In vivo intracellular recording and perturbation of persistent activity in a neural integrator

Transcription

1 rtiles 21 Nture Pulishing Group 21 Nture Pulishing Group In vivo intrellulr reording nd perturtion of persistent tivity in neurl integrtor E. Aksy 1,2, G. Gmkrelidze 1,2, H. S. Seung 1,3, R. Bker 2 nd D. W. Tnk 1 1 Biologil Computtion Reserh Deprtment, Bell Lortories, Luent Tehnologies, 7 Mountin Avenue, Murry Hill, New Jersey 7974, USA 2 Deprtment of Physiology nd Neurosiene, New York University Shool of Mediine, New York, New York 116, USA 3 Howrd Hughes Medil Institute, Brin nd Cognitive Sienes Deprtment, Msshusetts Institute of Tehnology, Cmridge, Msshusetts 2139, USA Correspondene should e ddressed to D.W.T. (dwtnk@luent.om) or R.B. (kerr1@endevor.med.nyu.edu) To investigte the mehnisms of persistent neurl tivity, we otined in vivo intrellulr reordings from neurons in n oulomotor neurl integrtor of the goldfish during spontneous sdes nd fixtions. Persistent hnges in firing rte following sdes were ssoited with step hnges in interspike memrne potentil tht were orrelted with hnges in eye position. Perturtion of persistent tivity with rief intrellulr urrent pulses designed to mimi sdi input only indued trnsient hnges of firing rte nd memrne potentil. When neurons were hyperpolrized elow tion potentil threshold, position-orrelted step hnges in memrne potentil remined. Memrne potentil flututions were greter during more depolrized steps. These results suggest tht sustined hnges in firing rte re supported not y either memrne multistility or hnges in pemker urrents, ut rther y persistent hnges in the rte or mplitude of synpti inputs. Persistent neurl tivity refers to sustined hnge of sodium tion potentil firing rte extending eyond rief sensory stimulus or motor ommnd. This tivity hs een oserved in diverse rin res 1 8, suggesting tht it is generl form of neurl dynmis. Beuse persistent tivity rries tre of pst event, it is thought to e losely relted to short-term or working memory 1,3,9. In this study, we otined intrellulr reordings in wke goldfish from neurons tht show sustined firing during eye fixtion, providing informtion out intrinsi neuronl exitility nd movement-ssoited memrne potentil hnges tht onstrins proposed mehnisms of persistent neurl tivity. The neurons reorded re loted in re I, ilterl premotor hindrin nuleus 7. During sequentil sdes nd fixtions omposing the k nd forth snning pttern of goldfish spontneous eye movements 1, these neurons dishrge s shown in Fig. 1 (refs. 7, 11). Trnsient inreses in firing rte re oserved during ipsilterlly direted (on) sdes, nd trnsient dereses our during ontrlterlly direted (off) sdes. When eye position is ove threshold vlue, there is sustined toni firing rte tht long outlsts the trnsient hnges in rte oserved during sdes (Fig. 1). This persistent neurl tivity vries linerly with eye position, with different position neurons showing different slopes nd thresholds in the firing rte/eye position reltionship 11. This trnsformtion of pulse-like sdi ommnd into step of sustined dishrge n e desried in terms of veloity-to-position neurl integrtor (VPNI), onept originlly introdued 12 to explin the time integrtion of hed-veloity signls into eye-position signls during the vestiulo-oulr reflex (VOR) 13. Lidoine intivtion of re I disrupts fixtions nd low-frequeny VOR, demonstrting tht this nuleus (onnetivity, Fig. 1) is n essentil omponent of the VPNI for horizontl eye movements 7,11. Hypothetil mehnisms for the persistent tivity of VPNI position neurons must ount for severl hrteristis 14. First, there is ontinuum of stle firing rtes 5,6,11, whih is more diffiult to explin thn istility. Seond, persistent tivity remins without either visul 11 or proprioeptive feedk 15, demonstrting entrl origin. Third, different liner reltionship etween firing rte nd eye position for eh neuron implies tht for eh eye position, onstrined pttern of stle tivity exists. At one extreme, persistent tivity my e purely single-ell phenomenon. For exmple, urst input ould trigger plteu potentils 16,17, with multiple dendriti genertion sites produing multiple firing levels 18. Alterntively, sdi ommnd neurons ould relese neuromodultors tht modify the frequeny of intrinsi pemker urrents 19 or lter sodium tion potentil threshold 2 in long-lsting wy. At the other extreme, persistent neurl tivity my e produed y reverertory tivity in positive feedk loop, network mehnism explored in neurl integrtor models 14, During intrellulr reording from individul re I position neurons, we used urrent injetion to pertur (stimulte or inhiit) with n rtifiil stimulus tht produed sde-like trnsient hnge of tivity. This provided diret test for voltgedependent regenertive events suh s plteu potentils; simultneously, it ddressed whether network of position neurons ould integrte the inresed firing rte of single neuron. Intrellulr reording ws lso used to revel persistent step hnges in interspike memrne potentil ssoited with endogenous hnges in firing rte nd to investigte their origin. Finlly, intr- 184 nture neurosiene volume 4 no 2 ferury 21

2 21 Nture Pulishing Group rtiles T 21 Nture Pulishing Group Eontr Eipsi sp/s Fextr Vextr uv 1 s Eipsi On urst ell Off urst ell Position ell 1 Position ell 2 Contr Position ell Fig. 1. Position neuron dishrge nd funtionl rhiteture of oulomotor pthwys. () Extrellulr voltge reording (V extr ) nd firing rte (F extr ) of position neuron in goldfish re I during one k nd forth yle of sdes nd fixtions (E ipsi, ipsilterl eye; E ontr, ontrlterl eye; T, temporl; N, nsl). () Shemti showing eye position, urst neuron dishrge nd dishrge of three position neurons during trnsitions in fixtion position. On urst ell tivity riefly preedes sdes nd produes urst dishrge of ipsilterl position neurons. This trnsient ommnd is trnsformed into persistent elevted dishrge of position neurons, supplying motoneurons with toni drive neessry to mintin new eye position. Off urst ell tivity initites sdes in the opposite diretion nd indues puses in dishrge followed y lowered persistent firing. Chnges in firing rte re opposite in sign for ontrlterl position neurons. () Shemti of urrent understnding of funtionl onnetions underlying horizontl sdes nd fixtions. Sdes re initited y urst neurons (). Position neurons (p) in re I re required for the toni dishrge of duens motoneurons () nd medil retus motoneurons (m; vi internuler ells, i) tht determine the new eye position (θ). Eh re I ontins 3 5 position neurons, some of whih my provide synpti onnetions to the ontrlterl re I (ref. 11). 1 degrees T N 5.1 mv re I p re I duens oulomotor m midline duens i lterl retus 1 ms medil retus θ N ellulr reording llowed hrteriztion of memrne potentil flututions ompnying hnges in persistent neurl firing, thus ddressing the role of synpti tivity. RESULTS Spike dishrge during intrellulr reording In vivo intrellulr reordings were otined from neurons in hindrin re I of wke hed-restrined goldfish performing snning yles of spontneous sdes nd fixtions (Fig. 1). Reordings from 18 position neurons were nlyzed. Oulomotor ehvior ws quntified either diretly y reording eye position (Fig. 2 nd ), or indiretly y extrellulr reording of seond position neuron in re I or motoneuron in the duens nuleus (Fig. 2). Intrellulrly reorded position neurons (n = 18) injeted with wek negtive holding urrent (, to 1 ) dishrged with qulittively the sme urst-toni pttern oserved in extrellulr reordings (Fig. 1). On-diretion steps in ipsilterl fixtion position were ompnied y persistent inreses in firing rte (Fig. 2 nd ). Likewise, off-diretion steps were ompnied y persistent dereses in rte (Fig. 2). On-sdes were ssoited with rief (5 1 ms) ursts of dishrge (Fig. 2 nd ), wheres off-sdes were ssoited with rief (5 1 ms) undershoots in rte (Fig. 2). Trnsitions etween different persistent levels of firing were similr in mplitude ( 5 5 ) to those reorded with extrellulr eletrodes 11. The similrities in dishrge pttern etween reording methods suggest tht position neurons funtioned normlly during intrellulr reording. In ddition, spontneous sodium tion potentil width t hlf-mximum verged.47 ±.16 ms (men ± s.d.; n = 18), omprle to vlues seen for reordings with extrellulr eletrodes 11. The popultion verge for spontneous tion potentil mplitude ws 51 ± 14 mv (n = 18), nd pprent resting memrne potentil (men over snning yle) verged 61 ± 7.3 mv (rnge, 7 to 5 mv; n = 8). Position-ssoited memrne potentil hnges Severl forms of vrition in urrent nd/or ondutne ould explin the step-like hnges of intrellulr firing rte seen in Fig. 2. For exmple, the hnges in toni tion potentil frequeny ould e produed y hnges in the frequeny either of periodi synpti urrents tht drive individul spikes 25 or of suthreshold osilltory pemker urrents 26. Alterntively, the hnges in firing rte ould e produed y stedy-stte hnges in memrne urrent or ondutne tht depolrize or hyperpolrize the spike genertion zone. Interspike memrne potentil (V is ) during dishrge hd step-like hnges onsistent with step-like hnges in verge urrent nd/or ondutne (Fig. 2). V is ws ssessed y verging setions of memrne potentil fter exluding tion potentils (Fig. 3 nd ). Trnsitions in rte ssoited with steps in fixtion position were lwys ompnied y similrly direted trnsitions in the verge vlue of V is ; inreses in rte were oupled with inreses (depolriztions) in potentil (Fig. 2 nd ; n = 18), nd dereses in rte were ompnied y dereses (hyperpolriztions) in potentil (Fig. 2; n = 18). This reltionship etween memrne potentil nd rte during spontneous sdes nd fixtions ws quntified for om- nture neurosiene volume 4 no 2 ferury

3 plete yle of the snning pttern (4 1 fixtions). Within one yle, vrition in men V is etween nsl nd temporl extremes verged 4.7 ± 2.5 mv (n = 18), nd ould e s high s 1 mv. Firing rte inresed with inresing V is, with the highest rte nd potentil vlues t temporl fixtions (Fig. 3 nd d; n = 18). All orreltion oeffiients (r) for the rte V is reltionship were greter thn.7 (men,.9; p <.5; n = 18). The slope (g spont ), mesure of the sensitivity of rte to fixtion-relted hnges in men potentil, verged 8.2 ± 5.6 /mv (n = 18), rnging from 2.2 to 23 /mv ross reordings. In ddition, V is ws orrelted with fixtion position, s ssessed y eye position mesurement (Fig. 3e; r >.7; men,.87; p <.5; n = 3/3) nd neuronl tivity (Fig. 3f; r >.8; men,.91; p <.5; n = 7/7). If the steps in potentil were indeed indued y steps in memrne urrent or ondutne, then they should e present in the sene of tion potentil dishrge. We tested this y hyperpolrizing with diret urrent injetion through the reording eletrode (n = 8). Addition of negtive urrent resulted in hyperpolriztion nd redued dishrge rtes t every fixtion position (dt not shown); strong negtive urrent ( < 1.2, reltive to originl holding level) prevented the toni dishrge of tion potentils ltogether (Fig. 4 nd ; n = 8/8). In the sene of tion potentils, persistent hnges in memrne potentil ( ) were still present, ourring in ssoition with hnges in fixtion position (Fig. 4 nd ; n = 8/8). During suppression of dishrge, when quntified over omplete yle of the snning pttern, ws orrelted with fixtion position, s ssessed y eye position mesurement (Fig. 4; r =.87, p <.5; n = 1/1) nd neuronl tivity (Fig. 4d; r >.7; men,.9; p <.5; n = 5/5). Vrition in men etween nsl nd temporl extremes verged 3.7 ± 2.2 mv (n = 8), nd ould e s high s 1 mv. The ove results suggest tht steps in memrne urrent nd/or ondutne produing persistent steps in verge memrne potentil ontriuted to fixtion-relted steps in firing rte. To determine if this ontriution is suffiient, we ompred the sensitivity of rte to vrition in memrne potentil for two distint situtions: when vrition of rte nd potentil ws ssoited with spontneous sdes nd fixtions (sensitivity g spont ), nd when vrition ws indued y urrent injetion through the reording eletrode (sensitivity g inj ). If sensitivity vlues were signifintly less with eletrode-generted hnge, this would st dout upon the suffiieny of memrne urrent hnges in explining the steps in rte. We lulted sensitivity g inj s the slope of the reltionship etween firing rte nd memrne potentil hnges indued y urrent steps or rmp-like urrent injetion profiles pplied during single fixtions (Fig. 5; n = 12; see Methods). The popultion verge for sensitivity g inj ws 12.3 ± 4. /mv (n = 12), rnging from 6.8 to 2. /mv, with n verge error estimted to e ±2% due to ridge-lne imperfetions. These sensitivity vlues lwys either mthed or exeeded those determined from hnges ssoited with spontneous sdes nd fixtions: sed on ell-y-ell omprison, g inj ws 6.4 ± 15.1% higher thn g spont (n = 12). This result suggests tht step-like hnges in verge memrne urrent polrizing the spike initition zone would e suffiient to explin the fixtion-relted hnges in rte. We lso tested whether ell s sensitivity g spont vried over the ourse of minutes. The reltionship etween firing rte nd V is ws mesured for five yles of the snning pttern done within spn of two minutes, s eletrode holding urrent ws fixed (, to 1 ; n = 3). The verge sensitivities (± s.d.) for eh ell were 4.9 ±.3, 7.1 ± 2.3 nd 12.1 ± 1.6 /mv. The smll stndrd devitions in sensitivity suggest tht the spike generrtiles 21 Nture Pulishing Group 21 Nture Pulishing Group Eontr Eipsi Fintr Vis E ipsi F intr V is Iinj E ontr F extr F intr V extr V is 1 ms 1 ms 1 ms 5 mv 2 mv 5 degrees Fig. 2. Memrne potentil hnges during trnsitions in fixtion position. () Eye position, firing rte of n intrellulrly reorded neuron (F intr ), memrne potentil ( ), interspike memrne potentil (V is ), nd injeted urrent ( ) for on-diretion steps during intrellulr reording. Solid lines t lower left indite the verge vlue of V is during seprte fixtions. () Chnges during off-diretion steps of the ipsilterl eye. This segment is tken from the end of nsl temporl nsl yle tht strted with the trnsitions shown in (). () Rte nd potentil hnges for different neuron during n on-diretion step. The firing rte (F extr ) of seond extrellulrly reorded position neuron (V extr ) served s surrogte for eye position. In this reording, the fst fterhyperpolriztion following tion potentils ws olished y sustituting esium for potssium in the eletrode solution mv 2 mv 5 degrees mv 2 mv.2 mv 186 nture neurosiene volume 4 no 2 ferury 21

4 21 Nture Pulishing Group rtiles 21 Nture Pulishing Group V is 2 ms 2 ms 1 ms 5 mv 5 mv F intr () Fintr () d ell Vis (mv) ell V is (mv) -1 1 Fig. 3. Persistent hnge in firing rte nd eye position orrelted with persistent hnge in memrne potentil. () Chrteristi memrne potentil during dishrge. Averge interspike memrne potentil ws ssessed fter exluding the underlined segments of dt tht egn 1 ms efore the pek of the tion potentil nd ended 3 ms fterwrd. The dt shown re verges of thirty tion potentils tken from the sme fixtion; left nd right pnels re from two different neurons. () Memrne potentil nd interspike memrne potentil remining fter spike exlusion for two position neurons. Right pnels of () nd () disply dt from the sme ell. (, d) Fixtion-verged intrellulr firing rte plotted ginst fixtion-verged interspike memrne potentil. The set of points in eh figure orrespond to the fixtions mde during one nsl temporl nsl snning yle, with memrne potentil mesured reltive to the first fixtion (ell 1, r =.97, p <.1, sme ell s in Fig. 2 nd ; ell 2, r =.98, p <.1, sme ell s in Fig. 2). (e) Interspike memrne potentil plotted ginst eye position (r =.86, p <.1). (f) Interspike memrne potentil plotted ginst the firing rte of seond extrellulrly reorded position neuron (r =.98, p <.1). (g) Chrteristi tion potentil wveform nd threshold vrition with fixtion position (N, nsl position; M, mid-rnge position; T, temporl position; lk dot, pprent tion potentil threshold). V is (mv) V is (mv) e f ell 1 Eipsi (degrees) ell F extr () g T M N N M T 1 ms T M N 1 mv tion mehnism for position neurons is lrgely onstnt on the minutes time sle during spontneous sdes nd fixtions. It hs een proposed tht slow, tive ondutnes like the lium-dependent potssium type my e involved in lineriztion of spike genertion 27,28. Position neuron tion potentils displyed prominent fter-hyperpolriztion (AHP) tht ws onsistent with the presene of slow potssium ondutnes (n = 15/18, Fig. 3). AHPs, ssessed for tion potentils followed y n interspike intervl of t lest 6 ms, were 2 1 mv deep, 2 4 ms in durtion, nd, in 3/15 reordings, displyed distint seond minimum (Fig. 3, right). As previously presented, the suppression of tion potentil genertion did not stop fixtion-relted steps in memrne potentil (Fig. 4). However, AHPs nd dendriti invsion ould e importnt in modifition of memrne urrents nd ondutnes, possily influening dishrge rte. To explore this possiility, the reltionship etween potentil nd fixtions over omplete snning yle ws ompred (n = 5) for the situtions with dishrge (Fig. 3e nd f) nd without dishrge (Fig. 4 nd d). Bsed on pired t-test of the differene in slopes for ells where fixtion ehvior ws monitored y neurl reording, there ws no signifint hnge in sensitivity (n = 5, p >.5). In ddition to hnges in spike frequeny, vritions in V is were lso ssoited with hnges in spike shpe. Ation potentils simultneously deresed in pek-to-pek mplitude (Figs. 2 nd 3g) nd inresed in width t hlf-mximum (Fig. 3g) s V is, firing rte nd position inresed; vrition of spike mplitude with position ws lso seen with extrellulr reordings 11 (Fig. 1). Furthermore, the pprent threshold for tion potentil initition, mesured y determining the point on the spike rise where slope exeeded 15 mv/ms, ws orrelted with V is, inresing with temporl positions (Fig. 3g; r >.7, p <.5; n = 11/14). Visul inspetion of reordings with tion potentils suppressed y negtive urrent injetion reveled no indition of rhythmi osilltion in memrne potentil (Fig. 4 nd ; n = 8/8). Also, during low firing rtes (<1 ), when the men time etween spikes exeeded the durtion of spike AHP, V is did not show evidene of n intrinsi rhythmiity (dt not shown; n = 18). However, we oserved non-rhythmi memrne potentil flututions (nlysis in finl setion of Results). In ddition to reveling steps in, suppression of tion potentils lso exposed the presene of reltively lrge, trnsient (5 1 ms) depolriztions during on-sdes (Fig. 4; n = 7/8). Defletions hd pek potentils tht were 2 to 14 mv ove the susequent post-sdi potentil (n = 7/8). The durtion of these events losely mthed the durtion of ursts in firing rte seen t more depolrized levels (Fig. 2 nd ) nd during extrellulr reording 11 (Fig. 1; n = 7/8). During off-sdes in the sene of tion potentils, memrne potentil trnsitioned in rmplike fshion etween two levels (Fig. 4; n = 8/8). These trnsitions ourred typilly in less thn 1 ms, oiniding with the durtion of undershoots in firing rte t more depolrized levels (Fig. 2; n = 8/8). The firing rte in the first 5 ms fter the trnsient urst ompnying sdes frequently exhiited slide towrd toni level (Fig. 2). This pttern of tivity, possily importnt in overoming the viso-elsti properties of the oulomotor plnt, hs een previously oserved in extrellulr reordings of position nture neurosiene volume 4 no 2 ferury

5 When fixtions were ove ell s position threshold, urrent stimultion lso filed to result in fter-dishrge (Fig. 5; n = 6/6). Firing rte during stimultion either remined fixed or deyed slowly (< 1 /s). After stimultion, firing rte (F intr ) returned to level similr to tht efore perturtion ( 1 < F intr < 5 ; weighted men, 3.6 ; n = 6). The pplition of repeted steps in lose suession filed to eliit ny systemti inrese in F intr. Repeted depolrizing tringulr urrent injetions into one neuron were used to test for hysteresis in firing rte (n = 5 trils), ut none ws oserved (Fig. 5 nd d). When fixtions were ove ell s position threshold, negtive urrent pulses were dministered to suppress firing; ll of these injetions filed to generte persistent suppression of dishrge (Fig. 6 nd ; n = 3/3). After inhiition, firing rte returned to level similr to tht efore perturtion ( 1 < F intr < 1 ; weighted men, 2.1; n = 3/3); lrge-mplitude injetions typilly eliited rief post-inhiitory reound in dishrge rte (Fig. 6). The pplition of repeted steps in lose suession filed to eliit ny systemti derese in F intr. Following rpid (<.1 ms) termintion of injetions pplied elow position threshold, verged memrne potentil returned, within 5 ms, to within 1 mv of the pre-stimulus level (n = 3/3). We used repeted hyperpolrizing tringulr urrent injetions into one neuron during fixtions elow the position threshold to test for hysteresis in memrne potentil (n = 5 trils), ut none ws oserved (Fig. 6 nd d). For two reordings, esium ws sustituted for potssium in the intrellulr reording solution. Steps in memrne potentil were still oserved, even when the fst omponent of the AHP ws eliminted (Fig. 2). Under these onditions, with the eletrotoni dey onstnt inresed, rief urrent injetions into one ell ould not produe sustined hnge in dishrge rte (Fig. 6). The ove results of diret stimultion onstrin the role tht voltge-dependent regenertive mehnisms ould hve in genertion of the persistent firing exhiited y position neurons. Diret stimultion lso llowed n estimtion of pprent input impedne nd relxtion time. When neurons were t rest or slightly hyperpolrized (5 1 mv), pprent input impedne, quntified y pplying smll urrent steps (<.5 ) in the sene of dishrge, verrtiles 21 Nture Pulishing Group 21 Nture Pulishing Group Fig. 4. Step-like hnges in memrne potentil in the presene of hyperpolrizing urrent. () Suthreshold, step-like depolriztions of memrne potentil ompnying nsl to temporl steps of position during lokde of toni tivity with 1.2 of holding urrent. The seond sde eliited single (trunted) tion potentil. () Suthreshold step hyperpolriztion ompnying temporl to nsl trnsition in position. () Fixtion-verged memrne potentil plotted ginst eye position during suppression of dishrge, with potentil mesured reltive to the first fixtion of the snning yle used (r =.87, p <.5). (d) Memrne potentil for the ell in () plotted ginst the firing rte of seond extrellulrly reorded position neuron (r =.97, p <.1). neurons 11 (Fig. 1). Assoited with this slide in firing rte ws slow relxtion of memrne potentil, pprent t oth depolrized (Fig. 2) nd hyperpolrized levels (Fig. 4). The results presented so fr suggest tht steps in memrne urrent nd/or ondutne tht produed persistent steps in verge memrne potentil mde signifint nd possily suffiient ontriution to fixtion-relted steps of firing rte. The reminder of this study ddresses how persistent steps in urrent nd/or ondutne might e generted nd mintined in eh position neuron. Diret stimultion nd trnsient hnge in tivity In priniple, steps in memrne potentil nd firing rte ould e the result of plteu potentils. These persistent events n e initited with rief depolrizing urrent injetions nd stopped with rief hyperpolrizing injetions through n intrellulr reording eletrode 29. To investigte possile involvement of regenertive plteu events in position neurons, we pplied trnsient urrent injetions in n effort to initite nd/or terminte persistent hnges in dishrge rte (n = 1). The presene of persistent inrese in rte following depolrizing steps or persistent derese following hyperpolrizing steps would e onsistent with n involvement of plteu potentils. In ddition, we used tringulr urrent injetions to see if position neurons showed hysteresis in their urrent dishrge reltionship, nother feture of ells with plteu potentils 16. We mintined stle dishrge tivity (s in Figs. 1 nd 2) using wek holding urrent (, to 1 ). We ssessed the firing rte efore, during nd fter given urrent injetion within single fixtion. For depolrizing injetions, stimulus durtion ws vried etween.5 nd 2. s; stimulus mplitude ws vried etween.3 nd 3.. For hyperpolrizing injetions, durtion ws vried etween.1 s nd 1. s; mplitude ws vried etween.5 nd 1.. When fixtions were elow ell s position threshold, diret depolrizing injetions were dministered to eliit dishrge rtes of 3 to 3, mthing, nd in some neurons exeeding, the mplitude of firing rte ursts during on-sdes. All of these injetions filed to result in fter-dishrge (Fig. 5; n = 8/8). During stimultion, elevted firing rte fter the first two interspike intervls either remined fixed or, prtiulrly during lrge injetions (> 1. ), deyed slowly t rtes of less thn 1 /s. Following rpid (<.1 ms) stimulus termintion, verged memrne potentil returned to within 1 mv of the pre-stimulus level within 5 ms (n = 5/5). V extr V extr 5 ms 1 ms.4 mv 5 mv.4 mv mv E ipsi (degrees) 2 4 Fextr () (mv) (mv) d nture neurosiene volume 4 no 2 ferury 21

6 21 Nture Pulishing Group rtiles 21 Nture Pulishing Group F intr Fintr Iinj Fintr 5 ms 5 2 mv.5 1 ms 1 ms Fig. 5. Trnsient depolrizing urrent injetion during single fixtions did not indue persistent inrese of dishrge rte or memrne potentil. () Chnge in firing rte nd memrne potentil in response to intrellulr depolrizing urrent injetion during nsl fixtion. () Chnge in firing rte nd memrne potentil in response positive urrent injetion during fixtion ove position threshold. () Firing rte nd memrne potentil in response to depolrizing tringulr urrent pulse during nsl fixtion. (d) Firing rte plotted ginst injeted urrent for the rmps in (). ged 3.2 ± 16.3 MΩ (rnge, MΩ; n = 6). When deeply hyperpolrized with urrent injetion ( 8 > > 1 mv), impedne dropped to 9.5 ± 2 MΩ (n = 2). When neurons were depolrized to slightly ove rest with urrent injetion ( 5 mv), pprent input impedne, mesured with hyperpolrizing urrent steps (<.5 ) tht loked ll dishrge, verged 12.5 ± 3.5 MΩ (n = 2). We ssessed memrne relxtion time following smll urrent steps (<.5 ) in the sene of dishrge while neurons were t rest or slightly hyperpolrized (5 1 mv). Relxtion time ws estimted y determining the point t whih the post-stimulus potentil returned two-thirds of the wy to the pre-stimulus level. This vlue verged 16.3 ± 3.9 ms (rnge, ms; n = 5). d F intr () mv.5 2 sending 2 mv 1 () desending Position-ssoited memrne potentil flututions If position neurons reeive exittory feedk from eh other, then hnges in toni firing should e ompnied y hnges in the rte of exittory postsynpti potentils (EPSPs). Alterntively, hnges in toni firing ould result from hnges in the mplitude of inident PSPs from neurons with onstnt rtes. In prtie, it n e diffiult to resolve individul PSPs in vivo euse of their high rtes nd pssive filtering y dendriti les. Therefore, we relied on the vrine of memrne potentil 3 s n indiret mesure of hnges in the rte or mplitude of synpti input. Interspike memrne potentil ompnying fixtions showed pronouned flututions throughout the reording (, to 1 ). Two exmples from ells exhiiting prominent flututions re shown in Fig. 7 nd, in whih insets highlight inresed flutution mplitude t temporl eye positions. In 15 reordings, firing rte ws low enough so tht flututions during segments of interspike memrne potentil ould e ompred t different fixtions. Flututions were quntified over omplete yle of the snning pttern. Root men squre interspike memrne potentil (V is rms ) vlues for eh fixtion were determined y verging over 3-ms interspike segments within eh fixtion (see Methods). For the popultion of ells nlyzed, men V is rms ws.49 ±.19 mv (n = 15). In 4 of 15 ells, V is rms signifintly inresed with temporl position, nd with V is (r >.8; slope, 5 ± 2 µv/mv; p <.5; Fig. 7, nd ). Correltion etween V is rms nd V is ws not oserved in the other position neurons (p >.5). To explore the origin of flutution hnges, we suppressed tion potentils with hyperpolriztion (Fig. 7d; n = 8, inluding 3 tht showed orreltion etween V is rms nd V is ), whih llowed ssessment of flututions in the sene of ny fter-polriztions. Flututions were quntified over omplete yle of the snning pttern, with root men squre memrne potentil ( rms ) mesured using 1-ms segments. In two of eight ells, rms signifintly inresed with temporl position, nd with (r >.8; slope, 1 ± 4 µv/mv, p <.1). Correltion etween rms nd ws not oserved in the other position neurons (p >.5). The two neurons tht did show orreltion were of the set of three neurons whose V is rms nd V is were orrelted. The oserved vritions in flutution mplitude re onsistent with hnge in synpti tivity with fixtion position. However, the position dependene of flututions my hve een used simply y voltge dependene of intrinsi urrents. To explore this possiility, one of these two position neurons ws injeted with trin of rief urrent steps during trnsitions etween nsl nd temporl fixtions (Fig. 7d). Despite signifint eletrode-generted hnge in memrne potentil ( 1 mv), little hnge of flutution hrteristis ourred within fixtion. The flututions during the temporl fixtion were still lrger in mplitude thn during the nsl fixtion, even when the memrne potentil ws more hyperpolrized during the temporl fixtion (Fig. 7d). This oservtion ws quntified for 1 fixtion trnsitions. Diret omprison of the differene in rms etween fixtions ( rms ) ws mde for two different dt sets: first, set with equl to.3 during oth fixtions, nd seond, set with equl to.3 during the nsl fixtion nd 1. during the temporl one. For oth dt sets, rms ws plotted ginst hnge in verge memrne potentil etween temporl nd nsl fixtions, with equl to.3 (set 1, Fig. 7e, top; set 2, Fig. 7e, ottom). For oth sets, rms ws onsistently higher t temporl positions (set 1, rms / = 4 ± 2 µv/mv, p <.1; nture neurosiene volume 4 no 2 ferury

7 rtiles 21 Nture Pulishing Group Fintr 5 rte nd fixtion were ompnied y pronouned steps in memrne potentil. Finlly, in qurter of the reordings, nsl to temporl steps in ipsilterl eye position were ompnied y inresed root men squre (RMS) flutution of memrne potentil. 21 Nture Pulishing Group Iinj Fintr 5 ms 5 mv Fig. 6. Trnsient hyperpolrizing urrent injetion during single fixtions did not indue persistent derese of dishrge rte or memrne potentil. () Chnge in firing rte nd memrne potentil in response to intrellulr hyperpolrizing urrent injetion during fixtion ove position threshold. () Chnges during lrge mplitude, long durtion perturtions. () Memrne potentil in response to hyperpolrizing tringulr urrent pulse during nsl fixtion. (d) Memrne potentil plotted ginst injeted urrent for the rmps in (). set 2, rms / = 8 ± 4 µv/mv, p <.1; n = 1 fixtion trnsitions). This result demonstrtes tht the flutution hnges during spontneous eye movements nnot e explined y the voltge dependene of intrinsi urrents. DISCUSSION These results provide informtion out persistent tivity tht ws previously unttinle y nlysis of extrellulr reordings. A si finding ws tht rief urrent injetions produing rtifiil sde-like hnges in firing rte did not produe persistent hnges in neurl tivity. A seond finding ws tht steps in firing d (mv) ms 1 ms 2 mv mv 1 () Single-ell stimultion nd indution of persistene Intrellulr injetion of urrent did not generte sustined plteu potentils or terminte memrne potentil steps produed during spontneous eye movements. In ddition, the pplition of repeted steps in lose suession filed to eliit ny systemti inrese in firing rte, inditing the sene of slow uildup in rte, termed windup, tht hs een oserved in motoneurons 31. These results suggest tht plteu potentils re not involved in the genertion of persistent neurl tivity in position neurons. There re, however, severl vets. Beuse intrellulr reordings were proly lose to the som 11 nd distl dendrites ommonly extend five hundred mirons 11, dendriti le ttenution my hve isolted puttive distl plteu potentils from perturtion. However, when we repled potssium with esium s the reording solution tion to derese the eletrotoni length of the ell, we were still unle to lter memrne potentil steps. Plteu potentils my lso hve een trnsiently ltered y injeted urrent, only to e rpidly re-initited y synpti urrents when injeted urrent ws turned off. Continuous high-frequeny extrellulr stimultion of regions ner puttive VPNI sites produe ipsilterlly direted drift of the eyes over tens of degrees nd fst phses tht reset eye position 32,33. The slow drift hs roughly onstnt veloity, onsistent with temporl integrtion of onstnt mplitude stimulus. Why, then, does extrellulr tivtion of mny neurons, ut not single-ell, seem to e integrted? One possiility is tht the single ell stimultion hd wek effet on the VPNI tht my e detetle only y signl verging of hundreds or thousnds of trils. Other possiilities re tht single-ell stimultion did not surpss stimulus threshold or tivte ritil synpti inputs to the integrtor. Short-term potentition of synpti inputs rrying eye veloity signls hs een proposed s mehnism for oulomotor integrtion 34. This mehnism predits no persistent hnge following trnsient hnge of firing rte in position neurons, onsistent with our dt. Memrne potentil steps during eye movements Anlysis of extrellulr spike wveforms previously led to the suggestion tht re I position neuron dendrites support sodium spike propgtion 11. Suh dendriti spikes ould provide ellulr positive feedk mehnism y providing depolriztion neessry for voltge-dependent urrents suh s those of the NMDA reeptor 35. The results presented here, demonstrting tht steps in memrne potentil remined when sodium tion potentil initition ws loked y hyperpolrizing urrent injetion, suggest tht spike-sed depolriztion of the dendrites is not neessry for the genertion of step hnges in memrne potentil. Two other ellulr mehnisms for persistent neurl tivity were rendered unlikely y our experiments: persistent hnges in suthreshold pemker urrents, nd persistent hnges in tion potentil threshold. None of our reordings showed evidene of suthreshold pemker urrents, wheres tion potentil threshold inresed, rther thn deresed, during steps with higher firing rtes. Memrne potentil flututions A ler inrese in RMS flutution of memrne voltge ws oserved during more depolrized steps. The oneptul frme- 19 nture neurosiene volume 4 no 2 ferury 21

8 21 Nture Pulishing Group rtiles Temporl ell 1 Nsl Nsl ell 2 Temporl F extr 2 F extr 5 21 Nture Pulishing Group e rms V is (mv) rms (mv) (mv) 1 s ell 1 ell Vis (mv) Temporl, =.3 Nsl, =.3 rms V is (mv) rms (mv) Vis (mv) (mv) 2 mv Temporl, = 1. Nsl, =.3 d Fextr Iinj Temporl ell 1 1 s Nsl 5 ms 2 mv 2 1 mv.3 1 Fig. 7. Persistent hnges in the mplitude of memrne potentil flututions orrelted with steps in memrne potentil. (, ) Memrne potentil hnge during fixtion trnsition ompnied y hnge in the mplitude of memrne potentil flututions (insets). In (), fixtion position ws monitored y the tivity of ontrlterl duens motoneuron; step inrese of firing rte (F extr ) ws orrelted with step derese in memrne potentil. In (), fixtion position ws monitored y the tivity of seond ipsilterl position neuron. Sle rs, 5 ms nd 2 mv. () Root men squre interspike memrne potentil (V is rms ) plotted ginst memrne potentil for two position neurons. (d) Memrne potentil hnges for the position neuron in () during suppression of tion potentil dishrge. Trnsitions in fixtion position (dshed line) were still ompnied y hnges in flutution mplitude (insets). Different levels of hyperpolrizing urrent were used to investigte the voltge dependene of hnges in flutution mplitude (see results). Sle rs, 5 ms nd 2 mv. (e) Quntittion of hnge in the mplitude of memrne potentil flututions during tion potentil suppression. is the differene in memrne potentil etween fixtions with equl to.3, s indited in (d). work developed 3 for the study of inresed memrne potentil flututions t the neuromusulr juntion during depolriztion indued y etylholine my e pplile to understnding our results. Inresed flututions were ttriuted to n inresed rte of single moleule inding events: if the inding of n etylholine moleule produes shot effet trnsient depolriztion, then n inresed rte of inding during etylholine pplition would led to oth inresed men memrne potentil nd inresed memrne potentil vrine. Here we found inresed flututions t temporl fixtion positions (Fig. 7). By nlogy, this inresed flutution mplitude ould reflet n inresed rte of EPSPs. Beuse the dishrge rtes of other position neurons were lso inresing with temporl fixtion position, the flututions oserved were onsistent with exittory reurrent feedk within the VPNI. However inresed synhrony of synpti inputs, rther thn simply inresed rte of synpti events, ould lso led to inresed memrne potentil noise 36. In ddition, inresed RMS noise ould e produed y hnge in the mplitude of synpti events with no hnge in rte. Position neurons modulte their firing rtes during horizontl VOR, implying tht they reeive toni inputs from neurons rrying hed veloity signls. Sdi inputs my lter the mplitude of these toni inputs, leding to hnge in men memrne potentil nd RMS noise. Experiments tht resolve single postsynpti urrents ould differentite etween these possiilities. Only qurter of the reordings nlyzed exhiited signifint orreltion etween RMS flutution noise nd men memrne potentil. This rtio my simply reflet the proility tht reording site ws lose enough to dendriti regions tht reeived synpti inputs modulted with eye position. Other implitions for reurrent network models We found n pproximtely liner reltionship etween firing rte nd injeted urrent (Fig. 5 nd d). This supports the use of model neurons hving this property in reurrent network models of the VPNI 24. The oservtion of multiple-omponent slow nture neurosiene volume 4 no 2 ferury

9 rtiles 21 Nture Pulishing Group 21 Nture Pulishing Group AHPs following tion potentils is onsistent with possile involvement of slow potssium urrents in the genertion of liner reltionship etween firing rte nd injeted urrent 27,28. In existing network VPNI models 14,21 24, the persistene of tivity in single neuron is hrterized y ellulr time onstnt; tuned positive feedk through reurrent synpses oosts the time onstnt of persistent tivity in the network ove tht of the single ell. In generl, longer ellulr time onstnts redue the preision required in tuning feedk 24. If the pprent 15-ms memrne time onstnt we mesured in VPNI neurons determines the relevnt ellulr time sle for integrtion in re I, then feedk tuning requirements would e very stringent. Therefore, it my e importnt to look for slower ellulr proesses tht ould redue the preision required, suh s short-term synpti plstiity or slowly deying synpti ondutnes, s well s mehnisms for dptively tuning feedk sed on VPNI performne. METHODS Preprtion nd eletrophysiology. All experiments were done in ompline with the Guide for the Cre nd Use of Lortory Animls ( Speifi protools were pproved y the Institutionl Animl Cre nd Use Committee. Goldfish (Crssius urtus, 3 5 inhes from tip to pedunle, 25 5 g) were purhsed from ommeril supplier (Hunting Creek Fisheries, Thurmont, Mrylnd) nd kept t 2 22 C in 5-gllon qurium with dily exposure to light. The methods used for hed restrint, surgil preprtion, reording of extrellulr tion potentils, re I mpping, nd eye movement mesurements were s desried previously 11. Intrellulr reordings from neurons in re I were otined with shrp eletrodes or pth eletrodes in the urrent-lmp onfigurtion. Shrp eletrodes filled with reording solution (1 M KCl, 5 mm KH 2 PO 4, 1 mm Fst Green, ph 7.4) were eveled to resistne of 6 12 ΜΩ. In five nlyzed reordings, 1 2% Neuroiotin ws dded to the reording solution to visulize ell morphology 11 ; in two other nlyzed ses, esium ws sustituted for potssium. Shrp eletrodes were imed towrd the somt nd proximl dendrites of position neurons 11. Pth eletrodes (ule #4.5 5) were filled with intrellulr solution (115 mm K-methnesulfonte, 9.6 mm K-HEPES, 3 mm N 2 -ATP, 1 mm Fst Green,.3 mm N 3 -GTP,.3 mm MgCl 2,.1 mm K 2 -EGTA, ph 7.3 with KOH, 252 mosm) nd hd resistne of 6 1 MΩ. Pth eletrodes were kept free of deris during penetrtion to re I y pplition of 1 psi positive pressure; one within re I, king pressure ws redued to.1.2 psi to enle serh for inreses in resistne inditive of proximity to neuron. Intrellulr voltge signls were low-pss filtered (5 khz, 12 pole-bessel, Cygnus Tehnologies, Delwre Wter Gp, Pennsylvni) nd digitlly smpled t rtes of t lest 1. khz on Digidt 12B dt quisition system nd Clmpex 7. softwre (Axon Instruments, Foster City, Cliforni). Sdi nd fixtion ehvior ws ssessed diretly y monitoring eye movements with the slerl serhoil method 11,37. In 12 of 18 preprtions from whih nlyzed reordings of position neurons were otined, 4 µg gllmine triethiodide (Flxedil, Amerin Cynmid, Wyne, New Jersey) or 1 µg doxurium hloride (Nuromx, Glxo Wellome, Reserh Tringle Prk, North Crolin) ws intrmusulrly injeted to fford inresed stility of reording. In these situtions, eye movements were still present, ut ttenuted to the extent tht fixtion ehvior ws etter monitored y using the firing rtes of neurons orrelted with horizontl eye position. This ltter mesurement of fitive eye movements ws done with seond extrellulr reording miroeletrode positioned in either the duens nuleus 38 or re I. Comined intrellulr nd extrellulr reordings were otined y estlishing the extrellulr reording first. Intrellulr reordings were mintined for medin time of 5 min (rnge, 1 6 min). Anlysis. Neurons exhiiting step-like vrition in firing rte (, to 1 ) like tht in Fig. 1 were seleted for further nlysis s position neurons (n = 18 totl; n = 15 shrp, n = 3 pth). At lest one full yle ws nlyzed for eh ell. In seven of these reordings, fixtion ehvior ws independently monitored y extrellulr neurl reording, nd in three others, y the slerl serh-oil method. Eh nlyzed ell ws reorded in different preprtion. All dt were nlyzed in the Mtl environment (MthWorks, Ntik, Msshusetts). Sdes were determined y identifying rpid (<.2 s) nd lrge trnsitions in either eye position (> 2 ) or firing rte (> 5 ). Cells were identified s exhiiting sde-relted ursts or undershoots of rte s previously desried 11 ; ells were identified s exhiiting trnsient depolriztions if overshooting memrne potentil devitions tht were more thn 1 mv ove the susequent post-sdi potentil were oserved on 75% of sdes. Interspike memrne potentil during periods of dishrge ws mesured fter removl of dt segments tht egn 1 ms efore nd ended 3 ms fter the pek of n tion potentil. This proedure removed ll supr-threshold tivity nd prt of the fter-hyperpolriztion, whih in some reordings, ould lst pproximtely 4 ms. Averge fixtion prmeters were lulted over region tht egn 15 ms fter sde nd ended 15 ms efore the next sde. In Figs. 2, 4 nd 7, verge potentil inditors to the left of tres re lulted over entire fixtions using these onditions, nd not just the regions shown. Firing rte ws lulted y previously desried method using smoothing window of 15 ms (ref. 11) exept when rief urrent pulses (Figs. 5 nd 6) were pplied, in whih se rte ws lulted y finding the reiprol of the interspike intervl. Apprent tion potentil threshold within fixtion ws determined using n tion potentil profile found y verging the wveforms of the first 1 spikes in the fixtion. A yle of the snning pttern used for ssessment of the reltionship etween verge firing rte nd memrne potentil inluded fixtions immeditely elow position threshold when present. All tests for ssessing the signifine of orreltions were done using one-tiled t-test. All vlues given with ± inditor represent men ± s.d.. All nlyzed responses to injeted urrent pulses were from regions of dt tht did not inlude sdi trnsitions. Injetions pplied ove the position threshold were done without referene to the nsl temporl degree of fixtion position. Bridge-lne ws normlly mintined during the reording with the use of smll (<.3 ) test pulses; remining imlne ws orreted during nlysis y using s referene the pek height of tion potentils efore injetion. The sensitivity g inj ws ssessed using 2 1 dt points of firing rte nd memrne potentil evoked y injeted urrent pulses or rmps tht vried in length from.2 2. s nd did not overlp with sdes. Furthermore, mesurements were mde only for vritions in indued firing rte tht were similr to the vritions oserved during ssessment of the sensitivity g spont. Persistent effets of trnsient urrent injetion were ssessed using 5 1 injetion pulses per ell for eh polriztion diretion. The firing rte efore, during nd fter injetion pulses ws mesured over identil length windows tht were.15.5 s long. Chnges in intrellulr firing rte ( F intr ) were defined s post-step minus pre-step firing rte. Root men squre (RMS) nlysis ws done y first reking memrne potentil reords during fixtions into segments tht were either 3 or 1 ms long (see Results). If the fixtion period ws ompnied y dishrge, eh segment ws ontined within single interspike region. If memrne potentil trnsitioned etween two polriztions euse of diret urrent injetion, the initil 5 ms fter urrent step ws not used. Next, slope-sutrtion ws done y lulting est fit line (in the lest-squres sense) to the dt segment nd sutrting the ordinte vlues of this line. The RMS vlue for given fixtion ws lulted s the men RMS of ll segments tken from tht fixtion. ACKNOWLEDGEMENTS We thnk G. Mjor nd F. Helmhen for omments on the mnusript. RECEIVED 21 SEPTEMBER; ACCEPTED 18 DECEMBER 2 1. Fuster, J. M. & Alexnder, G. E. Neuron tivity relted to short-term memory. Siene 173, (1971). 2. Fuster, J. M. Memory in the Cererl Cortex (MIT Press, Cmridge, Msshusetts, 1995). 3. Goldmn-Rki, P. S. Cellulr sis of working memory. Neuron 14, (1995). 192 nture neurosiene volume 4 no 2 ferury 21

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