Corollary discharge circuits for saccadic modulation of the pigeon visual system

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1 28 Nture Pulishing Group Corollry dishrge iruits for sdi modultion of the pigeon visul system Yn Yng 1, Peng Co 1,2, Yng Yng 1,2 & Shu-Rong Wng 1 A sdi eye movement uses vriety of trnsient pereptul sequele tht might e the results of orollry dishrge. Here we desrie the neurl iruits for sdi orollry dishrge tht modultes tivity throughout the pigeon visul system. Sdes in pigeons used inhiition tht ws medited y orollry dishrge followed y enhnement of firing tivity in the telenephli hyperpllium, visul thlmus nd pretetl nuleus lentiformis mesenephli () with opposite responses in the essory opti nuleus (). Intivtion of thlmi neurons eliminted sdi responses in telenephli neurons, nd intivtion of oth the nd the olished sdi responses in thlmi neurons. Sde-relted omnipuse neurons in the rinstem rphe omplex inhiited the nd exited the, wheres intivtion of rphe neurons eliminted sdi responses in oth optokineti nd thlmi neurons. It seems tht sdi responses in telenephli neurons re generted y orollry dishrge signls from rinstem neurons tht re trnsmitted through optokineti nd thlmi neurons. These signls might hve importnt roles in visul pereption. Humns nd other fovete nimls use rpid, drting eye movements (sdes) to serh for or fixte trget of interest with the fove. A vriety of pereptul phenomen our in temporl reltion to the sde. These inlude reeptive field shifts tht remp the visul field nd mintin sptil onstny nd egin in dvne of the sde 1 3, s well s ompression of the pereption of time nd spe 4,5. Neurl responses in reltion to sdes hve een found in the lterl geniulte nuleus (LGN) 6 8 nd in ortil res tht reeive mgnoellulr inputs in mmmls Both the pereptul effets nd t lest some of the neurl responses seem to e driven y orollry dishrge relted to the sde, rther thn y the rpid smering of the visul sene during the sde. Furthermore, orollry dishrge through the thlmus ontriutes to the genertion of sptilly urte sdes in monkeys 3,12. Beyond this exmple, however, the neuronl iruits tht underlie the hnges in firing tivity of visul neurons round sde re unler. Our understnding of the vin visul system offers good opportunity to revel neurl iruits tht medite orollry dishrge from the sdi system. The telenephli hyperpllium (Wulst) in irds reeives input from the nuleus optius priniplis thlmi () (lso lled the LGN). In turn, reeives fferents from the nd the of the essory opti system 13,14. These visul strutures might e homologous to the strite ortex, LGN, the nuleus of the opti trt, nd the terminl nulei of the essory opti trt in mmmls, respetively (Fig. 1) 15,16. Both the nd the, nd their mmmlin homologs, re involved in generting optokineti nystgmus (OKN), whih onsists of slow nd quik phses for stilizing imges on the retin Furthermore, the reeives fferents from the rphe omplex in the rinstem retiulr formtion 2, whih in turn reeives input from the opti tetum 21,22.The tetum or superior olliulus nd retiulr formtion re involved in sdi eye movements in ll the speies of vertertes tht hve een exmined. Therefore, the optokineti nulei nd rinstem retiulr formtion seem to e suitle ndidtes for modulting perisdi tivity in telenephli neurons using the s rely sttion. To provide physiologil nd ntomil evidene tht the sdi responses of telenephli ells re modulted y orollry dishrges from the optokineti nulei nd the rphe omplex through the, we used extrellulr reording, tivtion nd intivtion tehniques in three steps. First, we reorded the responses of neurons in the telenephlon,,, nd rphe omplex to sdes, to ompre their firing ptterns nd time ourses. Seond, we used orthodromi nd ntidromi tivtion to revel funtionl onnetions etween optokineti nd rphe neurons. Third, we investigted the effets of thlmi lokde on telenephli responses, the effets of nd lokde on thlmi responses nd the effets of rphe intivtion on optokineti nd thlmi responses to sdes. These investigtions provided onsistent evidene tht the optokineti nulei nd rphe omplex medite the sdi responses of telenephli neurons vi orollry dishrge signls tht pss through the thlmus. RESULTS We investigted the responses of 32 neurons (Supplementry Tle 1 online) during sdes nd other interventions, onsistently showing tht the sdi responses of telenephli ells re driven y 1 Lortory for Visul Informtion Proessing, Stte Key Lortory of Brin nd Cognitive Sienes, Institute of Biophysis, Chinese Ademy of Sienes, 15 Dtun Rod, Beijing 111, Chin. 2 These uthors ontriuted eqully to this work. Correspondene should e ddressed to S.-R.W. (wngsr@sun5.ip..n). Reeived 12 Novemer 27; epted 1 Mrh 28; pulished online 6 April 28; doi:1.138/nn.217 NATURE NEUROSCIENCE VOLUME 11 [ NUMBER 5 [ MAY

2 28 Nture Pulishing Group Strite ortex LGN NOT Hyperpllium Telenephlon TN Retin Entopllium Nuleus rotundus Pulvinr pthwys tht originte in the sde-relted rphe omplex nd projet through two optokineti nulei to nd finlly to the telenephli hyperpllium. Sdi responses in telenephli nd thlmi ells The eletro-oulogrms (EOG) in Figure 2 show tht sde in the pigeon onsists of shift of eye position nd severl yles of osilltion When the pigeon fed sttionry grtings, its spontneous sde hd pek speed of 485 ± 6 deg s 1 nd durtion of 242 ± 36 ms with 6.4 ± 1. yles of osilltion (men ± s.d., n ¼ 6). As illustrted in Figure 2,, the firing rtes of ll telenephli neurons reorded egn to drop 73 ± 44 ms efore sde onset nd underwent n inhiition tht lsted 348 ± 5 ms followed y enhned firing tht egn t sde offset nd lsted for 385 ± 78 ms (n ¼ 2). Perisdi hnges in the firing rtes of ll ells exmined were in phse with those in telenephli ells. Although the onset times of inhiition nd enhnement in ells preeded those in telenephli ells y 2 3 ms, the durtions of inhiition nd enhnement were not different etween telenephli nd thlmi ells (P 4.6, n ¼ 2 eh). To evlute the possiility tht perisdi inhiition nd enhnement in telenephli ells originte from ells, we tested the effets of intivting on perisdi tivity in six telenephli ells tht hd spontneous tivity (24 ± 11 spikes s 1 ) nd visul responses. When the pigeon mde sdes ross sttionry grtings, the firing rtes in these ells egn to derese 67 ± 26 ms efore sde onset. The inhiition lsted 342 ± 65 ms with hnge rtio of 43 ± 11% nd ws followed y enhnement, whih strted 275 ± 42 ms fter sde onset nd lsted for 36 ± 22 ms with hnge rtio of 34 ± 9%. Sdi inhiition nd enhnement in ll six ells were OT Extrstrite SC Rphe nuleus RIP Figure 1 Visul strutures nd pthwys re similr in irds nd mmmls. Pired squres represent similr strutures in irds (light gry) nd mmmls (drk gry). Arrows symolize neurl projetions. In irds, the thlmofugl pthwy goes from the to the telenephli hyperpllium; the tetofugl pthwy projets from the opti tetum (OT) to the nuleus rotundus to the telenephli entopllium, nd the essory opti pthwy projets from the retin to the. These strutures might e homologous to the lterl geniulte nuleus (LGN), strite ortex, superior olliulus (SC), pulvinr, extrstrite ortex nd terminl nulei (TN) of the essory opti trt in mmmls, respetively. The pretetl nd the rinstem rphe omplex re omprle to the nuleus of the opti trt (NOT) nd the nuleus rphe interpositus (RIP), respetively, in mmmls. Other revitions re defined in the text. ompletely olished 1 5 min fter GABA ws injeted into the nd reovered in 5 1 min (Supplementry Fig. 1 online). The spontneous firing of these telenephli ells ws redued y B2% during lokde. We then exmined sdi responses in ells under different onditions. When the pigeon mde sdes ross sttionry grtings, sdi inhiition nd enhnement in ells lsted 34 ± 45 ms nd 388 ± 96 ms, respetively (n ¼ 2, Fig. 2,). The perisdi responses persisted when sdes were mde ross lnk sreen (n ¼ 2, durtion ¼ 34 ± 7 ms for inhiition nd 395 ± 72 ms for enhnement) nd during sdes in omplete drkness (n ¼ 2, durtion ¼ 345 ± 54 ms for inhiition nd 393 ± 65 ms for enhnement). One-wy ANOVA nlysis indited tht the durtions of sdi inhiition (F 2, 57 ¼.5, P ¼.95) nd enhnement (F 2, 57 ¼.5, P ¼.95) in ells were not different under the three visul onditions. The hnge rtios of firing rtes were 52 ± 15% for inhiition nd 61 ± 18% for enhnement during sdes ross sttionry grtings, 55 ± 11% nd 62 ± 18% during sdes ross lnk sreen nd 54 ± 19% nd 64 ± 21% during sdes in omplete drkness. The mgnitudes of inhiition (F 2, 57 ¼.13, P ¼.88) nd enhnement (F 2, 57 ¼.17, P ¼.84) in ells were lso not different under the three onditions, even though there were some smll differenes in spontneous firing rtes. During the slow phse of OKN tht is indued y the motion of the grtings, the firing rtes of telenephli nd ells were enhned reltive to those during stedy fixtion. However, sdes in the form of the quik phse of OKN were ompnied y inhiition nd TEL Sdes Figure 2 Firing ptterns nd time ourse of responses in visul neurons in four rin regions during spontneous sdes nd OKN. (,) Five rows of tres (from top to ottom) show eye movements mesured with EOG nd sdi responses of single neurons in the telenephli hyperpllium (TEL),, nd during spontneous sdes mde y the pigeon viewing sttionry grtings () nd the quik phse of OKN indued y motion (rrow) of the grtings (). () Time ourse of the hnge rtio of firing rtes efore, during nd fter spontneous (light gry) nd optokineti (drk gry) sdes. Numerls (from left to right) give the strt time for the first omponent nd the end time for the first nd seond omponents of the inhiition nd enhnement, respetively. Dt show verges for 2 neurons in eh orresponding struture. Error rs, s.e.m. Vertil lines limit sdi durtion. Sle rs t the ottom-right of represent 1 ms, 25 mv. Chnge rtio (%) , Time from sde onset (ms) 596 VOLUME 11 [ NUMBER 5 [ MAY 28 NATURE NEUROSCIENCE

3 28 Nture Pulishing Group Figure 3 Comprisons of responses of neurons during sdes ross sttionry grtings nd simulted sde-like motion of grtings. ( d) Responses of two neurons (, nd,d). From top to ottom, eh pnel shows n exmple EOG tre, histogrms of verge firing rte otined from 15 sdes or stimuli nd rster disply showing individul responses. When tested with smll hnd-held stimuli nd lrge field grtings for the properties of the visul reeptive fields, the neuron in pnels nd hd purely exittory field (open irle in ), wheres the neuron in pnels nd d hd exittory-inhiitory fields (onentri irles in ). The sene of n eye movement is shown y EOG tres t the top of pnels nd d, with horizontl short rs inditing the durtion of simulted sde-like motion. Vertil lines indite the onset nd offset of rel (,) or simulted sde-like motion (,d). Letter M in d represents men spontneous rte. (e,f) Quntittive summry of responses in six neurons (E) with exittory (e) nd four (EI) with exittory-inhiitory (f) reeptive fields. Grphs plot the hnge rtio during sde s funtion of tht fter the sde. Filled nd open symols show responses to rel or simulted sdes, respetively. enhnement of firing rtes tht were very similr to those seen in the sme neurons during spontneous sdes (Fig. 2,). The prmeters of sdes nd the neurl responses depended wekly on whether the sdes were prt of OKN or spontneous. The verge sde durtion ws redued from 242 ± 36 ms for spontneous sdes to 24 ± 33 ms (n ¼ 6) for optokineti sdes, nd the durtion of inhiition of firing rtes in telenephli nd thlmi ells (n ¼ 2 eh) ws redued y B4 ms. However, the durtion of enhnement in these ells ws the sme during spontneous nd optokineti sdes. Thus, omprison of responses for these neurons reveled more similrities thn differenes in the responses for spontneous sdes versus optokineti ones (Fig. 2). The insensitivity of sdi responses of ells to the four visul onditions (sttionry nd moving grtings, lnk sreen, nd drkness) implies tht sdi responses originte from entrl strutures rther thn from retinl imge motion. To further test whether the perisdi modultion of firing tivity origintes from the visul smer of retinl imges during sdes, we ompred the responses of 1 ells to sdes ross sttionry grtings with their responses to simulted sdes (osilltory grtings (25 Hz) tht were moved t sde-like speed (48 deg s 1 ) for the sdi durtion (24 ms) while the eyes were sttionry, s identified y stedy EOG signls). All ten ells showed typil sequenes of inhiition followed y enhnement of firing rtes during nd fter rel sdes (Fig. 3,), ut eh responded to simulted sdes depending on its reeptive field orgniztion (Fig. 3,d), s mpped previously 14. We oserved tht six of the ells (6%) inresed their firing rtes in response to simulted sdes during fixtion (Fig. 3,e). The sme ells responded to oth lrge-field grtings nd hnd-held trget, nd were thus hrterized y single exittory reeptive field. Another four ells (4%) responded to the motion of smll trget, ut not to the motion of lrge-field grtings, inditing tht they hd onentri exittory nd inhiitory reeptive fields. The lned omintion of exittory nd inhiitory suunits in the reeptive fields of the ltter neurons explined the lk of responses to simulted sde-like motion of grtings when the eyes were sttionry (Fig. 3d,f). In no se did the response of ell during simulted sdes show ny sign of the strong inhiition seen during rel sdes. Optokineti origin of sdi responses in ells We next evluted the pttern of synpti onnetions to ells from the optokineti nulei. Sdi inhiition nd enhnement of firing rtes in neurons prlleled those in the (Fig. 2). The inhiition egn 48 ± 38 ms efore sde onset nd lsted for e Chnge of firing rtes during sde or sde-like motion Firing rte Rster Time from sde or motion onset (ms) x = ~ y = 232 Rel sde Simulted sde 5 5 1, E EI Sde Sde-like 2 x = 68 x = ~ x = 68 y = 54 y = ~ y = Chnge rtio of firing rtes fter sde or sde-like motion (%) 298 ± 5 ms (n ¼ 2) until sde offset nd then ws followed y enhnement lsting 35 ± 58 ms (Fig. 2,). By ontrst, ells showed sdi responses with the opposite phse. Firing ws enhned from 115 ± 49 ms efore sde onset nd then inhiited from 17 ± 5 ms (n ¼ 2) fter sde onset for 385 ± 69 ms (Fig. 2,). The hnge rtio for sdi inhiition nd enhnement in these ells ws 5 6%. Similr to ells, the firing rtes of nd ells during the slow phse of OKN were enhned in omprison with the firing rtes during stedy fixtion, nd their response ptterns during the quik phse of OKN were similr to those during spontneous sdes, with the durtions of inhiition in ells nd enhnement in ells eing redued y 35 4 ms (n ¼ 2 in eh smple; Fig. 2,). The sdi responses of ells re in phse nd those of ells re out of phse with the sdi responses of ells; the - pthwy is exittory wheres the - pthwy is inhiitory 14. These fts indite tht the inputs from the nd might work together to reiprolly modulte the responses of ells during sdes. To evlute this possiility, we exmined the effets of reversile intivtion of the, or oth on the sdi responses of ells (Fig. 4). Blokde of using GABA redued the hnge rtio for sdi inhiition nd enhnement, respetively, in 12 ells y 57% nd 52% of ontrol vlues, nd delyed inhiition onset to 42 ms, rther thn 1 ms efore sde onset. There ws no hnge in enhnement onset (242 versus 238 ms; Fig. 4,d,g). The redued inhiition lteny is expeted euse the - pthwy is inhiitory 14 nd the sdi responses of ells re out of phse with those of ells (Fig. 2); eliminting the enhnement nd inhiition of responses should redue the inhiition nd enhnement of responses. On the other hnd, lokde of the redued the hnge rtio for sdi inhiition nd enhnement in n dditionl 12 ells y 43% nd 45% of ontrol vlues, respetively. The onset of enhnement in these ells ws dvned from 258 to 196 ms fter sde onset, nd d f M s.d. 2.5 s.d. NATURE NEUROSCIENCE VOLUME 11 [ NUMBER 5 [ MAY

4 28 Nture Pulishing Group Chnge rtio (%) Inhiition Before During d g After 1 lokde lokde Doule lokde e , 5 5 1, 5 5 1, Time from sde onset (ms) Doule h i Before During 1 After Enhnement hnge rtio (%) the onset of inhiition did not hnge (92 versus 96 ms; Fig. 4,e,h). This redution is lso expeted euse the - pthwy is exittory 14 nd the sdi responses of ells re in phse with those of ells (Fig. 2): eliminting the inhiition followed y enhnement of responses should redue the inhiition nd enhnement of responses. Finlly, simultneous pplition of GABA to oth the nd the resulted in totl elimintion of sdi responses in ells (n ¼ 1; Fig. 4,f,i). In ll ses, responses returned to ontrol levels fter time hd een llowed for the injeted GABA to e lered. These results were onfirmed y the loss of sdi responses in ells (n ¼ 12) fter eletrolyti lesions of oth nd (Fig. 5). It is noteworthy tht the pthwy from the retin to the ws intt while the or ws intivted, inditing tht hnges in sdi responses of ells under these irumstnes were used y ontriutions of the optokineti nulei. It seems tht oth nd re neessry for the sdi responses of ells nd tht the retin- pthwy is not suffiient. Further studies indited tht the sdi responses of optokineti neurons re in turn modulted y the sde-relted rphe omplex. Two types of omnipuse ells modulte optokineti ells The rinstem retiulr formtion ontins omnipuse neurons (OPNs) tht re tive t high rtes during fixtion nd puse firing f Figure 4 Effets of lokde of the optokineti nulei y GABA on sdi responses of neurons. From left to right, the three olumns show dt for intivtion of the, or oth. Top row, firing rtes of three ells ( ) efore, during nd fter lokde re olor-oded with sle (spikes per s) on the right. Middle row, time ourse of the hnge rtio of ells (n ¼ 12 in d,e; 1inf). Arrows point to the onset of delyed inhiition (d) or dvned enhnement (e) during lokde. Bottom row, quntittive summry of results from the full smple of neurons (g i). Eh grph plots the solute vlue of the hnge rtio for inhiition s funtion of tht for enhnement. Open, filled nd gry symols show dt efore, during nd fter lokde, respetively. efore nd during sdes in ll diretions 23,26,31. We reorded 49 ells from the rinstem rphe omplex, ll of whih pused firing during spontneous sdes in multiple diretions nd during optokineti sdes indued y motion of grtings in the four rdinl diretions. Anlysis of the timing of onset nd offset of the puses divided the rphe neurons into two groups (Fig. 6). Erly OPNs (53%) stopped firing 133 ± 37 ms efore sde onset nd reovered 72 ± 57 ms efore sde offset (n ¼ 2, Fig. 6,), nd lte OPNs (47%) pused 77 ± 14 ms efore sde onset nd reovered 4 ± 89 ms fter sde offset (n ¼ 2, Fig. 6,). The qulittive differene in the responses of the two groups of OPNs n e seen in the exmple perisdi histogrms of Figure 6. Antidromi stimultion of the nd reveled tht the two groups of OPNs projeted differently. Stimultion of the evoked spikes in six of ten erly OPNs with ltenies of 2.2 ± 1. ms, ut did not evoke spikes in ny of the ten lte OPNs exmined (Fig. 6,). By ontrst, stimultion evoked spikes in eight of ten lte OPNs with ltenies of 5.4 ± 1.2 ms, ut did not evoke spikes in ny of the ten erly OPNs exmined (Fig. 6,). Eletril stimultion of the rphe omplex reveled the signs of rphe projetions to the optokineti nulei (Fig. 7). Rphe stimultion deresed firing tivity in ll the 15 ells exmined. The ltenies of inhiitory responses were imodl: short-lteny nd long-lteny groups hd ltenies of 2.9 ±.5 ms (n ¼ 5) nd 19. ± 4.8 ms (n ¼ 1), respetively. The rphe- inhiition lsted 19.6 ± 14.3 ms (n ¼ 15) nd ws not different in 1 Figure 5 Effets of optokineti or rphe lesions on sdi responses of thlmi nd optokineti neurons. () Eletrolyti lesions of oth optokineti nulei nd olished sdi responses in ells (n ¼ 12). ( d) Eletrolyti lesions of the rphe omplex eliminted sdi responses in, nd ells (n ¼ 12 in eh smple). Eh grph plots the hnge rtio of firing rtes s funtion of the time from sde onset. Negtive vlues on the x xis indite the time efore sde onset; negtive vlues on the y xis indite redution in firing. Open nd filled symols show responses mesured efore or fter the lesions (Supplementry Fig. 2). Vertil lines delimit sdi durtion. Error rs represent s.e.m.; time in represents 5 ms. Chnge rtio (%) 1 1 d , 5 5 1, Time from sde onset (ms) Control Lesion + Rphe 598 VOLUME 11 [ NUMBER 5 [ MAY 28 NATURE NEUROSCIENCE

5 28 Nture Pulishing Group Figure 6 Clssifition of rphe neurons nd their responses to ntidromi stimultion of the optokineti nulei. () Superimposed histogrms nd rsters for rphe neurons tht strted nd ended perisdi puses erly (top, E-OPN) nd lte (ottom, L-OPN) reltive to the sde. The rsters nd histogrms show responses umulted ross 15 spontneous sdes in multiple diretions. () Antidromi spikes in the ell shown in the top pnel of evoked y stimultion (top) nd those in the ell shown in the ottom pnel of y stimultion (ottom). Five tres re superimposed. Sle rs represent 5 mv, 1 ms. () Summry of response ltenies for 2 E-OPNs (tringles) nd 2 L-OPNs (irles). Filled tringles nd irles show 6 E-OPNs nd 8 L-OPNs tht were tivted ntidromilly from the or. The strt time efore sde onset is plotted on log sle. durtion etween groups (Fig. 7,). Rphe stimultion inresed tivity in ll 15 ells, gin with imodl ltenies. The two groups of ells hd ltenies of 5.8 ± 1.3 ms (n ¼ 4) nd 32.7 ± 9. ms (n ¼ 11). The rphe- exittion lsted 15.5 ± 5.3 ms for the short-lteny group nd 45.9 ± 19.7 ms for the long-lteny group (Fig. 7,d). We found no topogrphi orgniztion in the rphe- nd rphe- projetions, nd oth long-lteny nd short-lteny responses in the or ould e eliited y rphe stimultion t single sites. Finlly, we exmined the effets of intivting the rphe omplex on sdi responses in thlmi nd optokineti neurons. Eletrolyti lesions of the rphe omplex used norml sdes to e repled y slow shifts of the eyes, whih ould e generted y the intt optokineti nulei. Alignment of neurl responses y shift onset showed tht rphe lesions resulted in omplete elimintion of sdi responses in ll, nd ells exmined (n ¼ 12 eh; Fig. 5 d), yielding effets similr to those otined y rphe lesions with kini id (two pigeons, five ells; t-test, n 1 ¼ 12, n 2 ¼ 5, t ¼ 1.19, P ¼.25). Moreover, the sdi responses of ells (n ¼ 12) fter rphe intivtion were sttistilly identil to those fter eletrolyti lesions of oth the nd the (n ¼ 12, Fig. 5) (t-test, n ¼ 12, t ¼.43, P ¼.67) nd simultneous intivtion of oth optokineti nulei y GABA (n ¼ 1, Fig. 4,f,i; t-test, n 1 ¼ 12, n 2 ¼ 1, t ¼ 1.44, P ¼.16). We onlude tht rphe neurons send orollry dishrge signls to neurons through oth optokineti nulei. Histologil verifition Fifty-five eletrode sites mrked were ll lolized in the telenephli hyperpllium (4), (15), (13), (14) nd rphe omplex (9). In ddition, the rphe omplex in five pigeons nd oth optokineti nulei in three pigeons were Firing rte (spikes per s) End time of firing puses fter sde , Time from sde onset 5 25 x = 77 ms y = 4 ms lesioned t the orret ples (Supplementry Fig. 2 online). These mrks vlidte the stereotxi oordintes we used nd provide ntomil support for our onlusions. DISCUSSION Our study provides severl lines of evidene tht sdi inhiition nd filittion of telenephli neurons in irds re driven y orollry dishrge signls, nd eluidted the pthwys for trnsmitting the signls from the rphe omplex through the optokineti nulei to the thlmus nd then to the hyperpllium. Figure 8 summrizes these physiologil nd ntomil results. To our knowledge, this is the first demonstrtion of neurl iruit mehnism for the medition y orollry dishrge of profound perisdi modultion of firing tivity in telenephli neurons, lthough previous studies hve desried different orollry dishrge iruit from the superior olliulus to the frontl eye fields 3,12. We demonstrted the full pthwy for orollry dishrge from the OPN in the rphe omplex to the telenephli hyperpllium, the vin homolog of the visul ortex. Telenephli ells reeive inputs from the nd their sdi responses originte from the, euse these responses re eliminted y intivtion of the. The sdi responses of ells re identil under different visul onditions, proving tht they do not depend on visul input 6,7. On the other hnd, sde-like motion of grtings does not produe suh sdi responses ut insted uses different visul responses only. All this indites tht the profound effet of sdes on the responses of pigeon visul neurons origintes from entrl ut not retinl soures. Beuse the sdi responses of ells re in phse with those in ells nd out of phse with those in Antidromi stimultion L-OPN E-OPN x = 133 ms y = 72 ms Strt time of firing puses efore sde t zero (ms) Durtion (ms) Rphe stimultion d ms Lteny of inhiition or exittion (ms) Figure 7 Responses of neurons in the nd to eletril stimultion of the rphe omplex. (,) Originl reordings show tht neuron in the ws inhiited y rphe stimultion nd one in the ws exited y stimultion of the rphe omplex. Ten repets were umulted. Sles: 5 mv, 1 ms. (,d) The durtion of responses evoked in 15 (filled irles) nd 15 (open irles) ells y rphe stimultion re plotted on log sle ginst response ltenies lso plotted on log sle. Averge ltenies for orthodromi stimultion of the rphe omplex nd ntidromi stimultion of the optokineti nulei re expressed in numerls eside solid nd dshed vertil lines, respetively. NATURE NEUROSCIENCE VOLUME 11 [ NUMBER 5 [ MAY

6 28 Nture Pulishing Group Telenephlon Hyperpllium Lte-OPN Erly-OPN Eye Entopllium N. rotundus Tetum Rphe ells, nd euse the - pthwy is exittory nd the - pthwy is inhiitory 14, intivtion of either or lone uses 5% redution of sdi responses in ells, wheres simultneous intivtion of oth optokineti nulei elimintes thlmi perisdi responses. The motor system origin of the sdi responses of optokineti neurons seems to e the rphe omplex. Omnipuse neurons in the pigeon s rphe omplex ehve like those found in the primte nuleus rphe interpositus 23,26,31 ut ould e divided into erly OPN nd lte OPN groups. The former send inhiitory signls to nd the ltter exittory ones to, therey enhning tivity nd reduing tivity perisdilly. Although we do not know the origin of the post-sdi inhiition in the nd enhnement in the nd, the totl elimintion of sdi responses in neurons y intivtion of the nd (nd in optokineti ells y rphe lesions) implies tht the seond omponent of eh response, like the first, rises from orollry dishrge. It seems tht the pthwy from the rphe to the through the ontins two inhiitory synpses wheres tht relyed y the ontins two exittory synpses, inditing tht orollry dishrge signls relted to sdes re sent in prllel through oth optokineti nulei to the nd then to the telenephlon. Importnt support for the ide tht the sdi responses reported here originte from orollry dishrge omes from the ft tht they egin efore sde onset in ll the five rin res we surveyed. Furthermore, the timing of sdi responses in the rphe,,, nd telenephlon strongly supports the notion tht the signls tht initite sdi responses in telenephli neurons originte from the motor ommnds of eye movements nd proeed through the hin of pthwys shown in Figure 8. Firing in erly OPNs nd lte OPNs egins to puse 133 ms nd 77 ms efore sde onset, respetively. These time ourses led to enhnement of tivity tht egins 115 ms efore sde onset nd inhiition of tivity tht egins 48 ms efore sde onset. The signls for enhnement nd inhiition trnsmitted through the - nd -thlmi pthwys would initite inhiition in ells tht strts 95 ms efore sde onset nd lsts until the end of the sde. Then, inhiition in ells would initite inhiition in telenephli ells 73 ms efore sde OM Figure 8 Summry digrm showing neuronl pthwys nd informtion flow relted to sdi responses in the strutures under study (light gry retngles; drk gry represents strutures not studied here). Visul neurons in the telenephli hyperpllium reeive input from the, whih in turn reeives exittory input from the nd inhiitory input from the. Menwhile, the reeives exittory input from the lte omnipuse neurons (OPN) nd the reeives inhiitory input from the erly omnipuse neurons in the rphe omplex. Dshed lines represent neuronl pthwys identified in mmmls 26,36 ut not yet identified in the pigeon from the pretetum nd the rinstem retiulr formtion to the oulomotor nulei (OM). Histogrms show sdi inhiition nd enhnement of single ells in their respetive strutures (15 repets, time in ¼ 5 ms). Vertil lines in histogrms delimit sdi durtion. Exittory pthwys or enhnement in histogrms is shown in red nd inhiitory pthwys or inhiition in lue. onset. It seems tht optokineti nd rphe neurons send motor ommnds to the oulomotor nulei 35,36 nd efferene opies to ells, whose sdi responses initite those in telenephli ells. This dul signling ould e omplished y two groups of motor neurons with one projeting to the oulomotor nulei nd the other to. Alterntively, the xons of optokineti nd rphe neurons ould ifurte nd send their ollterls to different regions. We found good greement etween the ltenies of the vrious neurl pthwys nd the effets of lesions on the timing of residul perisdi responses. Comprison of Figures 2 nd 4 indites tht lokde of the delys the inhiition onset of ells to 42 ms efore sdes, implying tht the signl for inhiition onset t 48 ms would tke 6 ms to reh the through the exittory pthwy, in greement with the time for - trnsmission 14. In ontrst, lokde of the dvnes the enhnement onset of ells to 196 ms, implying tht the signl for inhiition onset t 17 ms would tke 26 ms to reh the through the inhiitory pthwy s tken y - trnsmission 14. It is ler tht sdi responses of the re modulted y oth optokineti nulei. The pigeon is n exellent model in whih to desrie the neurl pthwys for modultion of the responses of visul neurons y orollry dishrge relted to sdes in multiple diretions. We expet tht similr orollry dishrge pthwys exist in mmmls nd tht they medite mny of the pereptul nd motor sequele to sdes. Indeed, mny studies in mmmls postulte tht orollry dishrges from the motor system re importnt in sensory nd motor proessing. For exmple, the sptil ury of sdes nd visul proessing depends on orollry dishrge pthwys from the superior olliulus through the thlmus to the frontl eye fields 3,12, nd urte sustined pursuit eye movements depend on orollry dishrge pthwys etween the rinstem nd ereellr floulus 37. It is lso likely tht the enhnement of ortil tivity is involved in the postsdi filittion of visul proessing, s seen during smooth pursuit eye movements in primtes 8,38,39. It must lso ontriute to the rempping of ortil reeptive fields to mintin n urte retinl representtion when sdes our nd to the sptil nd temporl distortions tht our round sdes. The sdi effets on pursuit seem to e driven y omnidiretionl orollry dishrge signls like those shown here, wheres the diretionl nture of the signls tht drive sptil nd temporl distortions remin unler. Anlysis of the neurl sis for these ehviors is etter done in primtes, ut we think tht the omplete orollry dishrge iruit we disovered in the pigeon should provide sis for identifying the mehnisms of ehviors tht rely on orollry dishrge in primtes. Although it remins ontroversil, there is some evidene in primtes 6 VOLUME 11 [ NUMBER 5 [ MAY 28 NATURE NEUROSCIENCE

7 28 Nture Pulishing Group for effets of orollry dishrge on neurons in the visul pthwys. For exmple, neurons in oth the visul ortex 9,1 nd LGN 6,7 show redution followed y enhnement of firing rtes perisdilly. The LGN of mmmls reeives inputs from the pretetum 36,4 42 nd from the dorsl terminl nuleus of the essory opti trt 4.Onthis sis, we expet tht the sme kinds of perisdi modultion we found in ll pigeon visul neurons will e present in suset of mmmlin visul neurons, nd tht it will prove to hve importnt funtions for ehviors tht depend on orollry dishrge relted to the exeution of sdes. METHODS Preprtion nd setup. The experiments were rried out on 7 pigeons (Colum livi) of either sex nd 3 45 g ody weight, nd omplied with the guidelines for the re nd use of nimls estlished y the Soiety for Neurosiene nd pproved y the Institutionl Animl Administrtion Committee. Eh pigeon ws nesthetized y injetion of ketmine (4 mg kg 1 )nd xylzine (5 mg kg 1 ) into petorl musles, nd the depth of nesthesi ws monitored y rething ptterns nd y the reflex from pinhing the toe. The pigeon ws pled in stereotxi pprtus nd its slp ws inised nd retrted, nd the left tetum, telenephlon nd/or ereellum were exposed with dentl drill nd surgil foreps. A lightweight steel hedpiee onneted to steel rod ws emented onto the skull to reple the er rs s mens to fix the pigeon s hed. After the opening in the slp ws losed nd treted with erythromyin ointment, the pigeon ws given iuprofen s n nlgesi (2 mg kg 1 ) for postopertive pin nd returned to its home ge to reover from nesthesi. In 1 12 h, the pigeon s stnding, wlking nd peking ehviors hd returned to norml. It then ws lightly nesthetized with ketmine, wrpped in g nd pled on fom ouh. The hedpiee ws reonneted to the rod for stereotxi fixtion. The slp ws retrted gin nd the dur mter overlying the telenephli hyperpllium,,, nd/or rphe omplex ws exised. The wound edge nd musles were periodilly infiltrted with lidoine. The left eye ws overed ut llowed to move freely, nd the right eye ws held open during reording. A sreen of 131 y 141 ws pled 4 m wy from the viewing eye. The horizontl xis of the visul field on the sreen ws rotted y 381 to mth the pigeon s norml onditions 3,43. The pigeon s dpttion to the restrint ws signled y the oservtion tht it st unruffled in the pprtus in 2 h. Then, oth single-ell tivity nd EOG were reorded simultneously. Visul onditions. A lrge squre-wve grting onsisting of equl-width lk nd white stripes with sptil frequenies of.8 or.5 yles per deg ws generted y omputer with grphis GeFore 795 (mximum refresh rte 4 Hz, Nvidi) nd rer-projeted onto the sreen y projetor (mximum refresh rte 2 Hz, XG-C58X, Shrp). The refresh rte of 2 Hz of the disply system ws verified with photoeletri devie (Si photodiode S BQ, Hmmtsu Photonis) nd n osillosope (54622A, Agilent Tehnologies). The luminnes of lk nd white in the grting were.1 nd 6.6 d m 2, respetively. The responses of neurons were reorded during spontneous sdes emitted while the pigeon viewed sttionry grtings, lnk sreen tht ws uniformly illuminted (6.6 d m 2 ) nd fetureless to humn oservers nd omplete drkness ( lux, RTR Optoeletronis Tehnology). Some neurons were lso exmined for responses to simulted sde-like motion of grtings (.8 yles per deg; veloity, 481 s 1 ;durtion, 24 ms; osilltion frequeny, 25 Hz) while the viewing eye ws sttionry s identified y stedy EOG signls. In other experiments, grtings of.5 yles per deg were moved t s 1 in the temporo-nsl diretion to eliit OKN for omprison of neuronl responses to spontneous nd optokineti sdes. Eye movements were monitored y EOG for ll visul onditions. The intervl etween onseutive trils ws 2 s to llow neuronl responses to reover fully from ny dpttion. Eletrodes nd reording. We used four types of eletrode. The first ws single miropipette (B2-mm tip dimeter) filled with 2 M sodium ette, nd 2% (wt/vol) pontmine skylue ws used for extrellulr reording nd mking mrks. Spikes were mplified, displyed nd stored in digitl dt reorder (RD-135T, TEAC) for off-line nlysis. Some reording sites were mrked s desried 3,43. The seond eletrode ws two-rrel miropipette tht we used for loking the, nd/or, or rphe omplex; one rrel ws filled s ove nd used to onfirm the eletrode lotion physiologilly nd ntomilly, nd the other rrel ontined GABA (1 mm, ph 3.3) or kini id (9.4 mm, ph 7.4, Sigm Chemil) nd ws onneted to pneumti piopump (PV8, WPI) for pplying GABA (8 12 nl) or kini id (2 3 nl) 14,17. The third eletrode ws tungsten ipolr eletrode tht we used for eletril stimultion of the, or rphe omplex. Its poles were seprted y 4 mm 14,44 nd were glss-oted with 6 mm oftip exposed. The fourth eletrode type tht we used were three steel eletrodes (27- guge syringe needles) for reording EOG, with two inserted into the nterior nd posterior regions of the right oritl rh nd one s referene in the oipitl one 29. In some experiments, two dditionl needles were pled ove nd eneth the orit to reord eye movements in vertil diretions. EOG signls were mplified (DC to 2 Hz) nd stored for off-line nlysis with neuronl spike dt. All miroeletrodes were dvned into rin strutures ording to the pigeon rin tls 45. When we exmined the effets of the nd/or on ell, we injeted GABA t up to three sites in the (A4.5 4., L1.8, H4.) nd/or (A5.7, L4., H ) to llow us to find orret site where GABA ould lok the tivity of optokineti ells projeting to the ell. Beuse the projets topogrphilly to the telenephli hyperpllium 32,34, injetions of GABA (8 12 nl) in the (A6.5 7., L , H7.) ould influene firing tivity in telenephli ells in topogrphi region (A , L2., H ). Stimultion nd lesions. For stimultion with retngulr pulses of 3 5-mA intensity nd 5 1-ms durtion, tungsten ipolr eletrode ws vertilly inserted into the with its poles in rostroudl rrngement to void stimulting the opti trt nery 14 or into the rphe omplex (A.5 P.5, L., H2. 3.). Its poles were rrnged dorsoventrlly nd inserted lterlly into the to void stimulting the tetum 14. To rete eletrolyti lesions y pssing positive urrents of 3 ma for 1 s, we inserted the ipolr eletrode into the, or rphe omplex to mke up to three lesions euse of their lrge size nd irregulr shpe. Similr to injetion of GABA, kini id (2 3 nl) ws injeted into three sites in the rphe omplex for 3 4 min to mke hemil lesions. Reordings were mde from the pigeons with hemil lesions fter 5 d of survivl. At the end of experiments, eh of the pigeons ws euthnized y intrperitonel injetion of n overdose of urethne (4 g kg 1 ). For those pigeons with mrk or lesion, the rins were removed from the skull nd histologilly proessed for susequent mirosopi oservtions of mrked nd lesioned sites 3,43. Dt nlysis. Spike dt nd eye positions were smpled t 8, Hz round eh sde. Spikes were olleted from 5 ms efore to 1, ms fter the sde for dt nlysis; spikes olleted in the intervl from 2 5 ms efore the sde were verged s ontrol. A sde ws used for dt nlysis only if it ws seprted from the previous sde y t lest 1.5 s to void n overlp of enhnement (inhiition) indued y the previous sde, lthough this proility ws negligile (Fig. 2). The onset nd offset of sde were determined with Cool Edit (Syntrillium Softwre) ording to their hrteristi osilltions. For lirtion purposes, eye movements were videogrphed y n infrred video mer (DCR-HC4E, Sony) simultneously with EOG reordings. The video imges were used to lirte EOG 29, reveling sensitivity of mv per deg. Spikes were olleted efore, during nd fter reversile intivtion, or efore nd fter lesions of the, nd or rphe omplex for 15 3 repets. Firing rtes were verged in 5-ms ins in eh tril. We onsidered firing rte to e redued or enhned during sdes if it ws lower or higher thn the ontrol rte y more thn 2.5 stndrd devitions. To normlize hnges in the responses of different ells, we lulted the hnge rtio of firing rtes, defined s R ¼ f2 f1 f 1,wheref 1 is the intersdi firing rte s ontrol nd f 2 is the firing rte during sdes. Note: Supplementry informtion is ville on the Nture Neurosiene wesite. NATURE NEUROSCIENCE VOLUME 11 [ NUMBER 5 [ MAY 28 61

8 28 Nture Pulishing Group ACKNOWLEDGMENTS We thnk S.G. Liserger of University of Cliforni Sn Frniso for help in editing the mnusript. This work ws supported y the Ntionl Nturl Siene Foundtion of Chin (9288) nd y the Chinese Ademy of Sienes (KSCX1-YW-R-32 nd Brin-Mind Projet). AUTHOR CONTRIBUTIONS Yn Yng onduted the experiments throughout, P.C. onduted the first hlf of the experiments, Yng Yng onduted the seond hlf of the experiments nd S.-R.W. supervised the projet nd wrote the mnusript. All o-uthors onduted the dt nlyses. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions 1. Tolis, A.S. et l. Eye movements modulte visul reeptive fields of V4 neurons.neuron 29, (21). 2. Kusunoki, M. & Golderg, M.E. The time ourse of perisdi reeptive field shifts in the lterl intrprietl re of the monkey. J. Neurophysiol. 89, (23). 3. Sommer, M.A. & Wurtz, R.H. Influene of the thlmus on sptil visul proessing in frontl ortex. Nture 444, (26). 4. 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Visul neurons in the pigeon rin enode the elertion of stimulus motion. J. Neurosi. 24, (24). 44. Wng, S.R. & Mtsumoto, N. Postsynpti potentils nd morphology of tetl ells responding to eletril stimultion of the ullfrog nuleus isthmi. Vis. Neurosi. 5, (199). 45. Krten, H.J. & Hodos, W. A Stereotxi Atls of the Brin of the Pigeon (Colum livi) (The Johns Hopkins Press, Bltimore, Mrylnd, 1967). 62 VOLUME 11 [ NUMBER 5 [ MAY 28 NATURE NEUROSCIENCE