive of the presene of n I h urrent (see Supplementry Fig. 1). The sg rtio, efine s the stey-stte versus pek efletion uring hyperpolrizing urrent pulse in the own stte, ws.5 ±.5 (men ± s.e.m.; n = 11 rt Purkinje ells). Aross the popultion, in ketmine-xylzine nesthetize rts, the verge vlues of memrne potentil in own n up sttes were 61.6 ±.7 mv n 44.5 ±.8 mv, respetively (n = 24 ells), generting voltge ifferene etween the two sttes of 17.1 ± 1.1 mv (n = 24 ells). Spontneous flututions in memrne potentil uner riturte nesthesi were similr to those oserve uner ketmine-xylzine nesthesi, with the verge memrne potentil in the own stte ( 61.7 ± 1.4 mv, n = 5 ells) n the up stte ( 44.9 ± 1.6 mv, n = 5 ells) not signifintly ifferent (P >.8; Fig. 1). This suggests tht imolity in Purkinje ells oes not epen on the type of nesthesi use. Similr imolity ws lso oserve in whole-ell reorings from Purkinje ells in ketmine- xylzine nesthetize guine pigs (n = 2). The urtions of up n own sttes were quntifie in rt Purkinje ells using long perios of ontinuous reoring. The time spent y 1 s 2 mv 2 s Frtion of time (%) Frtion of time (%) 3 2 1 7 6 5 4 3 Memrne potentil (mv) 3 2 1 7 6 5 4 3 Memrne potentil (mv) Figure 2 Memrne potentil istility is speifi feture of Purkinje ells within the ereellr ortex. ( ) Eletrophysiologil properties of rt ereellr grnule ells in vivo. () Voltge responses of grnule ell to hyperpolrizing ( 1 pa) n epolrizing (+3 pa) urrent injetion (R in = 872 MΩ). (,) Spontneous tivity of grnule ell reore t rest (, 59 mv) n the orresponing memrne potentil istriution () lulte from 2 s of reoring (in size 1 mv). ( f) Eletrophysiologil properties of rt moleulr lyer interneurons in vivo. () Voltge responses of moleulr lyer interneuron to hyperpolrizing ( 7 pa) n epolrizing (+65 pa) urrent injetion (R in = 13 MΩ). (e,f) Spontneous tivity of moleulr lyer interneuron reore t rest (e, 63 mv) n the orresponing memrne potentil istriution (f) lulte from 2 s of reoring (in size 1 mv). Pnels n re from ifferent ells, s re n e. Memrne potentil (mv) Down stte urtion (s) 4 5 6 7 8 5 4 3 2 1 D U D U Ket.-xyl. Briturte 1 2 3 4 5 Up stte urtion (s) Purkinje ell in given stte vrie onsierly etween n within ells (Fig. 1). The men urtions of own n up sttes (verge over the men vlues of eh ell) were 1.5 ±.25 s (rnge:.41 3.61 s) n 1.45 ±.28 s (rnge:.25 4.84 s), respetively (n = 17 ells; Fig. 1), n the orresponing oeffiient of vrine (CV) vlues were.74 ±.6 for own sttes n.58 ±.5 for up sttes. We lso exmine the time etween suessive trnsitions to the own stte. The men urtion etween two suessive up-to-own trnsitions ws 2.98 ±.48 s with orresponing CV of.54 ±.3 (n = 17 ells). These results suggest tht the reurrene of up n own sttes in Purkinje ells is rther irregulr n is thus unlikely to result from simple unerlying osilltory proess (see Supplementry Note). To etermine whether memrne potentil imolity is n intrinsi property of Purkinje ells or whether it reflets imol synpti input, we ompre the intrellulr tivity of Purkinje ells with tht of their input neurons: grnule ells n moleulr lyer interneurons. Grnule ells were ientifie y their epth within ereellr ortex, their high input resistne (795 ± 88.4 MΩ, n = 6 ells) n their highfrequeny non- ommoting spike ishrge in response to strong epolrizing urrent pulses (Fig. 2). Reorings from grnule ells Frtion of time (%) 3 2 1 Grnule ell 7 6 5 4 Memrne potentil (mv) Figure 1 Spontneous memrne potentil istility in Purkinje ells in vivo. () Left: whole-ell perisomti reoring of rt Purkinje ell showing memrne potentil flututions etween two istint sttes: hyperpolrize quiesent stte ( own stte ) n epolrize spiking stte ( up stte ). Right: the orresponing histogrm of the memrne potentil lulte from 84 s of reoring (in size 1 mv). () Presume istl enriti whole-ell reoring of rt Purkinje ell showing similr up n own stte trnsitions (left) s reflete in the imol istriution of the memrne potentil (right, 1 s of reoring, in size 1 mv). () Comprison of the verge vlues of the two memrne potentil sttes (own stte, D; up stte, U) otine uner ifferent nesthetis (ketmine-xylzine (Ket.-xyl.), n = 24 Purkinje ells; riturte, n = 5 Purkinje ells). () Reltionship etween the urtion of own n up sttes. Eh point orrespons to single Purkinje ell. Error rs represent s.e.m. 2 mv 5 pa 1 ms 5 ms 2 mv e f Frtion of time (%) 2 1 Interneuron 2 mv 1 pa 1 ms 5 ms 7 6 5 4 Memrne potentil (mv) 2 mv NATURE NEUROSCIENCE VOLUME 8 NUMBER 2 FEBRUARY 25 23
25 Nture Pulishing Group http://www.nture.om/ntureneurosiene Extrellulr Cell-tthe Whole-ell were hrterize y frequent spontneous exittory postsynpti potentils tht oul on osion trigger spike ishrge (Fig. 2). The memrne potentil istriution of grnule ells ws unimol (P >.7; n = 6 ells) with men memrne potentil of 64.3 ± 1.9 mv (Fig. 2). These properties re onsistent with those previously esrie in vivo for this ell type 17. In greement with previous stuies 18,19, moleulr lyer interneurons in vivo showe lower input resistne (224. ± 34.3 MΩ, n = 1 ells; Fig. 2) n spontneous spiking t rest (Fig. 2e), whih ws reflete in the unimol (P >.5, n = 8 ells; P >.1, n = 2 ells) istriution of their memrne potentil roun men vlue of 56.1 ± 1.4 mv (Fig. 2f). Grnule ells n interneurons i not show imolity even when reorings from imol Purkinje ells were otine in the sme preprtion (n = 5). The existene of two istint sttes of memrne potentil in Purkinje ells shoul e reflete in extrellulr mesurements of spontneous spiking. Inee, trnsitions etween quiesent n spiking sttes were oserve in our extrellulr reorings in vivo (Fig. 3). The firing pttern of guine pig Purkinje ells reore extrellulrly onsiste of high-frequeny trins of simple spikes lternting with quiesent perios (Fig. 3, left). This firing pttern resulte in imol Figure 4 Intrinsi origin of memrne potentil istility in vivo. () Whole-ell perisomti reoring of istle ehvior of rt Purkinje ell uner riturte nesthesi (left). The orresponing histogrm of memrne potentil istriution lulte from 33 s of reoring (right, in size 1 mv) shows two istint peks refleting the two sttes of memrne potentil. () Bimolity in the sme Purkinje ell ws olishe y injetion of negtive DC urrent ( 8 pa, left). Note the unimol istriution of memrne potentil (right, 3 s of reoring, in size 1 mv). The mplitue of spontneous omplex spikes hs een trunte. () Whole-ell reorings (upper tres) of rt Purkinje ell in vivo showing tht the neuron n e swithe from the own stte to the up stte n from the up stte to the own stte following the injetion of rief epolrizing (left) n hyperpolrizing (right) urrent pulses, respetively (lower tres; 4 ms,.16 na/.4 na). () Whole-ell reoring (upper tre) from guine pig Purkinje ell in vivo showing tht the sme hyperpolrizing urrent pulse (lower tre; 1 ms, 2 na) n inue upto-own n own-to-up trnsitions epening on the initil stte of the memrne potentil. 1 s Proility.85.1 1 2 Rte (Hz) 4 ms 2 pa 1 mv Figure 3 Memrne potentil istility is reflete in spike output pttern in vivo. () Left: extrellulr reoring from guine pig Purkinje ell in vivo. Firing pttern onsiste of high-frequeny ursts of simple spikes lternting with quiesent perios. Right, imol istriution of the instntneous frequeny of simple spikes lulte from 1 min of ontinuous reoring (in size 5 Hz). () Cell-tthe pth-lmp reoring showing the imol firing pttern of rt Purkinje ell in vivo. () Intrellulr tivity of the sme Purkinje ell otine immeitely fter the formtion of the whole-ell onfigurtion, showing tht the imol tion potentil ishrge oserve in the ell-tthe reoring reflets memrne potentil imolity. istriution of the instntneous frequeny of simple spikes (Fig. 3, right). The prolonge perios evoi of simple spikes ounte for the pek t Hz, wheres the pek roun 1 Hz orrespone to the firing rte uring the ursts of high-frequeny simple spike firing. Aross the popultion, the verge urtions of tive n quiesent perios were.9 ±.2 s n 3.4 ±.8 s, respetively (n = 28 ells). Similr spiking ptterns were lso oserve in ell-tthe reorings from rt Purkinje ells (n = 9 ells, Fig. 3). To ssess the effet of the whole-ell onfigurtion on the memrne imolity, we performe ell-tthe n whole-ell reoring in the sme Purkinje ells (n = 6 ells; Fig. 3,). The intrellulrly reore memrne potentil, otine immeitely fter the estlishment of the whole-ell onfigurtion, showe tht the imol simple spike ishrge oserve in the ell-tthe reoring reflete unerlying memrne potentil imolity (Fig. 3,). The men instntneous frequeny of simple spikes in the ell-tthe onfigurtion (12.1 ± 3.8 Hz) ws omprle to tht mesure in the whole-ell onfigurtion uring the up stte (11.7 ± 3.3 Hz; P >.5). Aross the popultion, no signifint ifferene in the men firing rte etween the two onfigurtions ws oserve (P >.6, 65mV 8 mv Rest Hyperpolrize 1 mv 5 ms Frtion of time (%) Frtion of time (%) 12 8 4 9 7 5 3 Memrne potentil (mv) 3 2 1 9 7 5 3 Memrne potentil (mv) 5 ms 5 s 2 mv 2 pa 2 mv 1 na 24 VOLUME 8 NUMBER 2 FEBRUARY 25 NATURE NEUROSCIENCE
25 Nture Pulishing Group http://www.nture.om/ntureneurosiene 57 mv CF stim. In vitro In vivo / extrellulr - urst en * - urst onset 1 mv 2 s * 1 ms.1.1 n = 6 ells), initing tht the whole-ell onfigurtion i not ffet the memrne potentil ynmis. To unerstn the origin of Purkinje ell memrne imolity, we exmine its voltge epenene. Spontneous up n own stte trnsitions (Fig. 4) oul e olishe y injetion of negtive DC urrent ( 38 ± 9 pa, n = 9 ells), whih hyperpolrize Purkinje ells to 8.6 ± 2.5 mv (Fig. 4). When Purkinje ells were hyperpolrize, only omplex spikes were reore (Fig. 4, left) n the memrne potentil histogrm (Fig. 4, right) showe ell-shpe istriution roun the men holing potentil. Similrly, onstnt epolrizing urrent resulte in single up stte (t not shown). Wheres stey urrent injetion oul prevent memrne potentil imolity, rief urrent injetions were le to inue trnsitions etween sttes. Short epolrizing urrent pulses (.16 1 na, 2 1 ms urtion) elivere uring the own stte swithe the neuron to the up stte with proility of 7.5 ± 8.3% (Fig. 4, left; n = 6 ells), wheres rief hyperpolrizing urrent pulses (.2 2 na, 2 1 ms urtion) elivere uring the up stte oul inue trnsition to the own stte with n effiieny of 81.5 ± 1.5% (Fig. 4, right, n Fig. 4; n = 6 ells). These relile n lmost eterministi flip-flop-like trnsitions re hrteristi feture of istle systems, where trnsient perturtions re suffiient to inue sustine hnges. Notly, hyperpolrizing urrent pulses elivere uring the own stte oul lso inue trnsition to the up stte (Fig. 4). In four of five ells, hyperpolrizing urrent pulses (.2 2 na, 2 1 ms urtion; n = 5 ells) relily inue own-toup trnsition with proility of 61.1 ± 14.% (in the remining neuron, the hyperpolrizing urrent pulse i not inue up sttes). These finings emonstrte tht the imolity of Purkinje ell memrne potentil is onsequene of its istle ynmis. Synpti ontrol of intrinsi istility Tht the sme urrent pulse n inue memrne potentil trnsitions in oth iretions rise the possiility tht in Purkinje ells, No. of events No. of events 1 5 5 ms 2 mv.1.1 Figure 5 Climing fier input n trigger trnsitions etween sttes. () Left: whole-ell reoring from guine pig Purkinje ell in vitro. Climing fier stimultion (CF stim., rrowhes) triggere trnsitions etween up sttes n own sttes in oth iretions. Right, expne tres (orresponing to the open rrowhes in the left pnel) showing the omplex spike-evoke trnsitions. () Temporl reltionship etween omplex spikes n simple spike ursts in extrellulr reorings from guine pig Purkinje ells in vivo. Left, representtive exmples of simple spike ursts followe y (top) or preee y (ottom) omplex spike (sterisk). Right, ross-orreltion of omplex spikes () with the en (top) n with the eginning (ottom) of ursts of simple spikes (eh in is 5 ms). the sme synpti input my inue trnsitions etween up n own sttes. To test this hypothesis, we first exmine whether liming fier tivtion, hieve y iret eletril stimultion, oul inue trnsitions etween quiesent n spiking sttes in guine pig slie preprtion. During perios of quiesene, liming fier tivtion resulte in trnsition to epolrize potentil ssoite with simple spike ishrge. Similrly, the sme liming fier tivtion uring n tive stte ws ple of inuing trnsition towrs the lower stle stte (Fig. 5), therey terminting the spontneous ishrge. A reful exmintion of the reors otine in vivo onfirme this result. In mny ses, the ursts of simple spikes reore extrellulrly were immeitely followe (Fig. 5, top; see Methos) or immeitely preee (Fig. 5, ottom) y omplex spike (sterisk). We performe ross-orreltions etween omplex spikes n the eginning or the en of simple spike ursts; we foun signifint orreltion (P <.5) etween omplex spikes n the en or the eginning of simple spike ursts in 75% (21 of 28) n 64% (18 of 28) of the ells, respetively (Fig. 5, right). The orreltion etween omplex spikes n stte trnsitions ws lso oserve in the in vivo whole-ell reorings. Aross the popultion (n = 18 ells), 73 ± 4% of the trnsitions were preee y omplex spike, ourring less thn 1 ms efore the trnsition (Fig. 6, top). A signifintly lower numer of trnsitions pprently ourre spontneously in sene of ny liming fier input (Fig. 6, ottom; P <.5, n = 18 ells). Trnsitions etween sttes were hrterize y slow voltge hnge strting immeitely fter the omplex spike (Fig. 6, top), similr to the trnsitions triggere y iret liming fier stimultion otine in the in vitro experiments (Fig. 5, right). The temporl orreltion etween omplex spikes n stte trnsitions for one smple ell is shown (Fig. 6). Up-to-own n own-to-up trnsitions typilly ourre 4 n 7 ms, respetively, fter omplex spike. These strong orreltions llowe us to ssume tht stte trnsitions ourring less thn 1 ms fter omplex spike were triggere y the liming fier tivtion. Aross the popultion (n = 18 ells), 62 ± 7% of the trnsitions to the own stte n 84 ± 5% of the trnsitions to the up stte were triggere y liming fier input (Fig. 6). We lulte the effiieny with whih liming fier input inue trnsition s the rtio of the numer of omplex spikes tht inue trnsitions to the totl numer of omplex spikes. The men frequeny of omplex spike firing ws.8 ±.1 Hz (rnge:.2 1.7 Hz, n = 18 ells) n the effiieny of omplex spike in inuing trnsition ws 76.8 ± 4.9% (n = 18 ells). We lso ssesse the liming fier input effiieny in given stte. Complex spikes ourring uring the up stte were followe y trnsition to the own stte in 66. ± 6.4% of the ses, wheres liming fier input in the own stte resulte in trnsition to the up stte in 88.2 ± 3.4% of the ses (n = 18 ells; Fig. 6). These results inite tht liming fier input is ple of moulting, on-line, simple spike firing pttern in vivo. NATURE NEUROSCIENCE VOLUME 8 NUMBER 2 FEBRUARY 25 25
25 Nture Pulishing Group http://www.nture.om/ntureneurosiene Spont. No. of events No. of events 45 mv 33 mv 1 5 5.1.1.1.1 2 mv 5 ms Purkinje ell istility moulte y sensory stimultion The strong orreltion etween omplex spikes n stte trnsitions suggests tht sensory stimuli tht eliit omplex spikes oul moulte the stte of the ell. To exmine the influene of sensory stimultion on stte trnsitions, we use ir puff stimultion of the virisse or other periorl res to tivte liming fier inputs. In this set of experiments, reorings were performe in the Crus I n II of the ereellr ortex, regions tht hve een shown to respon strongly to ttile stimultion of the periorl res in nesthetize rts 2. As illustrte y the rster plot in Figure 7, ir puff stimuli were effetive in evoking omplex spikes (fille irles). Aross ells, omplex spikes were evoke in 38 ± 4% of the trils (n = 7 ells), n omprle to previous finings 2, the lteny of evoke omplex spikes from the onset of the stimulus ws 7 ± 7 ms (rnge: 47.9 93.5 ms, n = 7 ells). The mjority of the evoke omplex spikes triggere up-to-own or own-to-up trnsitions, epening on the initil stte. In the ell shown in Figure 7, 93% of the evoke omplex spikes were foun to e effetive in triggering stte trnsitions, with 67% inuing trnsition to the own stte (Fig. 7, top) n 26% trnsition to the up stte (Fig. 7, mile). In only 7% of the ses i evoke omplex spikes not inue ny trnsition (Fig. 7, ottom). Aross the popultion, 66.4 ± 11% of the evoke omplex spikes were effetive in triggering trnsitions (n = 7 ells). The effiieny of omplex spike evoke uring the up stte in inuing up-to-own trnsitions ws 57.8 ± 15.5%, ompre to 81.6 ± 7.3% for the opposite trnsition (n = 7 ells; Fig. 7). In reorings proximl enough to the som of the Purkinje ell to llow relile etetion of simple spikes 6, we ssesse the temporl reltionship etween peristimulus histogrms of evoke omplex spikes Perentge of trnsitions effiieny (%) 58 mv 53 mv 1 8 6 4 2 1 8 6 4 2 Spont. Spont. Figure 6 Chrteriztion of the omplex spike inue trnsitions in vivo. () Whole-ell reoring from rt Purkinje ell in vivo, showing representtive trnsitions etween up sttes n own sttes. Most trnsitions (92% in this ell) re ssoite with omplex spikes (, top) n only minority our pprently in the sene of omplex spike (Spont., ottom). () Temporl reltionship etween omplex spikes n stte trnsitions in 45-min reoring from guine pig Purkinje ell in vivo. Cross-orreltion of omplex spikes () with the trnsition from up-toown (top) n from own-to-up (ottom; eh in is 5 ms). () Popultion t from 18 rt Purkinje ell whole-ell reorings showing the perentge of up-own n own-up trnsitions ssoite with omplex spikes (), or ourring spontneously (Spont.). () Poole t (n = 18 ells) showing the effiieny of omplex spike ourring uring the up stte in inuing trnsitions to the own stte (Up-Down) n the effiieny of omplex spike ourring uring the own stte in inuing trnsitions to the up stte (Down-Up). n simple spike firing (n = 6; Fig. 7). Evoke up-to-own n ownto-up trnsitions were ssoite with prolonge ereses (Fig. 7, top) n inreses (Fig. 7, mile) of simple spike ishrge, respetively. After the sensory-evoke up-to-own trnsitions, the men simple spike firing rte ws signifintly reue (P <.1), from 57 ± 3 Hz uring the seline to 22 ± 7 Hz uring the first 5 ms fter the stimulus (Fig. 7, top). Conversely, ssoite with the sensory-evoke own-to-up trnsitions, the men simple spike firing rte ws signifintly inrese (P <.1), from 5 ± 1 Hz to 53 ± 7 Hz (Fig. 7, mile). When evoke omplex spikes i not trigger stte trnsitions, we i not oserve ny signifint hnge in simple spike firing ompre to seline level (P >.5; Fig. 7, ottom). In the sme ells, when sensory stimuli i not evoke omplex spike in the reore Purkinje ell (Fig. 7e), only slight inrese (+ 7%, P <.5) in the men simple spike firing rte ws oserve (Fig. 7f), initing tht the sensory-evoke hnges in simple spike ishrge resulte minly from the stte trnsitions triggere y the tivtion of liming fier input. A moel for istility n stte trnsitions To explore the iophysil eterminnts of Purkinje ell memrne potentil ynmis, we onstrute simplifie moel tht emultes the funmentl fetures of the oserve ehvior: istility, iiretionl trnsitions inue y outwr urrent pulses (Fig. 8, left) n iiretionl trnsitions inue y liming fier like input (Fig. 8, right). This single-omprtment moel onsists of three ioni urrents: n instntneous, non-intivting inwr urrent (moele here s soium urrent), slow h-like urrent n voltge-inepenent outwr urrent (see Supplementry Note). The ynmi vriles of this moel re the memrne potentil (V) n the intivtion (h) of the h-like urrent. Their qulittive ehvior is epite in the V-h phse plne shown in Figure 8. Eh point in the phse plne orrespons to possile stte of the ynmi vriles of the ell moel (V, h). The re n lue soli lines orrespon to the ḣ = n the V = nulllines, respetively, n the two outermost points of intersetion (irles) orrespon to the two stle sttes (stle fixe points) of the system. This istility is primrily ue to the nonlinerity of the non-intivting inwr urrent, whih is responsile for the N-shpe of the V = nullline. In the hyperpolrize stte (left open irle), the non-intivting inwr hnnels re lose n the memrne potentil is etermine y the omine effet of the h-like urrent n the voltge-inepenent outwr urrent. In ontrst, these hnnels re open in the epolrize stte n will ontriute sustntilly to the memrne potentil vlue in the up stte. The sins of ttrtion of the epolrize n hyperpolrize sttes re enote y the lue n green res, respetively. The seprtrix, the 26 VOLUME 8 NUMBER 2 FEBRUARY 25 NATURE NEUROSCIENCE
25 Nture Pulishing Group http://www.nture.om/ntureneurosiene Figure 7 Sensory-evoke omplex spikes in Purkinje ells n trigger memrne potentil istility in vivo. An ir puff (4 ms) to the ipsilterl virisse ws use to evoke responses in single Purkinje ells. () Rster plot showing the temporl reltionship etween sensory stimuli, omplex spikes (fille irles) n simple spike firing (lines) in rt Purkinje ell whole-ell reoring in vivo (3 onseutive trils of 121). () Whole-ell reorings showing representtive exmples of sensory-evoke omplex spikes (fille irles) triggering up-to-own, own-to-up, n no trnsitions, respetively. Note tht sensoryevoke sttes oul e interrupte y spontneous trnsitions ssoite with spontneous omplex spikes (rrows) or ourring in the sene of omplex spikes (rosse rrow). Open irles inite spontneously ourring omplex spikes. () Poole t (n = 7 ells) illustrting the effiieny of n evoke omplex spike ourring uring the up stte in inuing trnsitions to the own stte () n the effiieny of n evoke omplex spike ourring uring the own stte in inuing trnsitions to the up stte (). () Temporl reltionship etween peristimulus histogrms of evoke omplex spikes (, r hrt) n simple spike firing (SS, otte lines) on the sme grph, seprte into up-toown trnsitions (top, n = 5 ells), own-to-up trnsitions (mile, n = 6 ells) n no trnsitions (ottom, n = 6 ells) (in size 5 ms). Note tht up-to-own n own-to-up trnsitions ssoite with evoke omplex spikes re ompnie y roust n prolonge hnges in simple spike firing. Conversely, the men simple spike firing rte ws not signifintly hnge ompre to the seline perio (1 ms seline, n = 2 ells; 5 ms seline for the remining ells) when evoke omplex spikes i not evoke stte trnsitions. (e) Exmple of Purkinje ell up stte in the sene of ny evoke omplex spikes. Open irles inite spontneously ourring omplex spikes. (f) Averge peristimulus histogrm (n = 6 ells) of simple spike firing (otte line) ompute for sweeps where sensory stimuli i not evoke omplex spikes. Trils where stimuli were elivere either uring n up or own stte of the Purkinje ell were poole together. Br hrts represent spontneously ourring omplex spikes. Results presente in, n e re from the sme ell. Clirtion rs in pply lso to e. Tril no. e 3 2 1 1 Down to up trnsitions No trnsitions.1.1.2.3.4.5 Up to own trnsitions 1 ms.6 2 mv f proility proility proility proility Evoke effiieny (%).6.4.2.6.4.2 SS.4.4.8.4.4.8.6.4.2.4.4.8.6.4.2 1 8 6 4 2.4.4.8 8 4 8 4 8 4 8 4 SS frequeny (Hz) SS frequeny (Hz) SS frequeny (Hz) SS frequeny (Hz) orer etween these res, psses through the mile, unstle, fixe point (open squre). Any voltge perturtion t either of the two fixe points tht is lrge enough to ross the seprtrix will eventully le to the onvergene of the voltge to the other fixe point. For exmple, rief epolriztion from the own stte to potentil tht is more epolrize thn 54.5 mv will ross the seprtrix n rive the system to the up stte (trjetory not shown). Notly, liming fier input (moele here s rief, lrge inrese in soium onutne) elivere uring the up stte n hve ounterintuitive effet. As result of the epolriztion, the vlue of the intivtion term h uring the input is erese suffiiently to llow the system to ross the seprtrix into the sin of ttrtion of the own stte (right she line). Thus, when the input is terminte, the ynmis onverge to the own stte (sh-otte line). Conversely, suffiiently lrge hyperpolrizing pulse from the own stte (left she line) inues sustntil eintivtion of the h-like urrent, leing the system into the sin of ttrtion of the epolrize stle point. Thus, fter the outwr urrent pulse the ynmis will onverge to the up stte (left sh-otte line). Vrying the properties of the outwr urrent oul ffet the ility of rief hyperpolrizing urrent pulses or epolriztions to inue trnsitions etween sttes. To stuy this effet, we seprte the voltgeinepenent urrent into lek n voltge-inepenent potssium omponent. A suffiient erese in the potssium onutne will ownshift the V = nullline (lue line), suh tht simulte liming fier input will inue n upwr trnsition ut not the opposite trnsition (Fig. 8). Conversely, n inrese in this onutne will upshift the V = nullline suh tht the simulte liming fier input will only generte trnsition to the own stte (Fig. 8). To explin the spontneous trnsitions etween up sttes n own NATURE NEUROSCIENCE VOLUME 8 NUMBER 2 FEBRUARY 25 27
25 Nture Pulishing Group http://www.nture.om/ntureneurosiene h h.6.4.2.4 1 8 6 4 2 2 4 V (mv) 2 mv.5 s 8 4 4 V (mv) 2 mv 1 s 8 4 4 V (mv) sttes oserve in vivo (see Fig. 6, ottom) n in vitro 6,15,21,22, we moifie the moel y repling the voltge-inepenent potssium urrent with slowly tivting potssium urrent. In the sene of liming fier input (similr to in vitro onitions), this moifition generte spontneous trnsitions etween the sttes, resemling those oserve in slie preprtions 6,15,21,22 (Supplementry Fig. 2). In the presene of liming fier like input, this moel reproue oth spontneous n liming fier evoke trnsitions, s well s the osionl filures of liming fier input to evoke trnsition (see Supplementry Note n Supplementry Fig. 2). DISCUSSION We hve emonstrte tht Purkinje ells show istility of memrne potentil n spike output in vivo, whih is onsequene of their intrinsi memrne properties. This istility n e iiretionlly triggere y sensory-riven synpti input, suggesting tht it my represent n importnt intrinsi ellulr mehnism for proessing of sensory informtion in the ereellr ortex. Bistility of Purkinje ell output in vivo Bistility of Purkinje ells in vitro is well-oumente n hs een oserve to our spontneously 6,21,23 or fter moultion of intrinsi onutnes 15. Our intrellulr reorings from Purkinje ells of rts n guine pigs nesthetize with ketmine-xylzine or pentoritl estlish tht this istility is retine in vivo in ifferent niml speies n uner ifferent nestheti onitions. We further emonstrte tht this istility of memrne potentil is mnifeste in two moes of firing tivity: toni firing of simple spikes n quiesent perios. The presene of istility in Purkinje ell spike output is supporte y previous reports showing intermittent firing ptterns where perios of high-frequeny, simple spike firing re intersperse with.4 2 mv.5 s Figure 8 A moel for istility n stte trnsitions. (,) Simultion of moel neuron inorporting non-intivting inwr urrent, slow h-like urrent, n voltge-inepenent outwr urrent. Trnsitions etween the two stle sttes in oth iretions n e inue y rief outwr urrent pulses (.1 s, 7,2 na m 2 ) (, left) or y simulte liming fier input (4 ms, 1,2 µs m 2 soium onutne) (, right). () Phse plne for the two ynmi vriles in the moel, the memrne potentil V n n intivtion term h. The soli lines (re, lue) re the ḣ = n the V = nulllines respetively; the irles enote the two stle sttes (fixe points) n the squre enotes the unstle fixe point, whih is lote on the seprtrix, the orer etween the sins of ttrtion of the up stte (lue) n the own stte (green). Arrows mrk the trjetory of the ynmi vriles (V, h) uring (she lines until the igonl rrowhes) n fter (sh-otte lines) n outwr urrent injetion (left) or simulte liming fier-like input (right). () Deresing the potssium onutne (see Results for etils) shifts the V = nullline own (ottom) suh tht the trjetory uring omplex spike-like epolriztion (she line) oes not ross the seprtrix. Thus, liming fier input will only inue own-to-up n not n up-to-own trnsition (top). () Inversely, inresing the potssium onutne shifts the nullline up suh tht the trjetory uring omplex spike-like epolriztion (she line) rosses the seprtrix twie (ottom). In this se, liming fier input will only inue n up-to-own n not own-to-up trnsition (top). perios of quiesene in nesthetize 24 n eererte nimls 25 27. Similr irregulr lterntions etween quiesene n high-frequeny simple spike tivity hve een oserve in wke reorings from vrious nimls, inluing frogs 28, ts 18,29,3, squirrel monkeys 31 n rhesus monkeys 32,33. In ontrst, other ereellr stuies hve not reporte similr phenomen n typilly fin ontinuous firing of Purkinje ells rther thn imol ishrge, suggesting tht in these ses the ynmis of Purkinje ells re not istle. The resons for this isrepny re still unler. As in other strutures of the entrl nervous system showing intrinsi istility, inluing spinl or 8, thlmus 34 n olftory ul 7, this my reflet heterogeneity in the expression of istility within prtiulr ell type. Furthermore, the existene of istility in given ell is likely to epen on neuromoultion 15,23 n on the lne of exittion n inhiition in the lol network, whih my epen oth on the ereellr region n the ehviorl stte. Our fining tht omplex spikes triggere y liming fier input n swith Purkinje ells etween up sttes n own sttes my help to resolve the isrepny in the literture regring the effet of omplex spikes on simple spiking. Complex spikes hve een shown to e ssoite with oth inreses 26,35,36 n ereses 18,24,37 in simple spike tivity. Our t suggest tht the effet of omplex spike on simple spike ishrge epens on the stte of the Purkinje ell just efore the omplex spike. Thus, Purkinje ells preominntly in the own stte will ten to respon with n inrese in simple spike tivity ssoite with the omplex spike, wheres Purkinje ells preominntly in the up stte will ten to respon with erese. This is in greement with the prolonge enriti plteus ssoite with inrese spiking reporte to e triggere y liming fier input in intrellulr n extrellulr reorings from nesthetize ts 38. Mehnisms of istility Bistility is n intrinsi memrne property of Purkinje ells in vivo, s trnsitions etween sttes n e triggere y rief hyperpolrizing n epolrizing urrent pulses n olishe y suffiiently strong negtive or positive onstnt urrent injetion. Further eviene tht up sttes in Purkinje ells re not sustine y synpti tivity is provie y our reorings from interneurons of the moleulr lyer (Fig. 2e,f) n grnule ells (Fig. 2,), the soure of 28 VOLUME 8 NUMBER 2 FEBRUARY 25 NATURE NEUROSCIENCE
25 Nture Pulishing Group http://www.nture.om/ntureneurosiene prllel fier input to oth interneurons n Purkinje ells. Neither interneurons nor grnule ells showe istility, n thus it seems unlikely tht up or own sttes re mintine y imol tivity in the prllel fier or interneuron input. Bistility in Purkinje ells is thus very ifferent from the up n own sttes esrie in mny ortil n stritl neurons in vivo. In these neurons, the up stte is thought to result minly from ontinuous rrge of synpti exittion refleting wiespre tivity ross the network 3,39. In ontrst, in Purkinje ells, the persistene of the two sttes is mintine y intrinsi voltge-epenent mehnisms. The trnsition etween sttes in Purkinje ells n e triggere y the tivtion of the sme single (leit lrge) synpti input. Notly, reent in vitro stuy 3 hs shown tht the sme eletril stimulus pplie to the white mtter of the ererl ortex n turn on n off network-inue up stte in ortil neurons. Although the ioni n synpti urrents involve in sustining up sttes n own sttes in these ells re ifferent from those in Purkinje ells, our ynmil moel my serve s oneptul frmework for unerstning this phenomenon. Previous simplifie moels for neuronl istility in generl 4 n Purkinje ell istility in prtiulr 41,42, s well s the present moel, inite tht neither etile interply etween lrge numer of onutnes nor omplex enriti geometry re require to qulittively generte this ehvior. In our ynmil moel, istility relies on non-intivting inwr urrent. However, mny onutnes re likely to influene the urtion of sttes n the proility of trnsitions etween sttes. Inee, previous in vitro stuies hve shown tht Purkinje ell istility oul result from intertions etween non-intivting soium n lium onutnes with potssium onutnes 6,43. The role of the h-urrent (I h ) in generting the istility is more ontroversil. Some in vitro stuies hve reporte istility in Purkinje ells in the presene of I h 6,21, wheres others hve shown tht its ownregultion, for instne y serotonin, n unmsk or enhne istility 15. Our experiments lerly show tht I h is present in istle Purkinje ells in vivo (e.g. see Supplementry Fig. 1; see lso the epolrizing sg in Figs. 1,, 3 n 4,). This urrent my lso hve n importnt role in setting the voltge of the sttes n in ontrolling the trnsitions etween the sttes. In prtiulr, the ility of outwr urrent pulses to inue iiretionl trnsitions requires reoun response, whih is likely to result from the slow etivtion of the h- urrent (see Supplementry Note). Purkinje ell istility n olivo-ereellr ynmis The long timesle ssoite with Purkinje ell istility llows lrge numer of ifferent ynmil sttes to e sustine in the ereellr ortex for extene perios. Eh of these sttes is ssoite with speifi onfigurtion of up n own sttes in ifferent Purkinje ells. These network sttes n e use to mintin speifi funtionl networks within the olivry nuleus: the sustine firing stte of Purkinje ell inhiits neurons in the eep ereellr nulei, whih in turn remove the inhiition from the eletrilly ouple enrites of the inferior olive neurons, prouing synhronize rhythmi tivity in suset of olivry neurons. Notly, reorings of eep ereellr nuleus neurons show prolonge (up to.5 s) inhiitory responses to rief sensory stimultion (N.C. Rowln n D. Jeger, So. Neurosi. Astr. 75.1, 23), whih my orrespon to synhronize up sttes in presynpti Purkinje ells tivte y sensory stimultion. Computtionl implitions of istility The presene of istility my hve importnt implitions for integrtion of mossy fier input to the ereellr ortex meite y the prllel fier pthwy. First, the responsiveness of the Purkinje ell to sensory-evoke prllel fier input will epen on whether previous liming fier input hs swithe the Purkinje ell into the up stte or own stte. This suggestion is onsistent with previous work showing tht the gin of Purkinje ell responses to sensory-riven mossy fier input epens on the reent history of liming fier tivity on the time sle of hunres of milliseons 44. Seon, given tht istility is lso oserve in the istl enrites, the tivtion of voltge-gte enriti onutnes 6 will lso e influene. In prtiulr, tivtion of enriti lium spikes y prllel fier input is strongly voltge n time epenent 45. Given tht lium spikes hve een implite in the inution of long-term epression (LTD) t prllel fier synpses 46, the ft tht sensory-evoke liming fier inputs n inue prolonge epolriztions provies possile ellulr sustrte for liming fier epenent forms of prllel fier plstiity in whih ro time winow for inution (severl hunre milliseons) hs een oserve 38. Purkinje ell istility my therefore ontriute to etermining the rules for LTD inution in the intt ereellr network. Chemil synpses hve tritionlly een lssifie s either exittory or inhiitory oring to their effet on the firing rte of the postsynpti neuron. In this stuy we hve emonstrte tht the sme synpse n ply oth roles. The tivtion of liming fier synpses n either inrese or erese the firing rte of the Purkinje ell, epening on its initil stte. This property, in whih the sme input inues oth trnsitions in istle element, is known in eletronis s toggle swith, whih is wiely use in eletril evies. We suggest tht toggling of the Purkinje ells my serve s higher-orer reflex tht genertes n immeite n reflexive response of the system to the ourrene of n error y shifting the Purkinje ell tivity wy from its urrent erroneous operting stte. METHODS The re n experimentl mnipultion of the nimls ws rrie out in orne with the regultions of the Herew University of Jeruslem n the U.K. Home Offie. In vivo reorings. Sprgue-Dwley rts (P18 P27) or Dunkin-Hrtley guine pigs (18 3 g) were nesthetize y intrperitonel injetion of ketmine (5 mg kg 1 )-xylzine (3 mg kg 1 ) or pentoritl (6 mg kg 1 ). The level of nesthesi ws routinely monitore y oserving whisker movements or the response to noxious stimulus to the hin lims, n itionl oses of nestheti were e s neee. The nimls were ple in stereotxi pprtus, n the oipitl one n the ur mter were remove, exposing smll region of the ereellr vermis or hemispheres. The expose re ws overe with physiologil sline or gr (3% in physiologil or sline solutions). Sensory responses were evoke y n ir puff (3 7 ms, 4 psi) time y ustom pressure evie n elivere to the ipsilterl periorl surfe. Cell-tthe n whole-ell pth-lmp reorings were me with Multilmp 7A or n Axolmp 2B mplifier (Axon Instruments) using previously esrie tehniques 47. The pipette solution ontine 13 mm potssium methnesulfonte, 7 mm KCl, 1 mm HEPES,.5 mm EGTA, 2 mm MgATP, 2 mm N 2 ATP n.5 mm N 2 GTP, ph 7.2, in reorings from rts, or 14 mm potssium gluonte, 4 mm NCl, 1 mm HEPES, 4 mm MgATP, 1 mm EGTA n.1 mm CCl 2, ph 7.4, for guine pig reorings. Extrellulr reorings of guine pig Purkinje ell single-unit tivity were otine using glss pipette eletroes, pulle to DC resistne of 1 2 MΩ n fille with 2 M NCl. A ifferentil AC mplifier (DP-31; Wrner Instruments) ws use for monitoring ongoing tivity. Reorings were filtere t 3 1 khz n smple t 2 5 khz using n Instruteh (ITC-18) or Ntionl Instruments (PCI-MIO- 16XE-1) nlog to igitl or. Slie reorings. The in vitro experiments were performe on 3 µm thik sgittl slies of ereellr vermis from guine pigs (18 2g). Preprtion of slies n reoring tehniques re esrie elsewhere 48. Reorings were NATURE NEUROSCIENCE VOLUME 8 NUMBER 2 FEBRUARY 25 29
25 Nture Pulishing Group http://www.nture.om/ntureneurosiene performe t 27 3 C or t room temperture (22 25 C) in physiologil solution ontining 124 mm NCl, 5 mm KCl, 1.3 mm MgSO 4, 1.2 mm KH 2 PO 4, 26 mm NHCO 3, 1 mm gluose, n 2.4 mm CCl 2. Anlysis. Correltion etween omplex spikes n stte trnsitions in intrellulr reorings ws performe in Purkinje ells where the ientifition of omplex spikes ws unmiguous. The response of the memrne potentil to liming fier input uring the up n own sttes oul iffer onsierly (see Figs. 5 n 6). During the own stte, omplex spikes were hrterize y lrge-mplitue spike followe y urst of smller spikes n long epolriztion 6. During the up stte, omplex spikes were hrterize y stereotypi wveform, ompose of severl fst istint peks. The rte of these omplex spikes ws inepenent of the stte of the ell. Simple n omplex spikes reore extrellulrly were sorte offline oring to their mplitue n shpe, using n ptive templte routine in MATLAB (version 6., Mthworks). To ontrol the qulity of sorting, ll simple spikes n omplex spikes were superimpose n exmine visully. Cells were isre from the nlysis if the estimte numer of errors in sorting exeee 2%. Histogrms of memrne potentil istriution were onstrute for eh Purkinje ell, n Gussin fits were pplie to the iniviul peks. The mol vlue of eh Gussin fit ws tken s the men memrne potentil for the up stte n the own stte. To mesure the urtion of eh iniviul ste, trnsitions etween the up n own sttes were etete using the following thresholing proeure: two threshols were set t one-fourth n three-fourths of the istne etween the peks of the memrne potentil istriution. When the memrne potentil rose ove the lower threshol, own-to-up trnsition ws registere; when the memrne potentil fell elow the upper threshol, n up-to-own trnsition ws registere. To voi efining noise or iniviul omplex spikes s short stte, we remove pirs of lower n upper threshols generting time ifferene of less thn 1 ms. Up n own stte urtions were quntifie in Purkinje ells where the urtion of the reorings ws long enough to llow mesurement of t lest 15 up n own sttes. The evition from unimolity of the memrne potentil of Purkinje ells, s well s tht of grnule ells n moleulr interneurons, ws teste using ip test 16, using softwre R (version 1.9.1; http://www.r-projet.org). For eh ell, the test ws performe using 5 rnomly hosen vlues of memrne potentil. In extrellulr n ell-tthe reorings, we efine urst of simple spikes s n event tht is flnke y perios of t lest 1 ms evoi of simple spikes (qulittively similr results were otine using other riteri). Correltions etween omplex spikes n the eginning of urst were lulte using winow of 5 ms, strting 25 ms fter the omplex spike. Correltions etween omplex spikes n the en of urst were lulte using winow of 4 ms preeing the omplex spike. The use of ifferent winows i not proue qulittive ifferene in the results. Input resistne (R in ) ws lulte from stey-stte voltge efletions uring (4 5 ms) step hyperpolrizing urrent injetions (grnule ells, 5 1 pa; moleulr lyer interneurons, 2 7 pa). Aitionl t nlysis ws rrie out using Igor Pro (Wvemetris). Dt re men ± s.e.m. unless otherwise inite. Sttistil omprisons were me using Stuent s pire or unpire t-test. Note: Supplementry informtion is ville on the Nture Neurosiene wesite. ACKNOWLEDGMENTS We thnk H. Meiri, E. Chorev n P. Mnn-Metzer for exellent tehnil ssistne, J.T. Dvie for help with progrmming, n J.I. Simpson n T. Mrgrie for enourgement n helpful isussions. This work ws supporte y grnts from the Europen Commission (M.H. n Y.Y.), Wellome Trust (M.H n S.M), Gtsy Fountion (M.H), JSPS (K.K.), US-Isrel BSF (Y.Y.), the Isrel Siene Fountion (Y.Y.), the Isrel Siene Fountion Center of Exellene 86- (H.S.) n the Yeshy Horowitz Assoition (Y.L.). COMPETING INTERESTS STATEMENT The uthors elre tht they hve no ompeting finnil interests. 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