Millisecond-timescale,genetically targeted optical control of neural activity
Edward S Boyden1,Feng Zhang1,Ernst Bamberg2,3,Georg Nagel2,5&Karl Deisseroth1,4
Temporally precise,noninvasive control of activity in well-
defined neuronal populations is a long-sought goal of systems neuroscience.We adapted for this purpose the naturally occurring algal protein Channelrhodopsin-2,a rapidly gated
light-sensitive cation channel,by using lentiviral gene delivery in combination with high-speed optical switching to photostimulate mammalian neurons.We demonstrate reliable,millisecond-timescale control of neuronal spiking,as well as control of excitatory and inhibitory synaptic transmission.This technology allows the use of light to alter neural processing at the level of single spikes and synaptic events,yielding a widely applicable tool for neuroscientists and biomedical engineers.
Neural computation depends on the temporally diverse spiking pat-terns of different classes of neurons that express unique genetic markers and demonstrate heterogeneous wiring properties within neural net-works.Although direct electrical stimulation and recording of neurons in intact brain tissue have provided many insights into the function of circuit subfields(for example,see refs.1–3),ne
urons belonging to a specific class are often sparsely embedded within tissue,posing funda-mental challenges for resolving the role of particular neuron types in information processing.A high–temporal resolution,noninvasive, genetically based method to control neural activity would enable elucidation of the temporal activity patterns in specific neurons that drive circuit dynamics,plasticity and behavior.
Despite substantial progress made in the analysis of neural network geometry by means of non–cell-type-specific techniques like glutamate uncaging(for example,see refs.4–7),no tool has yet been invented with the requisite spatiotemporal resolution to probe neural coding at the resolution of single spikes.Furthermore,previous genetically encoded optical methods,although elegant8–10,11,have allowed control of neuronal activity over timescales of seconds to minutes,perhaps owing to their mechanisms for effecting depolarization.Kinetics roughly a thousand times faster would enable remote control
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Figure1ChR2enables light-driven neuron
spiking.(a)Hippocampal neurons expressing
ChR2-YFP(scale bar30m m).(b)Left,inward
current in voltage-clamped neuron evoked by1s
of GFP-wavelength light(indicated by black bar);
right,population data(right;mean±s.d.plotted
throughout;n¼18).Inset,expanded initial
phase of the current transient.(c)Ten overlaid
current traces recorded from a hippocampal
neuron illuminated with pairs of0.5-s light pulses
(indicated by gray bars),separated by intervals
varying from1to10s.(d)Voltage traces showing
membrane depolarization and spikes in a current-
clamped hippocampal neuron(left)evoked by
1-s periods of light(gray bar).Right,properties
of thefirst spike elicited(n¼10):latency to
spike threshold,latency to spike peak,and
jitter of spike time.(e)Voltage traces in
response to brief light pulse series,with light
pulses(gray bars)lasting5ms(top),10ms
(middle)or15ms(bottom).
Published online14August2005;doi:10.1038/nn1525
1Department of Bioengineering,Stanford University,318Campus Drive West,Stanford,California94305,USA.2Max-Planck-Institute of Biophysics,Department of Biophysical Chemistry,Max-von-Laue-Str.3,D-60438Frankfurt am Main,Germany.3Department of Biochemistry,Chemistry and Pharmaceutics,University of Frankfurt, Marie-Curie-Str.9,60439Frankfurt
am Main,Germany.4Department of Psychiatry and Behavioral Sciences,Stanford School of Medicine,401Quarry Road,Stanford, California94305,USA.5Present address:Julius-von-Sachs-Institut,University of Wu¨rzburg,Julius-von-Sachs-Platz2–4,D-97082Wu¨rzburg,Germany.Correspondence should be addressed to K.D.(deissero@stanford.edu).
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individual spikes or synaptic events.We have therefore devised a new strategy using a single-component ion channel with submillisecond opening kinetics,to enable genetically targeted photostimulation with fine temporal resolution.
Two rhodopsins in the unicellular green alga Chlamydomonas rein-hardtii were recently identified independently by three groups 12–15.One of them is a light-gated proton channel (Channelrhodopsin-1;ref.13),whereas the other,Channelrhododopsin-2(ChR2),is a light-gated cation channel 12.The N-terminal 315amino acids of ChR2are homologous to the seven-transmembrane structure of many microbial-type rhodopsins;they compose a channel with light-gated conductance (as proposed earlier 16).Inward currents in ChR2-expressing cells could be evoked within 50m s after a flash of blue light in the presence of all-trans retinal 12,suggesting the possibility that ultrafast neuronal stimulation might be possible with equipment commonly used for visualizing green fluorescent protein (GFP).ChR2therefore combines some of the best features of previous photostimulation methods,including the speed of a monolithic ion channel 9,and the efficacy of natural light-transduction machinery 11.
We found that ChR2could be expressed stably and safely in mammalian neurons and could drive neuronal depolarization.When activated with a series of brief pulses of light,ChR2could reliably medi
ate defined trains of spikes or synaptic events with millisecond-timescale temporal resolution.This technology thus brings optical control to the temporal regime occupied by the fundamental building blocks of neural computation.
RESULTS
Rapid kinetics of ChR2enables driving of single spikes
T o obtain stable and reliable ChR2expression for coupling light to neuronal depolarization,we constructed lentiviruses containing a ChR2-yellow fluorescent protein (YFP)fusion protein for genetic modification of neurons.Infection of cultured rat CA3/CA1neurons led to membrane-localized expression of ChR2for weeks after infection (Fig.1a ).Illumination of ChR2-positive neurons with blue light (bandwidth 450–490nm via Chroma excitation filter HQ470/40Â;300-W xenon lamp)induced rapid depolarizing currents,which
reached a maximal rise rate of 160±111pA/ms within 2.3±1.1ms
after light pulse onset (mean ±ported throughout paper,n ¼18;Fig.1b ,left).Mean whole-cell inward currents were large:496pA ±336pA at peak and 193pA ±177pA at steady-state (Fig.1b ,right).Light-evoked responses were never seen in cells expressing YFP alone (data not shown).Consistent with the known excitation spectrum of ChR2(ref.12),illumination of ChR2-expressing neurons with YFP-spectrum light in the bandwidth 490–510nm (300-W xenon lamp filtered with Chroma excitation filter HQ500/20Â)resulted in currents that were smaller (by 42%±20%)than those evoked with the GFP filters.Despite the inactivation of ChR2with sustained light exposure (Fig.1b and ref.12),we observed rapid recovery of peak ChR2photocurrents in neurons (Fig.1c ;recovery t ¼5.1±1.4s;recovery trajectory fit with Levenberg-Marquardt algorithm;n ¼9).This rapid recovery is consistent with the well-known stability of the Schiff base (the lysine in transmembrane helix seven,which binds retinal)in microbial-type rhodopsins,and the ability of retinal to re-isomerize to the all-trans ground state in a dark reaction,without the need for other enzymes.Light-evoked current amplitudes remained unchanged in patch-clamped neurons during 1h of pulsed light exposure (data not shown).Thus ChR2was able to sustainably mediate large-amplitude photocurrents with rapid activation kinetics.
We next examined whether ChR2could drive spiking of neurons held in current-clamp mode,with the
same steady illumination protocol we used for eliciting ChR2-induced currents (Fig.1d ,left).Early in an epoch of steady illumination,single neuronal spikes were rapidly and reliably elicited (8.0±1.9ms latency to spike peak,n ¼10;Fig.1d ,right),consistent with the fast rise times of ChR2currents described above.However,any subsequent spikes elicited during steady illumination were poorly timed (Fig.1d ,left).Thus,steady illumina-tion is not adequate for controlling the timing of ongoing spikes with ChR2,despite the reliability of the first spike.Earlier patch-clamp studies using somatic current injection showed that spike times were more reliable during periods of rapidly rising membrane potential than during periods of steady high-magnitude current injection 17.This is consistent with our finding that steady illumination evoked a single reliably timed spike,followed by irregular spiking.
In searching for a strategy to elicit precisely timed series of spikes with ChR2,we noted that the single spike reliably elicited by steady
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Figure 2Realistic spike trains driven by series of light pulses.(a )Voltage traces showing spikes in a single current-clamped hippocampal neuron,in response to three deliveries of a Poisson train (with mean interval l ¼100ms)of light pulses (gray dashes).(b )Trial-to-trial repeatability of light-evoked spike trains,as measured by comparing the presence or absence of a spike in two repeated trials of a Poisson train (either l ¼100ms or l ¼200ms)delivered to the same neuron (n ¼7neurons).(c )Trial-to-trial jitter of spikes,across repeated light-evoked spike trains.(d )Percent fidelity of spike
transmission throughout entire 8-s light pulse series.(e )Latency of spikes throughout each light pulse series (i)and jitter of spike times throughout train (ii).(f )Voltage traces showing spikes in three different hippocampal neurons,in response to the same temporally patterned light stimulus (gray dashes)used in a .(g )Histogram showing how many of the seven neurons spiked in response to each light pulse in the Poisson train.(h )Neuron-to-neuron jitter of spikes evoked by light stimulation.
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illumination had extremely low temporal jitter from trial to trial,as reflected by the small standard deviation of the spike times across trials (Fig.1d ,right;0.5±0.3ms,average of n ¼10neurons).This observation led us to devise a pulsed-light strategy that would take advantage of the low jitter of the single reliable spike evoked at light pulse onset.In order for this to work,the conductance and kinetics of ChR2would have to permit peak currents of sufficient amplitude to reach spike threshold,during a light pulse of duration shorter than the desired interspike interval.We found that multiple pulses of light with interspersed periods of darkness could elicit trains of multiple spikes (Fig.1e ;shown for a 25-Hz series of four pulses).Longer light pulses evoked single spikes with greater probability than short light pulses (Fig.1e ).In the experiments described here,we used light pulse durations of 5,10or 15ms.Thirteen high-expressing neurons fired reliable spikes,and five low-expressing neurons could reliably be depolarized to subthreshold levels.The ability to easily alter light pulse duration suggests that a straightforward method for eliciting spikes,even in multiple neurons having different ChR2current densities,would involve titrating the light pulse duration until single spikes were reliably obtained in all the neurons being illuminated.Modulation of light intensity would also allow for this kind of control.
Precise spike trains elicited by series of light pulses
The precise control described above raised the prospect of generating arbitrary spike patterns,even
mimicking natural neural activity.T o test this possibility,we generated series of light pulses,the timings of which were selected according to a Poisson distribution,commonly used to model natural spiking.A single hippocampal neuron could fire repeatable spike trains in response to multiple deliveries of the same Poisson-distributed series of light pulses (Fig.2a ;response to a repeated 59-pulse-long series of light pulses,with each pulse of 10-ms duration,and with mean interpulse interval of l ¼100ms).These optically driven spike trains were very consistent across repeated deliveries of the same series of light pulses:on average,495%of the light pulses in a series elicited spikes during one trial if and only if they elicited spikes on a second trial,for both the l ¼100ms series (Fig.2a )and a second series (mean interval l ¼200ms)comprising 46spikes (Fig.2b ;n ¼7neurons).We increased light pulse duration until reliable spiking was obtained:we used trains of 10-ms light pulses for four of the seven neurons and trains of 15-ms light pulses for the other three (for the analyses of Fig.2,all data were pooled).The trial-to-trial jitter for each individual spike was very small across repeated deliveries of the same Poisson series of light pulses (on average,2.3±1.4ms and 1.0±0.5ms for l ¼100ms and l ¼200ms,respectively;Fig.2c ).Throughout a series of pulses,the efficacy of eliciting spikes throughout the train was maintained (76%and 85%percent of light pulses successfully evoked spikes,respectively;Fig.2d ),with small latencies (Fig.2e ).As another indication of how precisely spikes can be elicited throughout an entire series of pulses,we measured the standard deviation of th
e latencies of each spike across all the spikes in the train.This ‘throughout-train’spike jitter was quite small (3.9±1.4ms and 3.3±1.2ms;Fig.2e ),despite presumptive channel inactivation during the delivery of a series of pulses.Hence,pulsed optical activation of ChR2elicits precise,repeatable spike trains in a single neuron,over time.
Even in different neurons,the same precise spike train could be elicited by a particular series of light pulses (shown for three hippocampal neurons in Fig.2f ).Although the large hetero-geneity of different neurons—for example,in their membrane capaci-tance (68.8±22.6pF)and resistance (178.8±94.8M O )—might be expected to introduce significant variability in their electrical responses to photostimulation,the strong nonlinearity of light-spike coupling dominated this variability.Indeed,different neurons responded in similar ways to a given light pulse series,with 80–90%of the light pulses in a series eliciting spikes in at least half the neurons examined (Fig.2g ).T o quantitatively compare the reliability of spike elicitation in different neurons,for each pulse,we calculated the standard deviation of spike latencies (jitter)across all the neurons.Remarkably,this across-neuron jitter (3.4±1.0ms and 3.4±1.2ms for the pulses in the l ¼100ms and l ¼200ms trains,respectively;Fig.2h )was similar to the within-neuron jitter measured throughout the light pulse series (Fig.2e ).Thus,heterogeneous populations of neurons can be controlled in concert,with practically the same precision observed for the control of single neurons over time.
Having established the ability of ChR2to drive sustained naturalistic trains of spikes,we next probed the frequency response of light-spike coupling.ChR2enabled driving of spike trains from 5to 30Hz (Fig.3a ;here tested with series of twenty 10-ms light pulses).It was easier to evoke more spikes at lower frequencies than at higher frequencies (Fig.3b ;n ¼13neurons).Light pulses delivered at 5or 10Hz could elicit long spike trains (Fig.3b ),with spike probability dropping off at higher frequencies of stimulation.For these experiments,the light pulses used were 5ms (n ¼1),10ms (n ¼9)or 15ms (n ¼3)in duration (data from all 13cells were pooled for the population analyses of Fig.3).As expected from the observation that light pulses generally elicited single spikes (Figs.1d and 2),almost no extraneous spikes occurred during the delivery of trains of light pulses (Fig.3c ).Even at higher frequencies,the throughout-train temporal jitter of spike timing remained very low (typically o 5ms;Fig.3d )and the latency to spike remained constant across frequencies (B 10ms throughout;Fig.3e ).Thus ChR2can mediate spiking across a physiologically relevant range of firing frequencies.
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Figure 3Frequency dependence of coupling between light input and spike output.(a )Voltage traces showing spikes in a current-clamped hippocampal neuron evoked by 5-,10-,20-or 30-Hz trains of light pulses (gray dashes).
(b )Population data showing the number of spikes (out of 20possible)evoked in current-clamped hippocampal neurons.(c )Number of extraneous spikes evoked by the trains of light pulses,for the experiment described in b .(d )Jitter of spike times throughout the train of light pulses for the
experiment described in b .(e )Latency to spike peak throughout the light pulse train for the experiment described in b .
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Remote activation of subthreshold and synaptic responses
For many cellular and systems neuroscience processes (and for nonspiking neurons in species like Caenorhabditis elegans )subthres-hold depolarizations convey physiologically significant information.For example,subthreshold depolarizations are potent for acti-vating synapse-to-nucleus signaling 18,and the relative timing of subthreshold and suprathreshold depolarizations can determine the
direction of synaptic plasticity 19.But compared with driving spiking,it is in principle more difficult to drive reliable and precisely sized subthreshold depolarizations.The sharp threshold for action potential production facilitates reliable ChR2-induced spiking,and the all-or-none dynamics of spiking produces virtually identical waveforms from spike to spike (as seen throughout Figs.1–3),even in the
presence of significant neuron-to-neuron variability in electrical properties.In contrast,subthreshold depolarizations,which operate in a more linear regime of membrane voltage,will lack these intrinsic normalizing mechanisms.Nevertheless,subthreshold depolarizations evoked by repeated light pulses were reliably evoked over a range of frequencies (Fig.4a ),with spaced repeated depolarizations resulting in a coefficient
of variation of 0.06±0.03(Fig.4b ;n ¼5).Thus,ChR2can be used to drive subthreshold depolarizations of reliable amplitude.Finally,synaptic transmission was also easily controlled with ChR2.
Indeed,both excitatory (Fig.4c )and inhibitory (Fig.4d )synaptic
events could be evoked in ChR2-negative neurons receiving synaptic input from ChR2-expressing neurons.
Expression of ChR2has minimal side effects
We conducted extensive controls to test whether simply expressing ChR2would alter the electrical properties or survival of neurons.Lentiviral expression of ChR2for at least 1week did not alter neuronal membrane resistance (212±115M O for ChR2+cells versus
239.3±113M O for ChR2Àcells;Fig.5a ;P 40.45;n ¼18each)or resting potential
(À60.6±9.0mV for ChR2+cells versus À59.4
±6.0mV for ChR2Àcells;Fig.5b ;P 40.60),when measured in the absence of light.This suggests that in neurons,ChR2has little basal
electrical activity or passive current-shunting ability.It also suggests that expression of ChR2did not compromise cell health,as indicated by electrical measurement of mem-brane integrity.As an independent measure of
cell health,we stained live neurons with the
membrane-impermeant DNA-binding dye propidium iodide.ChR2expression did not affect the percentage of neurons that took up propidium iodide (1/56ChR2+neurons ver-sus 1/49ChR2Àneurons;P 40.9by w 2test).
Neither did we see pyknotic nuclei,indicative of apoptotic degeneration,in cells expressing
ChR2(data not shown).We also checked for
alterations in the dynamic electrical properties
of neurons.In darkness,there was no differ-ence in the voltage change resulting from 100pA of injected current,in either the hyperpolarizing (À22.6±8.9mV for ChR2+neurons versus À24.5±8.7mV for ChR2Àneurons;P 40.50)or depolarizing (+18.9±
4.4mV for ChR2+versus 18.7±
5.2mV for ChR2Àneurons;P 40.90)directions.Nor was there any difference in the number of
spikes evoked by a 0.5-s current injection of +300pA (6.6±4.8for ChR2+neurons versus 5.8±3.5for ChR2–neurons;Fig.5c ;P 40.55).Thus,ChR2does not significantly jeopardize cell health or basal electrical properties of the expressing neuron.We also measured the electrical properties described above,24h after
exposing ChR2+neurons to a typical light pulse protocol (1s of 20-Hz
15-ms light flashes,once per minute,for 10min).Neurons expressing ChR2and exposed to light had basal electrical properties similar to non-flashed ChR2+neurons:cells had normal membrane resistance (178±81M O ;Fig.5a ;P 40.35;n ¼12and resting potential (À59.7±7.0mV;Fig.5b ;P 40.75).
Exposure to light also did not predispose neurons to cell death,as measured by live-cell propidium iodide uptake (2/75ChR2+neurons versus 3/70ChR2-neurons;P 40.55by w 2test).Finally,neurons expressing ChR2and exposed to light also had normal spike counts elicited from somatic current injection (6.1±3.9;Fig.5c ;P 40.75).Thus,membrane integrity,cell health and electrical proper-ties were normal in neurons expressing ChR2and exposed to light.500 ms 40 mV
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c transmission evoke
d via ChR2.(a )Voltag
e traces showing trains
of subthreshold depolarizations in a current-clamped hippocampal neuron in response to trains of light pulses (gray dashes).(b )Repeated light pulses induced reliable depolarizations.(c )Excitatory synaptic transmission driven by light pulses.The selective glutamatergic transmission blocker NBQX abolished
these synaptic responses (right).(d )Inhibitory synaptic transmission driven by light pulses.The
selective GABAergic transmission blocker gabazine abolished these synaptic responses (right).
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a b Figure 5Basal and dynamic electrical properties of neurons expressing
ChR2.(a )Membrane resistance of neurons expressing ChR2(black;n ¼18),not expressing ChR2(white;n ¼18)or expressing ChR2and measured 24h after exposure to a typical light-pulse protocol (gray;n ¼12).(b )Membrane resting potential of the same neurons described in a .(c )Number of spikes evoked by a 300-pA depolarization of the same neurons described in a .
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DISCUSSION
Combining the best aspects of earlier approaches that use light to drive neural circuitry,the technology described here demonstrates voltage control significantly faster than previous genetically encoded photo-stimulation methods 8–11.Notably,the ChR2method does not rely on synthetic chemical substrates or genetic orthogonality of the transgene and the host organism.Although the ChR2molecule does require the cofactor all-trans retinal for light transduction 12,no all-trans retinal was added either to the culture medium or recording solution for any of the experiments described here.Background levels of retinal may be sufficient in many cases;moreover,the commonly used culture medium supplement B-27used here (see Methods)includes retinyl acetate,and additional supplementation with all-trans retinal or its precursors may assist in the application of ChR2to the study of neural circuits in various tissue environments.
We used pulsed light delivery to take full advantage of the fast kinetics and high conductance of ChR2.This strategy was made possible with fast optical switches,but other increasingly common equipment,such as pulsed lasers,would also suffice.Unlike electrical stimulation,glutamate uncaging 20–22and high-powered laser excitation methods 23,ChR2can be genetically targeted to allow probing of specific neuron subclasses within a heterogeneous neural circuit,avoiding fibers of passage and the simultaneous stimulation of multiple cell types.
Because ChR2is encoded by a single open reading frame of only 315amino acids,it is feasible to express ChR2in specific subpopulations of neurons in the nervous system through genetic methods including lentiviral vectors (as we have done)and in transgenic mice,thus permitting the study of the function of individual types of neurons in intact neural circuits and even in vivo .Cell-specific promoters will allow targeting of ChR2to various well-defined neuronal subtypes,which will permit future exploration of their causal function in driving downstream neural activity (measured using electrophysiological and optical techniques)and animal behavior.ChR2also could be used to resolve functional connectivity of particular neurons or neuron classes in intact circuits in response to naturalistic spike trains (Fig.2)or rhythmic activity (Fig.3),for example,by using acute slice prepara-tions after intracranial viral injections.
Recent papers have explored the topics of static and dynamic microcircuit connectivity using calcium imaging of spontaneous activity 24,multi-neuron patch-clamping 25,26and glutamate uncaging 6.These studies have reported surprisingly refined and precise connec-tions between neurons.However,finer-scale dissection of micro-circuits,at the level of molecularly defined neuron classes (such as cannabinoid receptor–expressing cortical neurons,parvalbumin-positive interneurons or cholinergic modulatory neurons)would be greatly facilitated through use of a genetica
lly targeted,temporally precise tool like ChR2.This holds true also for recent microstimulation experiments that have demonstrated profound influences of a cluster of neurons in controlling attention,decision making or action 2,3,27,28.Understanding precisely which cell types contribute to these functions could provide great insight into how they are computed at the circuit level.Because the light power required for ChR2activation (8–12mW/mm 2)is fairly low,it is possible that ChR2will be an effective tool for in vivo studies of circuit maps and behavior,even in mammals.Finally,the efficacious and safe transduction of light with a single natural biological component also could serve biotechnological needs,in high-throughput studies of activity-dependent signal transduction and gene expression programs,for example,in guiding stem cell differentiation 29and screening for drugs that modulate neuronal responses to depolar-ization.Thus,the technology described here may fulfill the long-sought goal of a method for noninvasive,genetically targeted,temporally
precise control of neuronal activity,with potential applications ranging from neuroscience to biomedical engineering.METHODS
Plasmid constructs.The ChR2-YFP gene was constructed by in-frame fusing EYFP (Clontech)to the C terminus of the first 315amino acid residues of ChR2(GenBank accession number AF461397)via a Not I site.The lentiviral vector pLECYT was generated by PCR amplification of ChR2-YFP with pri-m
ers 5¢-GGCAGCGCTGCCACCATGGATTATGGAGGCGCCCTGAGT-3¢and 5¢-GGCACTAGTCTATTACTTGTACAGCTCGTC-3¢and ligation into pLET (gift from E.Wexler and T.Palmer,Stanford University)via the Afe I and Spe I restriction sites.The plasmid was amplified and then purified using MaxiPrep kits (Qiagen).
Viral production.VSVg pseudotyped lentiviruses were produced by triple transfection of 293FT cells (Invitrogen)with pLECYT,pMD.G and pCMV D R8.7(gifts from E.Wexler and T.Palmer)using Lipofectamine 2000.The lentiviral production protocol is the same as previously described 30except for the use of Lipofectamine 2000instead of calcium phosphate precipitation.After harvest,viruses were concentrated by centrifuging in a SW28rotor (Beckman Coulter)at 20,000rpm for 2h at 41C.The concentrated viral titer was determined by FACS to be between 5Â108and 1Â109infectious units (IU)per ml.
Hippocampal cell culture.Hippocampi of postnatal day 0(P0)Sprague-Dawley rats (Charles River)were removed and treated with papain (20U/ml)for 45min at 371C.The digestion was stopped with 10ml of MEM/Earle salts without L -glutamine along with 20mM glucose,Serum Extender (1:1000),and 10%heat-inactivated fetal bovine serum containing 25mg of bovine serum albumin (BSA)and 25mg of trypsin inhibitor.The tissue was triturated in a small volume of this solution with a
fire-polished Pasteur pipette,and B 100,000cells in 1ml plated per coverslip in 24-well plates.Glass coverslips (prewashed overnight in HCl followed by several 100%ethanol washes and flame sterilization)were coated overnight at 371C with 1:50Matrigel (Collaborative Biomedical).Cells were plated in culture medium:Neurobasal containing 2ÂB-27(Life T echnologies)and 2mM Glutamax-I (Life T echnol-ogies).The culture medium supplement B-27contains retinyl acetate,but no B-27was present during recording and no all-trans retinal was added to the culture medium or recording medium for any of the experiments described.One-half of the medium was replaced with culture medium the next day,giving a final serum concentration of 1.75%.
Viral infection.Hippocampal cultures were infected on day 7in vitro (DIV 7)using fivefold serial dilutions of lentivirus (B 1Â106IU/ml).Viral dilutions were added to hippocampal cultures seeded on coverslips in 24-well plates and then incubated at 371C for 7d before experimentation.
Confocal imaging.Images were acquired on a Leica TCS-SP2LSM confocal microscope using a 63Âwater-immersion lens.Cells expressing ChR2-YFP were imaged live using YFP microscope settings,in Tyrode solution containing (in mM)NaCl 125,KCl 2,CaCl 23,MgCl 21,glucose 30and HEPES 25(pH 7.3with NaOH).
Propidium iodide (Molecular Probes)staining was carried out on live cells by adding 5m g/ml propidium iodide to the culture medium for 5min at 371C,washing twice with Tyrode solution and then immediately counting the number of ChR2+and ChR2Àcells that took up propidium iodide.Coverslips were then fixed for 5min in PBS +4%paraformaldehyde,permeabilized for 2min with 0.1%Triton X-100and then immersed for 5min in PBS containing 5m g/ml propidium iodide for detection of pyknotic nuclei.At least eight fields of view were examined per coverslip.
Electrophysiology and optical methods.Cultured hippocampal neurons were recorded at approximately DIV 14(7d post-infection).Neurons were recorded by means of whole-cell patch clamp,using Axon Multiclamp 700B (Axon Instruments)amplifiers on an Olympus IX71inverted scope equipped with a 20Âobjective lens.Borosilicate glass (Warner)pipette resistances were B 4M O ,range 3–8M O .Access resistance was 10–30M O and was monitored throughout the recording.Intracellular solution consisted of (in mM)97
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