ARTICLES. Dendritic organization of sensory input to cortical neurons in vivo

Transcription

1 Vol 6 9 April 00 doi:0.08/nture0897 ARTICLES Dendriti orgniztion of sensory input to ortil neurons in vivo Hongbo Ji *, Nthlie L. Rohefort *, Xiowei Chen & Arthur Konnerth In sensory ortex regions, neurons re tuned to speifi stimulus fetures. For exmple, in the visul ortex, mny neurons fire predominntly in response to moving objets of preferred orienttion. However, the hrteristis of the synpti input tht ortil neurons reeive to generte their output firing pttern remin unler. Here we report novel pproh for the visuliztion nd funtionl mpping of sensory inputs to the dendrites of ortil neurons in vivo. By ombining high-speed two-photon imging with eletrophysiologil reordings, we identify lol subthreshold lium signls tht orrespond to orienttion-speifi synpti inputs. We find tht even inputs tht shre the sme orienttion preferene re widely distributed throughout the dendriti tree. At the sme time, inputs of different orienttion preferene re interspersed, so tht djent dendriti segments re tuned to distint orienttions. Thus, orienttion-tuned neurons n ompute their hrteristi firing pttern by integrting sptilly distributed synpti inputs oding for multiple stimulus orienttions. A growing mount of evidene indites tht informtion proessing in the brin involves the omputtion of eletril nd hemil signls in neuronl dendrites (for review, see ref. ). One of the most effetive wys for the nlysis of these dendriti signls relies on the imging of the dynmis of intrellulr C onentrtion (reviewed in ref. ). Thus, synpti input-relted dendriti C trnsients hve been identified nd studied in detil in vitro 6, while in vivo work hs explored tion-potentil-relted dendriti C signls 7 9. However, nothing is known bout the nture of subthreshold sensory evoked input signls in the dendrites of mmmlin ortil neurons. A detiled knowledge of sensory input signls would represent n importnt step forwrd in the understnding of dendriti omputtion,0. An intriguing open question is whether sensory inputs with similr fetures re lustered on the sme dendrite of neuron or dispersed throughout the dendriti tree. Clustered inputs re pble of generting dendriti spikes (reviewed in ref. ) nd my form neuronl omputtionl subunits in vivo, s they do under ertin experimentl onditions in vitro. Alterntively, sensory inputs tht re not lustered, but widely distributed, my underlie different rules of integrtion nd formtion of neuronl output signls like, for exmple, the liner summtion of exittory inputs. Visully evoked supr- nd subthreshold tivity For the funtionl nlysis of spiny dendrites in vivo by mens of twophoton lium imging, we seleted s n experimentl model neurons in lyer / of the mouse primry visul ortex (Fig. ). The somt of the neurons were loted pproximtely between 0 nd 00 mm below the ortil surfe. Their dendriti trees hd hrteristi pttern, onsisting of numerous bsl nd oblique dendrites but no pronouned pil trunk 6 8 (Fig. ). As in mny other mmmlin speies 9, lyer / neurons of the mouse primry visul ortex respond seletively to drifting grtings or brs with tion potentil firing. By performing whole-ell reordings involving the shdow-pthing pproh, we found tht stimultion with drifting grtings shifted the membrne potentil to the up-stte 6 for the entire durtion of the stimulus. An inresed probbility of up-sttes during drifting grting stimultion ws previously observed in the t visul ortex 7. The neuron illustrted in Fig. d fired tion potentils preferentilly during the presenttion of speifilly oriented drifting grtings nd ws identified s highly-tuned neuron (Fig. e left) on the bsis of its orienttion seletivity index (OSI), whih ws higher thn 0. (Methods). In our reordings, 6 out of 7 neurons were highly tuned for preferred orienttion (Fig. f left), 6/7 neurons were poorly tuned, while the rest of /7 neurons did not respond relibly to visul stimultion. For better ssessment of the stimulus-evoked subthreshold depolriztion in highly tuned neurons, we hyperpolrized them to subthreshold membrne potentils. In the exmple shown in Fig. d, the neuron ws hyperpolrized from its resting level of 6 mv to 70 mv. In ontrst to the highly tuned tion potentil pttern (Fig. e left), the stimulus-evoked subthreshold depolrizing responses were brodly tuned in this (Fig. e right) s well s in ll other highly tuned neurons (Fig. f right). Thus, in mouse visul ortex, s in t visul ortex 8,9, the high tuning level of tion potentil firing, the neuronl output signl, ontrsts with the low tuning level of the fferent subthreshold input signls. These observtions prompted us to ombine whole-ell reordings with two-photon lium imging to serh for dendriti signls ssoited with the fferent tivity. We performed high-speed two-photon imging t 0 full frmes per seond or 60 hlf frmes per seond using resonnt glvo-snners 0 nd, initilly, foused on the dendrites tht were visible in the plne of fous ontining the ell body (Fig. g). When stimulted with their preferred orienttion, neurons responded with tion potentil firing tht ws ssoited with globl dendriti lium trnsients in ll imged dendrites. In the neuron illustrted in Fig. g, ll five visible dendrites displyed lium trnsients tht were lrger when the neuron fired four tion potentils (Fig. h left) thn when it fired two tion potentils (Fig. h right). The dependene of the lium trnsient mplitude on the number of tion potentils nd the observtion tht similr lium trnsients were evoked by diret neuronl depolriztion through the reording pipette (Supplementry Fig. ) indite Institute of Neurosiene nd Center for Integrted Protein Siene, Tehnil University Munih, Biedersteinerstrsse 9, 8080 Munih, Germny. *These uthors ontributed eqully to this work. 07

2 ARTICLES NATURE Vol 6 9 April 00 L Primry visul ortex b µm 0 µm L/ 00 µm Pth pipette (OGB, Alex, bioytin) L d L 6 mv 0 mv s L6 70 mv e Spike rte Subthreshold depolriztion g Neuron, 7 µm f h Normlized spike rte 0 n=6 neurons Orienttion Normlized subthreshold depolriztion 0 n=6 neurons Orienttion 0. Δf/f Som s APs APs V m 0 mv Figure Visully evoked tion potentils, subthreshold depolriztions nd globl dendriti lium signls., Reonstrution of bioytin-filled lyer / (L/) neuron in mouse primry visul ortex (projetion long the ntero-posterior xis). Dt from the sme neuron re presented in b e. b, Mirophotogrph of spiny bsl dendrite., Projetion long the dorso-ventrl xis obtined in vivo from 69 setions (step size 0. mm) from Alex fluoresene. d, Whole-ell urrent-lmp reordings of responses to drifting grtings of different orienttions. Three trils were superimposed. Upper red tres, tion potentil responses t resting potentil (indited on the left); lower blue tres, subthreshold responses obtined fter hyperpolrizing the neuron to 70 mv. e, Polr plots of visully evoked responses, verge of eight trils. Red plot, spike rte; blue plot, mplitude of subthreshold depolriztion. f, Tuning properties of spiking nd subthreshold responses for six orienttion-seletive neurons, eh normlized to their preferred orienttion (noted s 0u). Error brs, s.d. g, h, Two-photon imging of dendriti lium signls in bsl nd oblique dendrites of nother lyer / neuron during tion potentil firing (eletril reording lower red tre) evoked by drifting grtings. g, Averge imge of 00 frmes reorded t 7 mm below the ortil surfe. Yellow dshed lines indite out-of-fous portions of the dendrites. Green dshed lines indite the regions of interest (ROIs). h,c -dependent fluoresene hnges (blk tres) reorded in the som nd in five dendrites (indited by numbers in g) nd the orresponding membrne potentil (V m, red tres) reordings, during two seprte trils. Light grey brs indite the stimultion period with oriented grtings. AP, tion potentil. tht suh globl dendriti signls re lrgely due to the tivtion of voltge-gted lium hnnels by bk-propgting tion potentils; this hs previously been shown for bsl dendrites in in vitro reordings from lyer / neurons,. The mplitude of the C trnsients t different dendriti sites showed ttenution long given dendrite (Supplementry Fig. d, e) nd vritions from tril to tril, but we were unble to obtin unmbiguous evidene for signl peks tht would orrespond to speifi synpti inputs, s found in tetl neurons of the tdpole. Therefore, we deided to serh for lium signls ssoited with subthreshold synpti 08 lium signlling in onditions in whih we tively hyperpolrized the neurons. Subthreshold lium signls in dendriti hotspots Figure illustrtes the results of n experiment in whih neuron ws hyperpolrized to 70 mv to prevent tion potentil firing. In these onditions, lium trnsient ws evoked in subregion of dendrite (Fig., b) by visul stimultion (drifting grting), but not deteted in the other dendrites or in the som. To ssess the sptil dimensions of suh lol lium trnsients, we nlysed experiments

3 NATURE Vol 6 9 April 00 ARTICLES b g Som 70 mv AP + Alex Single tril Avg. trils 0. Δf/f 0 mv s Control AP Wsh-out in whih long portions of the sme dendrite were disernible in the plne of fous of our reordings. In the neuron shown in Fig., we identified two dendriti hotspots of visully evoked lol lium trnsients. The systemti nlysis of segments with length of mm long the dendrite showed tht the lium signl ws present in three neighbouring segments with ler pek in the middle segment (Fig., d). Repetitive visul stimultion relibly evoked lol dendriti lium signls (Fig. e; men filure rte 6 8%, 6 hotspots in 7 neurons). The sptil extent of these dendriti hotspots hd n verge hlf-width t hlf-mximum (HWHM) of mm (n 0) (Fig. f). Wht is the mehnism underlying these lium signls? First, we notied tht the NMDA (N-methyl-D-sprtte) reeptor ntgonist D()--mino--phosphonovleri id (AP) used mjor ttenution of the lium trnsients, inditing their synpti origin (Fig. g, h). Seond, the dimensions of the hotspots were quite similr to the tive dendriti shft segments of pyrmidl neurons in vitro displying lium trnsients s result of the spred of lium from single tive spines,. In line with this possibility, we lso identified spines t the hotspot region when onstruting post ho superverge imges (Fig. bottom). Furthermore, previous reordings performed in bsl dendrites of neoortil neurons in vitro 0 0. f Δf/f 0. Δf/f s h Normlized Δf/f HWHM =.7 ± 0.8 µm (n = 0) 0 Distne (µm) 0 Super-verge d d d d d d d d d d d e Conseutive trils (red box) *** 0. Δf/f s 0 Control AP Figure Subthreshold lol dendriti lium signls evoked by drifting grting stimultion., Two-photon imge used for lium reordings in b. The imge is n verge of 00 frmes. Five ROIs re indited by green dshed retngles. b, Subthreshold C trnsients (blk tres) nd orresponding depolriztion (blue tres) evoked by drifting grting stimultion. Note the prominent C signl in dendrite. Left tres, single trils; right tres, verge (blk tre) of five individul trils (grey tres) from vrious dendriti sites nd som, s indited., Upper pnel, pseudoolour imge of lol dendriti C signls. Rtio of the verges of 0 frmes before nd 0 frmes during stimultion. Yellow rrows indite two sites of lol dendriti lium signls. Lower pnel, enlrged view of the dshed box indited in the upper imge, super-verge obtined from 00 frmes. d, Clium reordings from five neighbouring dendriti regions of mm length (d d, ROIs shown in ), verge of five trils. e, Clium signls evoked during five onseutive trils within the ROI indited by the red dshed line in. f, Amplitude distribution of lium signls within dendriti hotspots (n 0 hotspots, 7 neurons). Grey dshed lines indite the Gussin fitting to the mplitude distribution of lium signls within individul hotspots. Red line indites the Gussin fit to ll points. Averge hlf-width t hlf-mximum (HWHM) with stndrd devition (6) is indited. g, Left, two-photon imge of neuron (verge n 00 frmes) nd shemti representtion of drug pplition pipette ontining AP nd Alex-9. The red dshed lines indite the pproximte re of drug pplition. The green dshed box is the ROI for lium monitoring. Right, C reordings before, during nd fter the pplition of AP. Averge tre (blk line) of five individul trils (grey lines). h, Amplitude of lium signls before nd during the pplition of AP obtined from n 9 lol lium signls in four neurons. The mplitude of eh C signl ws normlized to the men mplitude of ll signls in ontrol ondition. Pired t-test, ***P, demonstrted tht folly evoked subthreshold exittory postsynpti potentils indue lium trnsients in dendriti omprtments of omprble dimensions. In our reordings, the men mplitude of hotspot lium trnsients (Methods) ws Df/ f (n 0). This vlue is omprble to the mplitudes of bk-propgting tion-potentils-dependent dendriti lium trnsients evoked by the preferred orienttion (Df/f , men number of tion potentils. 6., n 0; Supplementry Fig. ). Thus, the mplitude nd the sptil extent of these visully evoked lium trnsients resemble those generted by individul synpti inputs, but not those evoked by dendriti NMDA spikes, whih produe lium trnsients tht over lrger dendriti domins nd hve mnifold lrger mplitudes,6,7. Mpping dendriti distribution of sensory inputs To determine the sptil distribution of the dendriti hotspots refleting sensory inputs, we performed experiments in whih we ttempted to imge s mny fol plnes s possible in every neuron, requiring h of whole-ell reording. In eh fol plne, we determined the orienttion preferene of the lol lium signls by presenting drifting grtings (Methods). Figure illustrtes n experiment in whih four fol plnes were imged t vrious depths under the ortil surfe. A totl of hotspots were identified in this neuron. Figure b shows the response from three seleted hotspots (numbered, nd 7 in Fig. ) obtined during presenttion of eight diretions of drifting grtings. From these verged lium trnsients (n 6 trils), we onstruted polr plots for eh hotspot (Fig. ). A loser inspetion of the polr plots reveled the presene of multiple orienttion preferenes in this nd ll other neurons. The dendriti distribution of the orienttion preferenes did not pper to follow ny strit rule, but seemed to be rndomly distributed throughout the dendriti tree. Another remrkble finding is tht the lium trnsients of most lol dendriti hotspots displyed pronouned orienttion preferene. In order to quntify the tuning level, we lulted the OSI for eh hotspot nd found tht the mjority of hotspots (7%, n 0) were highly tuned for prtiulr orienttion (OSI. 0.). The verge OSI vlue for ll hotspots of the neuron shown in Fig. ws 0.6. Comprble results were obtined from 6 dditionl neurons (men OSI 0.9; n 0 09

4 ARTICLES NATURE Vol 6 9 April 00 µm b 7 0. Δf/f s µm 8 µm µm µm Figure Heterogeneity nd distribution pttern of orienttion-tuned dendriti hotspots., Four two-photon imges (eh n verge of n 00 frmes) of lyer / neuron obtined t different depths under the ortil surfe s indited. Red dshed boxes indite hotspots of lol dendriti lium signlling. b, Lol dendriti lium signls evoked by drifting grtings of different orienttions (verge of six trils) t three different dendriti sites indited in., Lotion of eh hotspot indited s red dot on the Z-projetion of the reonstruted dendriti tree. Red dshed lines point to the polr plot obtined for the orresponding lol C signls. The frme (grey dshed line) indites the re of imging. The output signl of the neuron ws tuned for the vertil orienttion. hotspots). An importnt finding ws tht dendriti hotspots were found in eh of the reorded neurons (7/7), regrdless of the tuning level of tion potentil firing nd even in the neurons tht were not relibly firing in response to drifting grtings. This suggests tht these hotspots represent generl nd highly relible feture of lyer / neurons of the primry visul ortex. Hotspots of the sme orienttion preferene in given neuron were found widely dispersed over vrious dendrites. For exmple, in the neuron shown in Fig. left, we identified six hotspots for the stimulus orienttion indited in the figure nd, remrkbly, eh hotspot ws on different dendriti brnh. The nlysis of lrger set of neurons (n 8, Fig. right) onfirmed this widespred distribution nd emphsized the bsene of ny lustering of inputs on single dendrites. Furthermore, hotspots oding for the sme orienttion were found throughout the entire three-dimensionl spe surrounding the ell body. A similr wide distribution ws observed for ll four orienttions tested (Supplementry Fig. ). The bsene of input 0 lustering beme prtiulrly obvious when we nlysed the inputs to individul dendriti brnhes. Figure b shows three exmples of dendriti brnhes with neighbouring hotspots tht hve different orienttion preferenes. In 9/0 dendriti brnh segments, lolized between two brnhing points, we enountered two or three hotspots tuned for different orienttion preferenes. In just one out of these 0 ses, neighbouring hotspots oded for the sme orienttion. The grph in Fig. right summrizes these results. The left pnel of Fig. shows tht ll full dendrites, tht is, individul dendrites together with ll their higher order brnhes, reeived inputs for multiple orienttions. Finlly, we ompred in neurons the tuning levels of the firing pttern, representing the output signl, with the tuning levels of lol dendriti lium responses, representing the synpti input signls. Experimentlly, the orienttion preferene of spike firing in these neurons ws determined during the initil 0 min of whole-ell reording during whih the lium inditor dye ws llowed to equilibrte in the dendrites. Then, the neurons

5 NATURE Vol 6 9 April 00 ARTICLES One orienttion, multiple dendrites Superposition of eight neurons the bove-mentioned observtion tht ll neurons hd input signls oding for multiple orienttions, indites tht the orienttion preferene of the output signl is the result of omputtionl proess tking ple on the level of individul neurons. 0 µm b One dendrite, multiple orienttions Dendrite, 9 µm Dendrite, 7 µm Dendrite, 7 µm d Input (dendriti C + signls) Output (spike rte) OSI men = 0.60 OSI men = 0.79 (Highly tuned neurons) Normlized proportion Normlized proportion 0 0. OSI men = OSI 0 were hyperpolrized nd we determined the orienttion preferene of lol dendriti lium signls. We ompred six highly tuned neurons (men OSI 0.79) with six brodly tuned ones (men OSI 0.9) (Fig. d right). Unexpetedly, the input signls hd very similr tuning levels for the two neuronl groups, with men OSI vlues of 0.60 nd 0.9, for highly nd brodly tuned firing ptterns, respetively. This disprity between input nd output signls, ombined with Proportion % % Full dendrite Brnh segment Mult. Sme Mult. Sme Orienttion(s) 0 0. OSI men = 0.9 (Poorly tuned neurons) 0 0. OSI Figure Sptil rrngement of dendriti hotspots nd input output reltion., Distribution of dendriti hotspots (red dots) tuned for the orienttion preferene indited in the upper left orner, in the dendriti tree of neuron (left pnel). Right pnel, summry of the results obtined for the sme orienttion in eight neurons. Cell bodies re indited by the grey-filled dshed irles (blk), dendrites re indited by dshed red lines (Zprojetions reonstruted from stks). b, Three two-photon imges of dendrites (eh n verge of n 00 frmes) t higher mgnifition with hotspots s indited. Yellow rrows indite the preferred orienttion of lol lium signl in the orresponding hotspot. Note tht the hotspots in dendrites nd s well s the two distl hotspots in dendrite were loted in brnh point-delimited segments., Summry grphs. Left pnel, the proportion of full dendrites (n ) with hotspots oding for multiple orienttions versus those with just one nd the sme orienttion ($ hotspots per full dendrite). Right pnel, the proportion of brnh segments (between two brnhing points) with hotspots oding for multiple orienttions versus those with the sme orienttion ($ hotspots per brnh, n 0). d, Input output reltions in highly tuned (upper) nd poorly tuned (lower) neurons. The red nd blue histogrms show orienttion seletivity indies (OSIs s indited) for the input (C signls in dendriti hotspots) nd the output (spiking rte) of highly nd poorly tuned neurons (n 6 for eh group), respetively. Conlusions Our results revel bsi insights into the dendriti orgniztion of sensory inputs to neurons of the visul ortex in vivo. First,weidentified disrete dendriti hotspots s synpti entry sites for speifi sensory fetures. These hotspots represent novel dendriti lium signls in vivo nd were found in ll lyer / neurons, irrespetive of their output firing pttern. Seond, we showed tht fferent sensory inputs with the sme orienttion preferene re widely dispersed over the dendriti tree nd do not onverge on single dendrites, s repetedly proposed in reent yers (see review in ref. nd referenes therein). Third, we found tht even neurons with highly tuned output signl reeive input signls tht re heterogeneous nd ode for multiple orienttions nd/or diretions. Thus, tken together, our results support neuronl integrtion model involving summtion of distributed inputs, rther thn models tht stress the role of onvergent inputs to single dendrites 6,. However, it is ertinly possible tht other types of ortil neurons, espeilly those with pronouned pil tufts 8 or neurons in other speies with olumnr orgniztion of the visul ortex 7,9, hve more lustered sensory inputs to the sme dendrite, pble of generting lrge mplitude dendriti spikes 6,. The pproh introdued in this study opens the wy to detiled nlysis of vrious types of neurons followed by the onstrution of funtionl wiring digrms of sensory pthwys with single input resolution in vivo. METHODS SUMMARY C7BL/6 mie (postntl dy (P)8 P) were prepred for in vivo two-photon lium imging nd whole-ell reordings under isoflurne nesthesi s desribed previously 9. Whole-ell pth-lmp reordings of lyer / neurons of primry visul ortex (monoulr region) were performed by shdow-pthing. Neurons were dilysed with pipette solution ontining the fluoresent C inditor Oregon green BAPTA- hexpotssium (OGB-; 00 mm), Alex-9 ( mm) nd bioytin ( mg ml ). Bsl nd oblique dendrites tht ppered on the sme fol plne were imged by high-speed two-photon mirosopy involving resonnt glvo-snner 0. Membrne potentil hnges nd C signls were simultneously reorded. The fol plne depth nd the imged re were hosen to ontin s mny dendrites s possible. At eh fol plne, we imged the tivity evoked by drifting squre wve grtings (0.0 yles per degree, Hz, eight diretions, stnding phse s, drifting phse s). Trnsient hnges in C fluoresene (Df/f) were systemtilly exmined by n dptive lgorithm, whih involved smll regions of interest (ROIs) of mm, noise filtering nd pttern mthing. The NMDA reeptor ntgonist AP (together with Alex-9) ws lolly pplied by pressure ejetion lose to the imged dendrites. The spred of the ejeted solution ws monitored by imging Alex fluoresene. The tuning level of lol dendriti lium signls with regrd to the orienttion of the drifting grting ws quntified by n OSI. Reeived Otober 009; epted Februry 00.. London, M. & Häusser, M. Dendriti omputtion. Annu. Rev. Neurosi. 8, 0 (00).. Bloodgood, B. L. & Sbtini, B. L. C signling in dendriti spines. Curr. Opin. Neurobiol. 7, (007).. Mrkrm, H. & Skmnn, B. Clium trnsients in dendrites of neoortil neurons evoked by single subthreshold exittory postsynpti potentils vi low-voltge-tivted lium hnnels. Pro. Ntl Ad. Si. USA 9, 07 (99).. Nevin, T. & Skmnn, B. Spine C signling in spike-timing-dependent plstiity. J. Neurosi. 6, 00 0 (006).. Yuste, R. & Denk, W. Dendriti spines s bsi funtionl units of neuronl integrtion. Nture 7, (99). 6. Häusser, M. & Mel, B. Dendrites: bug or feture? Curr. Opin. Neurobiol., 7 8 (00). 7. Helmhen, F., Svobod, K., Denk, W. & Tnk, D. W. In vivo dendriti lium dynmis in deep-lyer ortil pyrmidl neurons. Nture Neurosi., (999). 8. Murym, M. et l. Dendriti enoding of sensory stimuli ontrolled by deep ortil interneurons. Nture 7, 7 (009).

6 ARTICLES NATURE Vol 6 9 April Svobod, K., Denk, W., Kleinfeld, D. & Tnk, D. W. In vivo dendriti lium dynmis in neoortil pyrmidl neurons. Nture 8, 6 6 (997). 0. Johnston, D. & Nrynn, R. Ative dendrites: olorful wings of the mysterious butterflies. Trends Neurosi., 09 6 (008).. Lrkum, M. E. & Nevin, T. Synpti lustering by dendriti signlling mehnisms. Curr. Opin. Neurobiol. 8, (008).. Ohki, K. & Reid, R. C. Speifiity nd rndomness in the visul ortex. Curr. Opin. Neurobiol. 7, 0 07 (007).. Mgee, J. C. Dendriti integrtion of exittory synpti input. Nture Rev. Neurosi., 8 90 (000).. Polsky, A., Mel, B. W. & Shiller, J. Computtionl subunits in thin dendrites of pyrmidl ells. Nture Neurosi. 7, 6 67 (00).. Csh, S. & Yuste, R. Liner summtion of exittory inputs by CA pyrmidl neurons. Neuron, 8 9 (999). 6. Bnnister, A. P. Inter- nd intr-lminr onnetions of pyrmidl ells in the neoortex. Neurosi. Res., 9 0 (00). 7. Hirsh, J. A. & Mrtinez, L. M. Lminr proessing in the visul ortil olumn. Curr. Opin. Neurobiol. 6, 77 8 (006). 8. Svobod, K., Helmhen, F., Denk, W. & Tnk, D. W. Spred of dendriti exittion in lyer / pyrmidl neurons in rt brrel ortex in vivo. Nture Neurosi., 6 7 (999). 9. Hubel, D. H. & Wiesel, T. N. Reeptive fields, binoulr intertion nd funtionl rhiteture in the t s visul ortex. J. Physiol. (Lond.) 60, 06 (96). 0. Hubel, D. H. & Wiesel, T. N. Reeptive fields nd funtionl rhiteture of monkey strite ortex. J. Physiol. (Lond.) 9, (968).. White, L. E. & Fitzptrik, D. Vision nd ortil mp development. Neuron 6, 7 8 (007).. Dräger, U. C. Reeptive fields of single ells nd topogrphy in mouse visul ortex. J. Comp. Neurol. 60, (97).. Métin, C., Godement, P. & Imbert, M. The primry visul ortex in the mouse: reeptive field properties nd funtionl orgniztion. Exp. Brin Res. 69, 9 6 (988).. Niell, C. M. & Stryker, M. P. Highly seletive reeptive fields in mouse visul ortex. J. Neurosi. 8, (008).. Kitmur, K., Judkewitz, B., Kno, M., Denk, W. & Häusser, M. Trgeted pthlmp reordings nd single-ell eletroportion of unlbeled neurons in vivo. Nture Methods, 6 67 (008). 6. Kerr, J. N., Greenberg, D. & Helmhen, F. Imging input nd output of neoortil networks in vivo. Pro. Ntl Ad. Si. USA 0, (00). 7. Anderson, J., Lmpl, I., Reihov, I., Crndini, M. & Ferster, D. Stimulus dependene of two-stte flututions of membrne potentil in t visul ortex. Nture Neurosi., 67 6 (000). 8. Crndini, M. & Ferster, D. Membrne potentil nd firing rte in t primry visul ortex. J. Neurosi. 0, 70 8 (000). 9. Bringuier, V., Chvne, F., Gleser, L. & Fregn, Y. Horizontl propgtion of visul tivity in the synpti integrtion field of re 7 neurons. Siene 8, (999). 0. Rohefort, N. L. et l. Sprsifition of neuronl tivity in the visul ortex t eyeopening. Pro. Ntl Ad. Si. USA 06, 09 0 (009).. Gordon, U., Polsky, A. & Shiller, J. Plstiity omprtments in bsl dendrites of neoortil pyrmidl neurons. J. Neurosi. 6, (006).. Koester, H. J. & Skmnn, B. Clium dynmis ssoited with tion potentils in single nerve terminls of pyrmidl ells in lyer / of the young rt neoortex. J. Physiol. (Lond.) 9, 6 66 (000).. Bollmnn, J. H. & Engert, F. Subellulr topogrphy of visully driven dendriti tivity in the vertebrte visul system. Neuron 6, (009).. Kovlhuk, Y., Eilers, J., Lismn, J. & Konnerth, A. NMDA reeptor-medited subthreshold C signls in spines of hippompl neurons. J. Neurosi. 0, (000).. Noguhi, J., Mtsuzki, M., Ellis-Dvies, G. C. & Ksi, H. Spine-nek geometry determines NMDA reeptor-dependent C signling in dendrites. Neuron 6, (00). 6. Holthoff, K., Kovlhuk, Y., Yuste, R. & Konnerth, A. Single-shok LTD by lol dendriti spikes in pyrmidl neurons of mouse visul ortex. J. Physiol. (Lond.) 60, 7 6 (00). 7. Mjor, G., Polsky, A., Denk, W., Shiller, J. & Tnk, D. W. Sptiotemporlly grded NMDA spike/plteu potentils in bsl dendrites of neoortil pyrmidl neurons. J. Neurophysiol. 99, 8 60 (008). 8. Lrkum, M. E., Nevin, T., Sndler, M., Polsky, A. & Shiller, J. Synpti integrtion in tuft dendrites of lyer pyrmidl neurons: new unifying priniple. Siene, (009). 9. Stosiek, C., Grshuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon lium imging of neuronl networks. Pro. Ntl Ad. Si. USA 00, 79 7 (00). Supplementry Informtion is linked to the online version of the pper t Aknowledgements We re grteful to B. Skmnn for disussions nd to Y. Kovlhuk for help in the initil experiments. This work ws supported by grnts from the DFG (to A.K.) nd the Friedrih Shiedel Foundtion. A.K. is Crl von Linde Senior Fellow of the Institute for Advned Study of the TUM. H.J., N.L.R. nd X.C. were supported by the DFG (IRTG 7). Author Contributions H.J., N.L.R. nd X.C. rried out the experiments. H.J., N.L.R. nd A.K. performed the nlysis. A.K. designed the study nd wrote the mnusript with the help of ll uthors. Author Informtion Reprints nd permissions informtion is vilble t The uthors delre no ompeting finnil interests. Correspondene nd requests for mterils should be ddressed to A.K. (rthur.konnerth@lrz.tum.de).