Simultaneous All-Optical Manipulation and Recording of Neural Circuit Activity with Cellular Resolution in Vivo

© 2015 Nature America, Inc. All rights reserved. London, London, UK. Correspondence should be addressed to M.H. ( 1 readout of neural activity are associated with a range of limitations, of patterns activity natural manipulating and generating for crucial is photostimulation patterned with concurrently neurons multiple ref. see (but tively effec combined to be yet have optogenetics two-photon and ing imag calcium two-photon However, channels. and stimulation imaging the the both in precision single-spike with and goal a single-cell such achieve to required ratio signal-to-noise and ( at the spatial and temporal resolution simultaneously at which they function neurons many of activity the manipulating recording for and desirable highly is approaches experimental resolution cellular with neurons of hundreds from activity neural of readout quantitative permit neurons of of at precision scale populations the spatial tion with millisecond manipulations of neural activity, enabling activation and inactiva experimental revolutionizing rapidly are actuators Optogenetic resolution in the mouse brain definedneural circuits with single-cell and single-spike flexibleand long-term optical interrogation of functionally with genetic or viral approaches, enabling high-throughput, extends the optogenetic toolkit beyond the specificity obtained ensembles based on their functional signature. during different behavioral states and targeting of neuronal demonstrate interrogation of the same neuronal population Proof-of-principle experiments in mouse barrel cortex of the manipulation with negligible optical cross-talk. calcium imaging provides high-resolution network-wide readout concurrent optogenetic activation, while simultaneous fast selected neurons to be targeted for spatiotemporally precise calcium indicator. A spatial light modulator allows tens of user- coexpression of a red-shifted opsin and a genetically encoded two-photon optogenetic activation and calcium imaging by with cellular resolution manipulating and recording the activity of multiple neurons We describe an all-optical strategy for simultaneously Adam M Packer neural circuit activity with cellular resolution S 140 Received Wolfson Institute for Biomedical Research, University College London, London, UK. Fig. 1 Fig. Articles Previous implementations of simultaneous manipulation and and manipulation simultaneous of implementations Previous imultaneous all-optical manipulation and recording of

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© 2015 Nature America, Inc. All rights reserved. A two-photon targeted cell-attached patch-clamp recording was obtained from neuron i. This neuron was targeted for optogenetic stimulation in a in stimulation optogenetic for targeted was neuron This i. neuron from obtained was recording patch-clamp cell-attached targeted A two-photon bar, 100 scale (pink; C1V1-2A-mCherry and ( tubes. photomultiplier PMT2, PMT1, filters; F1–3: module; scanning resonant RSM, galvanometers; GM2, GM1, block; order zero ZB, modulator; light spatial SLM, lenses; L1–4, plate; HWP, half-wave shutter; S, cell; Pockels PC, ( stimulation. Stim, recording. simultaneous during iv–vi neurons adjacent of immediately stimulation without i–iii neurons user-selected in photostimulation reliable and robust Bottom, photostimulation. enables opsin an and of activity, readout optical an generates sensor Top, a calcium goal. experimental 1 Figure ( target photostimulation the shifting 12 was µ Methods) Online (see galvanometers mm 6 using tion neuron, ( C1V1 and GCaMP6s photostimulated expressing also the resulting neurons in neighboring in trial, not but transients every on calcium large neuron in recorded the in generated 20 ms (see Online Methods). Single action potentials were reliably fashion spiral a in scanned was which spot, photostimulation single a generate to SLM the ( cortex somatosensory mouse the of 2/3 layer in recordings patch-clamp cell-attached simultaneous by performing approach all-optical our ( calibrated nm 1,064 at excitation two-photon via neurons 200 × 200 of using a resonant system scanning at view 920 nm and of photostimulated field a over movies imaging calcium opsin a red-shifted encoded genetically calcium indicator patterned green-fluorescent an ‘ultrasensitive’ GCaMP6s, with ( co-infected and stimulation imaging to order optogenetic two-photon in simultaneous microscope provide scanning resonant dual-beampath animal behaving awake, ( an in network local the functional and these in neurons their stimulation to on response the based observe then and neurons identity groups targeted activate selectively individually to of able be to was goal design Our photostimulation C RESULTS time histogram are shown. Bottom, calcium imaging recordings obtained simultaneously from neurons i–iii in in i–iii neurons from simultaneously obtained recordings imaging calcium Bottom, shown. are histogram time ( pattern. spiral Fig. 1 Fig. ombining calcium imaging and single-cell single-cell and imaging calcium ombining m laterally and ~24 ~24 and laterally m a | Fig. 1 Fig. ). We incorporated an SLM into a two-photon two-photon a into SLM an incorporated We ). Single-cell two-photon optogenetic photostimulation and single-action-potential readout readout single-action-potential and photostimulation optogenetic two-photon Single-cell b ) Optical layout of the SLM-based two-photon patterned photostimulation, two-photon resonant scanning, moving moving scanning, resonant two-photon photostimulation, patterned two-photon of SLM-based the layout ) Optical e e ) Top, electrophysiological recording during photostimulation trials (pink bar). Single sweep (from trial 2), raster plot, and peristimulus peristimulus and plot, raster 2), trial (from sweep Single bar). (pink trials photostimulation during recording ) Top, electrophysiological ). The spatial resolution of spiral photostimula spiral of resolution spatial The ). 2 in vivo in 1 ( Fig. 1 Fig. µ n m axially, measured by incrementally incrementally by measured axially, m = 3 experiments). We programmed programmed We experiments). 3 = i. 1 Fig. c 2 ). We recorded high-speed (30 Hz) (30 Wehigh-speed ). recorded 2 over the neuronal cell body for for body cell neuronal the over b ). We visualized neurons neurons visualized We ). µ 2 0 m). ( m). , , and C1V1-2A-mCherry, Supplementary Fig. 1 Fig. Supplementary d ) Inset from a large field of view (200 × 200 × 200 (200 of view field a large from ) Inset Fig. 1 Fig. in d ). We ).

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© 2015 Nature America, Inc. All rights reserved. neurons are circled in black. 300 total neurons, 120 trials, mean inferred action potentials post-photostimulation = 1.0 1.0 = post-photostimulation potentials action inferred mean trials, 120 neurons, total 300 black. in circled are neurons panel from field the in photostimulation of strength the ( circles). (white bar, 100 (scale GCaMP6s and C1V1-2A-mCherry with colabeled cortex somatosensory 3 Figure jitter low with neurons individual in potentials action generating optically reliable for a strategy found and parameters of range a with pho tostimulating while mice five in neurons 19 from recorded we total, In half-maximum; at (full-width axially the 22 was moving neuron, the by to relative measured pattern galvanometers, mm 3 with pattern trials; 18.1 tials, (3.8 properties electrophysiological intrinsic its from expected as potentials, action more and latency shorter a with although fashion, similar a in responded which neuron, same photostimulation onparadigm the a fast-spiking putative inter tested small also we the comparison, in For volume). available photostimulation opsins of population restricted the ing exhaust desensitization opsin of result a as (perhaps action potentials more in result not did mination 3b Fig. Supplementary s.e.m.; are bars Error ms. 34 or 16 11, lasting photostimulations spiral and patterns photostimulation spot 20 ( jitter. and latency low showing data raw overlaid Right, bar). pink (photostimulation, trials ten over delivery stimulus around times of spike Middle, raster of one targets. the in recorded electrophysiologically was generation potential action while photostimulated were locations multiple which in configuration recording patch-clamp cell-attached showing 100 bars, (scale mirrors galvanometer and SLM the using locations multiple to patterns photostimulation spiral targeting for a protocol present right, 100 bar, (scale C1V1-2A-YFP expressing neurons 2/3 layer cortex of of somatosensory view ( neurons identified of multiple 2 Figure 142 legend). in arrowhead black c a C1V1-2A-mCherry GCaMP6s Articles a ) Metrics evaluating performance for 10 and 10 for performance evaluating ) Metrics ) The leftmost panel shows an example field field example an shows panel leftmost ) The The spatial resolution of photostimulation using the grid grid the using photostimulation of resolution spatial The

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is directed on a sample, but distributed over a larger area larger a over distributed but sample, a on directed is power more much whereby strategy alternate an to opposed as over multiplexed was beamspot a spatiotemporally small neuron, a in density power high a (i.e., required was beamlet per power less as targeted, be to neurons more allowed group a as beamlets for targets many ( photostimulation providing simultaneous tissue, cortical of territories large across neurons in expression strong showed C1V1-2A-mCherry concurrently neurons so as multiple to activate beamlets targeted spatially ual, We the used SLM to into split laser the individ photostimulation S ably ably generated one at potential action latencies (1.2 similar ( neuron a over recordings cell-attached when just one of targeted the selected ten targets was two-photon positioned simultaneous via potentials action generated strategy photostimulation our that confirmed imultaneous photostimulation of multiple selected neurons

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© 2015 Nature America, Inc. All rights reserved. cantly with the expression of C1V1 ( C1V1 of expression the with cantly signifi not but slightly correlated photostimulation to response The analysis. subsequent from excluded be could neurons these responses to photostimulation ( previously observed as strongly, very work previous others’ ( responded neurons neighboring of proportion small a Only mice. three in ( photostimulation to responses reliable and strong showed neurons target Individual neurons we the ( 10 photostimulated selected neously imaging these and the surrounding 290 neurons at 30 Hz, simulta While neurons. of ten a cluster to targeted beamlets ten populations neural how precisely controlled photostimulation inputs are integrated ( by neurons specified multiple of stimulation ( approach all-optical the Wecombined N programmed photostimulation. of patterns using neurons selected individually multiple, in show experiments These potentials action timed precisely generate can approach our that animals. seven in neurons 11 from work with low jitter comparable (results to from those previous potentials action generated optically summary, we demonstrated for trials tostimulation six recorded neurons in In three animals). 5.6 ± on 88% potentials one or more action potentials, (1.3 latency in increase slight a in photostimulated neurons from 10 to 20 reduced the of power laser per spot, resulting number the Increasing respectively. body, cell the cover to it took time longer the and time exposure lation photostimu increased of the because presumably animals), four 11.3 87% on potentials action 1.5 spiral: ms (34 latency tion increased the number of action potentials generated and their in six neurons in four animals; deviation of the latency), 107 respectively; photostimulation trials latency, 18.8 98% and 1.2 exact comparisons; *, ** and *** denote behavioral states ( spike responses to photostimulation (mean and running speed. (Regression line in gray.) ( ( speed. of running neurons.ten Bottom, photostimulation simultaneous ( imaging. calcium high-speed with observed view were simultaneously and These neurons the ofsurrounding across field photostimulation. for were chosen in (marked white) cortex of 2/3 somatosensory mouse ( Styrofoam treadmill. ( 4 Figure In summary, we were able to optically invoke spatiotemporally spatiotemporally invoke optically to able were we summary, In d c a 0% of 30.1 trials, etwork readout during targeted multineuron stimulation multineuron targeted during readout etwork ) Top, mean calcium transient of all stimulated ) neurons Top,to transient in response of stimulated mean calcium all ) Mice were head-fixed and allowed to run freely on a fixed-axis on a freely fixed-axis to run and ) allowed Mice were head-fixed ) Correlation between mean perturbation of responses to photostimulation ± 0.8 ms jitter for 16 ms and 34 ms spirals, respectively; 70 pho ± ± P 23 values in the text). Anes., anesthetized. 3.3 ms jitter, 93 photostimulation trials in six neurons in in neurons six in trials photostimulation 93 jitter, ms 3.3 3% and 88% 88% and 3% , | 24 ± Dependence of network perturbations on behavioral state. on behavioral ofperturbations Dependence network 0.05 action potentials, one or on potentials, more 0.05 action potentials action , 2 6 ) in 10–20 selected neurons, confirmed by recording recording by confirmed neurons, selected 10–20 in ) Supplementary Fig. 5 Fig. Supplementary ± 17.1 and 8.0 n = 3 mice, 576 imaged neurons; Tukey’s test for multiple ± b in ) ) A of field neurons view ten in which in layer 8.5 and 35.9 ±

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© 2015 Nature America, Inc. All rights reserved. of indicator sensitivity and lack of two-photon optogenetics and calcium The imaging. combination spectral overlap between the optoge same neurons with cellular resolution in mice and the recording for activating strategy an all-optical developed allow this to be achieved in a minimally invasive manner. We have approaches Optical behavior. drive to together work neurons of how to is populations understanding crucial function they which at resolution temporal and spatial the at circuits neural Probing DISCUSSION animal. of behaving awake, the in neurons ensembles defined functionally activating for approach our two in respectively, view; of fields neurons, sensory-responsive weakly and 30% (maximum groups sensory-responsive weakly and groups) (both responsive the strongly sensory- between tion different was not significantly running on a Styrofoam treadmill. The response to photostimula and awake was animal the while groups three the of each within ( tions orienta stimulation particular to weakly or strongly that responded neurons revealed barrel the within stimulation sensory to Next, two-photon imaging of the responses of neurons individual ( cortex somatosensory the in C2 barrel identify to ing properties. To achieve this goal, first we performed intrinsic imag functional individual their of basis the on population the within neurons of groups different target to us allowed approach Our O states. behavioral different and activity ongoing during networks of neuronal perturbing for approach robustness our the demonstrate results These per mouse). trials per 20 state minimum states; behavioral between distributed n Tukey’sawake versus anesthetized; test for multiple comparisons; awake; ( neurons network local of for non-stimulated states the responses compari sons). We multiple a observed significant difference between for all behavioral test Tukey’s anesthetized; versus awake (ANOVA); ( anesthetized were photostimulation ( photostimulation of five neurons responding strongly to a givenphotostimulated sensory stimulation(pink line). or ( weakly to both stimuli revealed similarstimulation responses (gray shading)(mean were simultaneously responded differently or not at all to sensory caudal whisker stimulation. Five neurons that on their response to dorsal-ventral and rostro- neurons were selected for photostimulation based see somatosensory cortex. (For definitions of symbols, 2A-mCherry (pink) in the C2 barrel of mouse C1V1- and (green) GCaMP6s coexpressing bar, 50 (scale view signatures functional individual their on based manipulation 5 Figure 144 minimizes wavelengths emission GCaMP6s and excitation netic P = 3 mice, 576 imaged neurons, 30 stimulated neurons, 280 trials Articles ptogenetic manipulation targeted to functional ensembles to functional targeted manipulation ptogenetic = 4.2 × 10 × 4.2 = b

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© 2015 Nature America, Inc. All rights reserved. This work was supported by grants from the Wellcome Trust, the Gatsby Gatsby the Trust, Wellcome the from grants by supported was work This AAVdj virus. to access and plasmids for University) (Stanford Deisseroth K. of and AAVdj; use on advice for NS069375-01A1) P30 no. (grant Core Virus and Vector Gene Neuroscience Stanford the at Lochrie M. of microscope; the customization enabling for Technologies) Prairie (formerly Corporation Bruker at staff the manuscript; the on comments and discussion for Peterka D. and B. Judkewitz Smith, S. Bianco, I. B. Clark, Roth, A. Schmidt-Hieber, C. Wilms, C. software; and routines analysis discussion, helpful for Pettit N. and London M. Stringer, Turaga, C. S. Pachitariu, M. We thank Ackno online ve Note: Any Information Supplementary and Source Data files are available in the of version and are Methods references any in available the associated M circuits provide should approach neural in processing information into insights new this fundamental using work Future behaviors. a exhibiting population a specific functional signature within during sensory processing or defined neurons those only in code sion than previously possible, enabling investigation of the neural preci greater far with targeted be to manipulation optogenetic allow will This population. in the ensembles of specific targeting enable to stimuli sensory different to neurons of response characterization the of functional detailed a use to able were we that showing experiments proof-of-principle describe we Here sion. preci temporal lacks and of activity readout indirect a very only alone ana identity tomical and/or genetic on based targeted be could manipulations optogenetic Previously, neurons. the of signature functional the unprecedented of basis the the on manipulation with optogenetic target us to opportunity provides neurons of population approach. the of side readout methods imaging complex more and faster in advances addition, In here. the into integration their enable will excitation, two-photon their of characterization including of optrodes use the as such optogenetics, and electrophysiology of bination on relying the com strategies and readout manipulation taneous are downstream any confusion about which cells are directly stimulated and which removes precision single-cell with neurons target and locations data. Additionally,crucial neuronal the ability to observe directly of loss the in resulting photostimulation, during readout the of excitation on one-photon relying approaches all-optical in previous observed problem talk recent most the indicator of calcium ‘ultrasensitive’ imaging calcium high-speed with readout approaches different these among choice the dictate will addressed being question biological underlying the that expect We method. this with sequence a in rates high at performed be can which of neurons, among ensembles activity of synchronous active development, our approach is well suited for the generation photostimulate neurons. Thus, all while of to are these technologies currently under strategy scanning fast a using and beam the Charitable Foundation, the European Commission (Marie Curie International International Curie (Marie Commission European the Foundation, Charitable ethods Our ability to read out and manipulate activity in the same same the in activity manipulate and out read to ability Our fast combining approach two-photon dual all-optical, Our w rsion rsion of the pape ledgments in 38– the pape the

vivo 11– 4 0 n analysis and 1 . 3 14 . Further development of new molecular tools molecular of new development . Further , 1 5 r . , , which has been a problem with other simul r . 4 2 , , or on 5– 7 4 . These approaches require blocking blocking require approaches . These 1 could be incorporated into the the into incorporated be could c-fos 2 0 drastically reduces the cross- the reduces drastically expression in

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6. 4. 3. 2. 1. c R The authors declare no competing financial interests. COM A.M.P., L.E.R., H.W.P.D. and M.H. designed the study and wrote the paper. A.M.P., L.E.R. and H.W.P.D. performed experiments and analyzed data. A Council. Research European the and Council Research Medical the Organization, Biology Molecular European the 328048), no. grant Fellowship Incoming 14. 13. 12. 11. 10. 9. 8. 7. 5. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. om/reprints/index.ht eprints and permissions information is available online at UTHOR

P (2012). networks inhibitory cortical distinct by (2013). optogenetics. method of tagging neuronal populations for identification during for populationsneuronal tagging of method animal. behaving the in circuits neural local of recording and perturbation light-assisted mice.moving freely in control neurons single from recordings (2010). 18720–18731 illumination. light structured with photo-stimulation (2011). patterning. lightholographic andremote-focusing by photostimulation and imagingdimensional Neurosci. Mol. Front. optogenetics. with combination and imaging activity neural multi-color channel. light-sensitive program by a small brain area in zebrafish. zebrafish. in area brain small a by program mice.moving freely in behaviour learned Neurosci. Nat. fields. firing cell place of imaging and perturbation optical resolution (2012). 862–885 circuits. modulation. bistable and excitation saturation. at dysfunction. social and processing activity. (2009). window.cranial chronic a through neocortex (2008). modulators.light spatial with photostimulation microscopy.fluorescence (1997). neurons. pyramidal neocortical in dynamics calcium 1511 during subtypes neuron projection striatal recording.electrophysiological Wilson, N.R., Runyan, C.A., Wang, F.L. & Sur, M. Division and subtraction and Division M. Sur, & F.L.Wang, C.A., Runyan, N.R., Wilson, Huber,D. cellular- D.W.SimultaneousTank, & K. Rickgauer,J.P.,Deisseroth, neurons. in calcium Imaging A. Konnerth, & Grienberger,C. for photons andTargeting neurons Hausser,M. & B. Roska, Packer,A.M., Lima, S.Q., Hromadka, T., Znamenskiy, P. & Zador, A.M. PINP: a new a PINP: Zador,A.M. Znamenskiy,T.,P.& Hromadka, S.Q., Lima, S. Royer, P.Anikeeva, Y.LeChasseur, M. Maschio, Dal V.Three- Emiliani, & D. Ogden, A., Begue, C., Ventalon,F., Anselmi, J. Akerboom, a using plasticity synaptic ofinduction Optical Oertner,T.G. Y.P. Zhang,& Fajardo, O., Zhu, P. & Friedrich, R.W. Control of a specific motor motor specific a of Control R.W. Friedrich, & P. Zhu, O., Fajardo, Packer,A.M. R. Prakash, channelrhodopsin-2 of excitationD.W.Two-photon Tank, Rickgauer,J.P.& O.Yizhar, T.W.Chen, A. Holtmaat, V.Nikolenko, scanning W.W.Webb,laser Two-photon& W.,Strickler,J.H. Denk, D.W. Tank, & D. Kleinfeld, W., Denk, K., Svoboda, identification of Optogenetic Kreitzer,A.C. & S.F. A.V.,Owen, Kravitz, ETING

CONTRI , 21–32 (2013). 21–32 , F Nat. Methods Nat. Nature IN et al. et et al. et et al. et n et al. et A B et al. et a NCI et al. et et al. et UTIONS et al. et ture methods

et al. et et al. et Nat. Neurosci. Nat. Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. 17 et al. et Multi-array silicon probes with integrated optical fibers: optical integrated with probes silicon Multi-array

Sparse optical microstimulation in barrel cortex drives cortex barrel in microstimulationoptical Sparse Neocortical excitation/inhibition balance in information in balanceexcitation/inhibition Neocortical 499 Ultrasensitive fluorescent proteins for imaging neuronal imaging for proteins fluorescent Ultrasensitive A et al. et Two-photon optogenetic toolbox for fast inhibition, fast for toolbox optogeneticTwo-photon , 1816–1824 (2014). 1816–1824 , L Eur. J. Neurosci. J. Eur. Two-photon optogenetics of dendritic spines and neural and spinesdendritic of optogeneticsTwo-photon Optetrode: a multichannel readout for optogenetic for readoutmultichannel a Optetrode: m Long-term, high-resolution imaging in the mouse the in imaginghigh-resolution Long-term,

SLM microscopy: scanless two-photon imaging and imaging two-photon scanless microscopy: SLM Genetically encoded calcium indicators for indicators calcium encoded Genetically INTERESTS A microprobe for parallel optical and electrical and optical parallel for microprobe A , 295–300 (2013). 295–300 ,

l . 6

Simultaneous two-photon imaging and imaging two-photon Simultaneous , 2 (2013). 2 , 9 , 1202–1205 (2012). 1202–1205 , Nat. Methods Nat. Science

16 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc.

PLoS ONE PLoS Nat. Neurosci. Nat. | , 805–815 (2013). 805–815 , in vivo in O.2 NO.2 VOL.12

248

31 Nature Nat. Methods Nat.

, 73–76 (1990). 73–76 , , 2279–2291 (2010). 2279–2291 , 4 . , 139–141 (2007). 139–141 , Nat. Methods Nat.

in vivo in

106 4

Nature 477 , e6099 (2009). e6099 , in vivo in

, 15025–15030 (2009). 15025–15030 , Nat. Protoc. Nat. Front. Neural Circuits Neural Front. 15 , 171–178 (2011). 171–178 , . Nature | Front. Neural Circuits Neural Front.

, 163–170 (2011). 163–170 , FEBRUARY 451

recordings. 9 In vivo In , 1171–1179 (2012). 1171–1179 ,

, 61–64 (2008). 61–64 , Nature Opt. Express Opt. 8

, 319–325 (2011). 319–325 ,

488 http://www.nature. Articles 108

4 dendritic , 1128–1144 , , 343–348 ,

, 19504–19509 , Neuron 385

Brain Res. Brain 2015

, 161–165 161–165 ,

in vivo in 18

7 73

| , , 67 67 ,

145 , 5 , ,

(2009). system. Tet the and transfer gene viral using (2014). cortex. auditory the in discharge corollary for cortex. visual mouse in state 156 (2009). indicators. calcium GCaMP improved with cortex. perturbations to stimulation channelrhodopsin-2. optogenetics. with control Zhu, P.Zhu, basis circuit and synaptic A Mooney,R. & A. Nelson, Schneider,D.M., behavioral by responses visual of Stryker,M.P.Modulation & C.M. Niell, Y. Fu, L. Tian, P.E.Sensitivity Latham, & Hausser,M. L., Beeren, A., Roth, M., London, single-neuron by microcircuits cortical of DissectionY. Dan, & A.C. Kwan, Papagiakoumou,E. neural precise toward scalpel: to cudgel From K. Svoboda, & S. Peron, | O.2 NO.2 VOL.12 , 1139–1152 (2014). 1139–1152 , et al. et Nature et al. et et al. et A cortical circuit for gain control by behavioral state. behavioral by control gain for circuit cortical A in vivo in Optogenetic dissection of neuronal circuits in zebrafish in circuits neuronal of dissection Optogenetic Imaging neural activity in worms, flies and mice and flies worms, in activity neural Imaging

466 in vivo in | . FEBRUARY , 123–127 (2010). 123–127 , et al. et Curr. Biol. Curr. Nat. Methods Nat. implies high noise and suggests rate coding in coding rate suggests and noise high implies Scanless two-photon excitation of excitation two-photon Scanless Nat. Methods Nat.

Neuron 22

2015 , 1459–1467 (2012). 1459–1467 ,

7 , 848–854 (2010). 848–854 ,

65 |

, 472–479 (2010). 472–479 , n 8 Nat. Methods Nat. , 30–34 (2011). 30–34 , a ture methods Front. Neural Circuits Neural Front. Nature

513

6 , 875–881 , , 189–194 ,

3 Cell , 21 ,

43. 42. 41. 40. 39. 38. 37. 36. 35. 34.

fear memory recall. memory fear systems. neural in Optogenetics Methods Nat. dimensions.three in activity neural microscopy.light-field using activity zebrafish. in rhodopsins.microbialengineered using tissue. scattering inside deep neurons.pyramidal CA1 hippocampal in activity neuronal related theta of mechanisms USA Sci. Acad. Natl. Proc. light. sculpted by activity neuronal of controloptogenetic single-cell Liu, X. Liu, K. Deisseroth, & M. Mogri,T.J., Davidson, L.E., Fenno, O.,Yizhar, J. Freeman, of imaging Simultaneous R. Yuste, & D.S. Peterka, J., Jackson, S., Quirin, R. Prevedel, M.B.Ahrens, D.R. Hochbaum, Papagiakoumou,E. Network J.C. Magee, & A. B.V.,Zemelman,Vaziri, Losonczy,A., Two-photon A. Vaziri, & B.V.,J. Andrasfalvy,Zemelman,Tang,B.K., et al. et et al. et Optogenetic stimulation of a hippocampal engram activates engram hippocampal a of stimulationOptogenetic et al. et

Nature et al. et 11 , 941–950 (2014). 941–950 , et al. et Mapping brain activity at scale with cluster computing. cluster with scale at activity brain Mapping Simultaneous whole-animal 3D imaging of neuronal of imaging 3D whole-animal Simultaneous Brain-wide neuronal dynamics during motor adaptation motor duringdynamics neuronal Brain-wide

et al. et Nat. Neurosci. Nat. Nature 485 All-optical electrophysiology in mammalian neuronsmammalian in electrophysiology All-optical , 471–477 (2012). 471–477 , Functional patterned multiphoton excitationmultiphoton patterned Functional

107

484 Nat. Photonics Nat. , 11981–11986 (2010). 11981–11986 , , 381–385 (2012). 381–385 , Neuron

13 Front. Neural Circuits Neural Front. , 967–972 (2010). 967–972 , Nat. Methods Nat.

Nat. Methods Nat. 71 , 9–34 (2011). 9–34 ,

7 , 274–278 (2013). 274–278 ,

11 , 727–730 (2014). 727–730 , , 825–833 (2014). 825–833 ,

8 , 29 (2014). 29 ,

© 2015 Nature America, Inc. All rights reserved. Titration of calcium indicator expression. indicator calcium of Titration 4 and of 0.7 administration 7 days with post-operative cement. by dental place in fixed and (VetBond) cyanoacrylate of layer thin a by skull the to sealed Norland craniotomy, the into (NOR-61, press-fit were cement Adhesive) Optical optical UV-curable with Optics) circular mm (UQG 3 coverslip glass a square to 2 mm cemented a coverslip glass of composed windows (Sugi, Cranial Sugi-sponges Kettenbach). with stanched or solution with away external washed sterile was was bleeding any craniotomy which circular during mm 3 performed a above), (see injection virus After LED. green a with visualized as pattern vessel blood the to from the brain indicated thereflectance in barrel,increase An which surface. couldthe be illuminated localized(LED) relativediode light-emitting red a while Technologies) Vision Allied F-032b, (Pike camera device) (charge-coupled CCD a with imaged and CaCl mM 2 HEPES, mM 10 KCl, mM 2.5 NaCl, mM (150 solution external sterile with perfused was brain The interval. s interstimulus a 20 with s 4 for Hz 10 at oscillate to programmed was actuator The glass pipette glued to a piezoeletric actuator (Physik Instrumente). in barrel certain experiments. C2 The C2 the whisker wasto threaded intosite a trimmed injection the localize further to formed imaging Intrinsic before injection. virus Sun-Medical) with cortex C&B, cement dental (Super-Bond ing somatosensory overly skull the to fixed was well imaging circular mm 5 a with from the midline to the temporalis muscles, and a metal headplate containing mixture a 0.1 with injected were animals Co-injected visible 24 days later. Animals singly injected received 1 were performed through the same craniotomy site, which was still 1 also injections, Subsequent Vetbond (3M). with closed was 1 with injected first were 2/3 (~300 0.1 of rate a at injected and pipette injection AAV1-Syn-GCaMP6s-WPRE-SV40 AAVdj-CaMKIIa-C1V1(E162T)-TS-P2A-mCherry-WPRE (AAV2-CaMKIIa-C1V1(E162T)-p2A-EYFP, Virus Apparatus). beveled apparatus hydraulic injection driven by a pump ~15 syringe (Harvard pipettes diameter: (inner injection point sharp a Calibrated to diameter). in mm 0.5 of bregma, mm lateral and 3.5 from 2 mm posterior hemisphere, Craniotomies were drilled (NSK UK Ltd.) installation. over barrel window cortex (right chronic and headplate injections, Viral procedures. surgical g mg 0.01 an used with were sexes anesthetized mixture (0.1 and of both injection a intraperitoneal ketamine-xylazine were of Animals old randomization. months 3 without to weeks 4 imately ( mice C57/BL6 1986. Act Procedures) from (Scientific license Animal the with under accordance in out Office Home UK the carried were procedures surgical All ONLINE doi: the on heavily depended approach all-optical our in readout the Following all surgical procedures, animals recovered for at least bilaterally removed was scalp the experiments, chronic For 10.1038/nmeth.3217 µ l GCaMP6s virus and 0.9 0.9 and virus GCaMP6s l µ l ml l

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a reduction in virus titer to 10% of the original level (i.e., 10 (i.e., level original the of 10% to was titer virus parameter in reduction a crucial The implantation. window chronic by introduced artifacts confounding potential to minimize in order on the (depending extent of Acute expression). was imaging used intervals from the imaging). cortical surface to a maximum depth of for 500 0.5% and at 10 taken were site infection the surgery tiling Two-photon images for 1.5% induction, anesthesia for isoflurane (5% under below) (see imaging two-photon enable to performed was craniotomy acute an points of time these each At post-injection. weeks 3 and weeks 2 time week, 1 three points: of each for used be could animals two cohort, each within that such divided then was cohort viral-titer six-animal Each dilutions. above the of one with cohort each injected and each mice six of cohorts three into sexes both of mice C57/BL6 300 bregma, of min NaCl, 0.001% pluronic F-68). 100 nl of was virus atinjected 0.1 mM 140 8.0, Tris,pH mM (20 solution buffer a in diluted liliter 10 × 3.22 of solutions titer three at 10 × 3.22 titers: virus viral GCaMP6s with animals injected we expression, expression of GCaMP6s. To assess the stability of calcium indicator performed by raster scanning a femtosecond-pulsed laser laser femtosecond-pulsed resonant or a standard via Coherent) II, Ultra scanning (Chameleon beam raster by performed photostimulation. and Imaging (PackIO Bessel a software custom with kHz 20 at with recorded and kHz 4 above filter filtered Devices), a on (Molecular 700a amplified were MultiClamp Signals solution. external with filled and resistance) pipette M (~5 glass borosilicate from pulled pipettes Two-photon above. recordings patch-clamp described targeted as performed was recordings. installation patch-clamp targeted Cell-attached wavelength. emission on and GCaMP6s based separated both were which C1V1-2A-mCherry, spectrally easily with neurons weeks, of 5 co-labeling After interest. observed of we tissue cortical the to surgical access optical single a during long-term animals to provide installed was window a chronic procedure, the into injected viruses of been mixture had this After above). (see C1V1-2A-mCherry expressing virus the with directly it dilute could we meant virus ( classifier the based we on which neuron held-out one including not 20, ref. in (94% ( spikes of number inferred the and spikes of number true the correlation between the of strength the by defined as performance best the in resulted window ms 433 a over inference this of result the deconvolution algorithm the of results the integrated we when reported reliably be could window ms 250 a within occurring burst a in spikes of number 60 Hz ( at imaging while recordings cell-attached targeted two-photon performing by resolution single-action-potential confirmed we ( post-injection weeks as 12 as expression long stable in resulted which milliliter), per genomes The need to reduce the titer of the GCaMP6s-producing GCaMP6s-producing the of titer the reduce to need The −1 ± into L2/3 barrel cortex (2 mm and posterior 3.5 mm lateral 3%) and false positive rate (7% positive 3%) and false n Supplementary Fig. 9c Fig. Supplementary 4 = 4 neurons; 9 ) written in LabView (National Instruments). (National LabView in written ) Supplementary Fig. 9d Fig. Supplementary µ m below dura). We divided 18 adult (P60–P80) (P60–P80) adult 18 We dura). divided below m 13 Supplementary Fig. Supplementary 9a genomes per milliliter of stock and lower lower and stock of milliliter per genomes 4 6 ( 12 Supplementary Fig. 9b Supplementary and 3.22 × 10 × 3.22 and ). We also quantified the hit rate rate hit the quantified We also ). 47 Supplementary Fig. 8 Fig. Supplementary , Two-photon imaging was was imaging Two-photon 4 8 were obtained using glass glass using obtained were ). ± 6%) on as neurons three 11 ). ). We found that the genomes per mil per genomes n a ture methods ). ). Integrating Headplate Headplate ). Next, Next, ). µ µ µ m m 12 - l

© 2015 Nature America, Inc. All rights reserved. G) algorithm (GS) Gerchberg-Saxton The Coherent). fs; 100 width, pulse watts; 2.3 fs; Fianium Ltd.) 250–400 or width, pulse nm output, 1,055 (total laser fixed at femtosecond-pulsed 1,064 nm (total output, 5 watts; ( coverslip a to glued foil of zero at order was beam blocked of the focus L3 with a piece small The SLM. the of area active crystal liquid the driving voltages to values pixel converted table lookup Systems). manufacturer-supplied The Nonlinear Optics/Boulder (Meadowlark driver SLM the electronics to connected cable interface visual digital and card graphics Ti 660 GTX GeForce NVIDIA an via mask SLM the on phase the displayed Systems) Nonlinear Optics/Boulder (Nikon). 16×/0.8-NA = Objective (Hamamatsu); tube plier multi-alkali = photomultiplier tube (Hamamatsu); PMT1 PMT2 = GaAsP photomulti Chroma); (607/45m-2P, filter bandpass Chroma); (HQ575dcxr, 575LP F2 = = 4 525/70 Dichroic nm mm; bandpass 180 filter length (525/70m-2P, Chroma); nm ORCA-Flash4.0 Tube mm; 75 = focal length lens 675/67 = focal F3 lens = Scan (Hamamatsu); = = camera 607/45 F1 sCMOS nm follows: (Semrock); as filter are bandpass detectors and lenses filters, additional The removed. is Chroma) T700lpxxr-xxt, long-pass, metal-oxide nm 3 (700 Dichroic when only camera (sCMOS) semiconductor complementary in scientific beamlets the SLM on the mode image widefield to used be can Chroma) 660LP, Technology). An optional beam-combining dichroic (Dichroic 2, 660 nm long-pass, The GM2. T1030SP,1, (Dichroic Chroma onto short-pass nm a 1,030 is dichroic relayed Corp.) Bruker by Technology, integrated (Cambridge (RSM) mirror galvanometer scanning resonant a by axis fast the along scanned be also could and Corp.) Bruker by Technology, integrated Cambridge mm, 6 (GM2, path mirrors galvanometer of imaging pair second a The with scanned lens. was first the of positioned focus the foil) of with location the (coverslip at (ZB) block order zero a with f doublet, achromatic inch 1 (L3, telescope a via Technologies)) dual beam Ultimate microscope by Bruker Corp. (formerly Prairie into galvanometer integrated Technology, Cambridge mm, 6 (GM1, or 3 pair, mirror galvanometers photostimulation the to relayed was SLM The Thorlabs). (WPH10M-1064, (HWP) plate maxi half-wave a with SLM polarization the from and efficiency area diffraction for mized active SLM the fill to Thorlabs) ( (L1 telescope beam-expanding a using resized Associates), (Vincent to refer (PC (acronyms cell Pockels a by controlled was power The relative sample. the moves to telescope, expansion beam and SLM microscope, entire including the that difference significant the with but scope using a lightpath micro similar to that the for to an coupled was Systems) Nonlinear Optics/Boulder active area, 512 × 512 pixels, optimized for 1,064 nm, Meadowlark nm. at 765 imaged was mCherry and imaging, calcium during nm of 920 wavelength an excitation with imaged was GCaMP6s experiments. all for used was (Nikon) objective (Bruker Corporation, formerly Prairie Technologies). moving a with scanning raster A galvanometer 16×/0.8-NA n displayed on the SLM that would result in the desired pattern with = 400 or 250 mm; L4, 2 inch achromatic doublet, doublet, achromatic inch 2 L4, mm; 250 or 400 = a h ectto suc fr h pootmlto pt ws a was path photostimulation the for source excitation The (Meadowlark 1.3 Version BNS_DVI running computer A mm 7.68 × (7.68 (SLM) modulator light spatial reflective A ture methods f 5 m) n L ( L2 and mm) 50 = 5 0 was used to calculate phase masks to be be to masks phase calculate to used was Fig. 1 Fig. b Fig. 1 Fig. f ) = 200 mm), plano-convex lenses, lenses, plano-convex mm), 200 = ) (Conoptics Ltd.) and shutter (S) (S) shutter and Ltd.) (Conoptics ) b ). in

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© 2015 Nature America, Inc. All rights reserved. 44. All MATLAB. or Prism mean are values with performed were tests statistical time step deconvolution non-negative fast algorithm, for a which the only required parameter with was the imaging performed infer was Spike ence channels. C1V1 and GCaMP both using hand by memory reducing and selected were somata neuronal defining Contours speed requirements. increasing matrix- a thus with DFT, upsampling multiply selective via images two between algorithm doi:

10.1038/nmeth.3217 J. Virol. J. viruses. adeno-associated ofretargeting and interbreedingmultispecies Grimm, D. Grimm, 46

5 82 . . Analyses were performed with MATLAB and ImageJ; 1 . This algorithm computes the cross-correlation cross-correlation the computes algorithm This . et al. et , 5887–5911 (2008). 5887–5911 , ±

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image registration algorithms.registration image Optik pictures. plane diffraction and image from phase the of determination computation velocity angular for circuit neural a and neocortex in inhibition of architecture microcircuit canonical nonspecific, dense, machine: processing (TPTP) patching targeted neurons unlabeled of electroporation single-cell and recordings patch-clamp frompopulation calcium imaging. Nature signals.intrinsic of imaging optical by revealed cortex of architecture Guizar-Sicairos, M., Thurman, S.T. & Fienup, J.R. Efficient subpixel Efficient J.R. Fienup, & S.T.Thurman, M., Guizar-Sicairos, the for algorithm practicalW.O. A Saxton, R.W.& Gerchberg, Packer,A.M. Margrie,T.W.Two-photon& M. P.,Brecht, W.,Osten, Denk, S., Komai, TargetedHausser,M. W.& Denk, M., Kano, B., Judkewitz, K., Kitamura, Vogelstein,J.T. FunctionalT.N. Wiesel, & C.D. Gilbert, R.D., Frostig, E., Lieke, A., Grinvald, (

Stuttg. 324 in vivo in , 361–364 (1986). 361–364 , . PhD thesis, Columbia University (2011). University Columbia thesis, PhD . Understanding the nervous system as an information an as system nervous the Understanding ) . 35 et al.et Nat. Methods Nat. , 237–250 (1972). 237–250 , Fastnonnegative deconvolution forspiketrain inference in vivo in

5 Opt. Lett. Opt. , 61–67 (2008). 61–67 , . J. Neurophysiol.J. Nat. Protoc. Nat.

33 , 156–158 (2008). 156–158 ,

, 647–652 (2006). 647–652 , 104 , 3691–3704, (2010). n a ture methods

CORRIGENDA Corrigendum: Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo

Adam M Packer, Lloyd E Russell, Henry W P Dalgleish & Michael Häusser Nat. Methods 12, 140–146 (2015); published online 22 December 2014; corrected after print 6 February 2015

In the version of this article initially published, the size of the scale bar reported in the legend of Figure 3a was incorrect. The correct size is 100 μm, not 50 μm. In addition, the volume of injected virus in the Online Methods section “Titration of calcium indicator expression” had the incorrect unit. The correct volume is 100 nl, not 100 μl. The errors have been corrected in the HTML and PDF versions of the article. Nature America, Inc. All rights reserved. America, Inc. © 201 5 Nature npg

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