Optogenetic control of striatal dopamine release in rats

Optogenetic control of striatal dopamine release in rats

Caroline E Bass1, Valentina P Grinevich1, Zachary B Vance2, Ryan P Sullivan2, Keith DBonin2, and Evgeny A Budygin1,*

1 Medical Center Boulevard, Department of Physiology and Pharmacology, Wake ForestUniversity School of Medicine, Winston-Salem, North Carolina 27157, USA2 1834 Wake Forest Road, Olin Physical Laboratory, Department of Physics, Wake ForestUniversity, Winston-Salem, North Carolina 27109

AbstractOptogenetic control over neuronal firing has become an increasingly elegant method to dissect themicrocircuitry of mammalian brains. To date, examination of these manipulations onneurotransmitter release has been minimal. Here we present the first in-depth analysis ofoptogenetic stimulation on dopamine neurotransmission in the dorsal striatum of urethane-anesthetized rats. By combining the tight spatial and temporal resolution of both optogenetics andfast-scan cyclic voltammetry we have determined the parameters necessary to control phasicdopamine release in the dorsal striatum of rats in vivo. The kinetics of optically induced dopaminerelease mirror established models of electrically evoked release, indicating that potential artifactsof electrical stimulation on ion channels and the dopamine transporter are negligible. Furthermorea lack of change in extracellular pH indicates that optical stimulation does not alter blood flow.Optical control over dopamine release is highly reproducible and flexible. We are able torepeatedly evoke concentrations of dopamine release as small as a single dopamine transient (50nM). A U-shaped frequency response curve exists with maximal stimulation inducing dopamineeffluxes exceeding 500 nM. Taken together, these results have obvious implications forunderstanding the neurobiological basis of dopaminergic-based disorders and provide theframework to effectively manipulate dopamine patterns.

KeywordsVoltammetry; dopamine release and uptake; channelrhodopsin

The mesolimbic and nigrostriatal pathways originate in the ventral tegmental area (VTA)and substantia nigra (SN) and project to the ventral and dorsal striatum, respectively.Imbalances in dopamine neurotransmission (deficient or excessive) are thought to contributeto a number of neurogenerative and psychiatric diseases including Parkinson’s disease,schizophrenia, depression, and addiction. The effectiveness of many pharmacologicalinterventions in treating these disorders results from their ability to modulate the release,uptake, synthesis and degradation of dopamine in specific subregions of the mesostriatalpathways (Olanow 2004; IsHak et al. 2009; Maggio et al. 2009).

Attempts to selectively modulate these circuits using deep brain stimulation (DBS) havebeen made with some success for the treatment of Parkinson’s Disease (PD) symptoms,depression and obsessive compulsive disorder to name a few. The advantage of this

*Corresponding Author: Evgeny A. Budygin, Department of Physiology and Pharmacology, Wake Forest University School ofMedicine, Medical Center Blvd., Winston-Salem, NC 27157, Tel: (336) 716-8530, Fax: (336) 716-8501, [email protected].

NIH Public AccessAuthor ManuscriptJ Neurochem. Author manuscript; available in PMC 2011 September 1.

Published in final edited form as:J Neurochem. 2010 September 1; 114(5): 1344–1352. doi:10.1111/j.1471-4159.2010.06850.x.

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approach is that the region of interest can be spatially isolated by placement of the electrode.However, new genetic approaches promise to expand the utility of DBS by enhancing thespatial resolution of the effect and genetically isolating the neuronal subtypes affected by thetreatment. The most promising approach thus far has been optogenetics, a technique inwhich light induces activation via a light-sensitive ion channel (e.g. channelrhodopsin-2,ChR2), which can be used to genetically isolate the effect to neuronal subtypes (Gunaydin etal.; Gradinaru et al. 2009; Sohal et al. 2009; Tsai et al. 2009).

While several groups have characterized the electrophysiological parameters surroundingoptical control over neurons (Gunaydin et al.; Boyden et al. 2005; Lin et al. 2009), to dateonly one study has actually confirmed neurotransmitter release from stimulated dopamineneurons (Tsai et al. 2009). Before this methodology is employed on a wider basis, severalfundamental issues must be addressed. First, since light diffusion is limited in brain tissue,does the depolarization of the subset of neurons within the relatively small opticalstimulation zone result in substantive neurotransmitter release at the level of the terminalfield? Secondly, the electrophysiological properties of optically stimulated ChR2 suggestthat there are limitations to this protein, including the induction of extra spikes and aninability to stimulate past the 40 Hz frequency range. Finally, while the effect can berestricted temporally, spatially, or to various neuronal subtypes this is most readily achievedusing mouse models. For example, Tsai et al used cre/lox recombination to restrict the effectto dopaminergic neurons by delivering a cre-sensitive ChR2 virus into the VTA of a tyrosinehydroxylase-cre mouse line. While mouse models can provide the necessary genetic controlover expression, they have limited viability in many complex behavioral studies(Oleson etal. 2009a). In order for this methodology to reach its full potential, it is essential to evaluatethe parameters of optical stimulation on dopamine release, including resultingconcentrations and reuptake dynamics, as well as determine the feasibility of applying thisapproach to existing rodent (particularly rat) models without the need to produce or obtaincostly transgenics.

In our study, we have produced the first in depth analysis of in vivo optically induceddopamine release in real time using fast-scan cyclic voltammetry (FSCV) in rats. We electedto use a generalized non-restricted promoter which will stimulate a variety of neuronalsubtypes so that our results can be more readily compared to electrical stimulation. We havecharacterized dopamine release and uptake changes following different light stimulationparameters. Our results indicate that optical control over dopamine release is highly flexible,reproducible and in some ways surpasses that elicited by electrical stimulation. Our findingsdemonstrate that optogenetics can be easily incorporated into an existing rodent model andprovides the basis for future studies using optogenetics to modulate dopamine release.

Materials and methodsAdeno-associated virus (AAV) packaging

The EF1α-ChR2-EYFP AAV plasmid was a kind gift from K. Deisseroth. AAV2 invertedterminal repeats flank the transgene cassette which consists of the EF1α promoter followedby a ChR2-EYFP fusion gene, woodchuck postregulatory element (WPRE) and humanGrowth Hormone polyA signal sequence, respectively. Packaging of the EF1α-ChR2-AAV10 was carried out according to a standard triple transfection protocol to create helpervirus-free pseudotyped AAV2/10 virus (Xiao et al. 1998). An AAV2/10 rep/cap plasmidprovided the AAV2 replicase and AAV10 capsid genes (Gao et al. 2002; De et al. 2006),while adenoviral helper functions were supplied by pHelper (Stratagene, La Jolla, CA).Briefly, AAV- 293 cells (Stratagene, La Jolla, CA) were transfected with 10 μg of pHelper,and 1.15 pmol each of AAV2/10 and AAV vector plasmids via calcium phosphate

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precipitation. The cells were harvested 72 hours later and the pellets resuspended in DMEM,freeze-thawed three times and centrifuged several times to produce a clarified viral lysate.

Stereotaxic virus injectionMale Sprague-Dawley rats (300-350 g; Charles River, Raleigh, NC) were anesthetized withketamine hydrochloride (100 mg/kg, i.p.) and xylazine hydrochloride (20 mg/kg, i.p.) andplaced in a stereotaxic frame. The scalp was shaved, swabbed with iodine and a centralincision made to expose the skull. Two small holes were drilled and 2 scull screws wereplaced in to secure a cement cap. A third hole was drilled above the right SN (from bregma:anterior-posterior, 5.6 mm; lateral, 2 mm) and 2 μl of EF1α-ChR2-AAV10 was slowlyinjected into SN (dorsal-ventral, 7.6 mm) over three minutes via a Hamilton syringe. Thetissue was allowed to rest for two minutes before it was slowly retracted. The exposed scullwas coated with dental cement secured by scull screws and upon drying the animals werereturned to their home cages for recovery. All protocols were approved by the InstitutionalCare and Use Committee at Wake Forest University School of Medicine. All experimentsconformed to international guidelines on the ethical use of animals.

FSCV experimentFor ChR2 stimulation, rats were anesthetized with urethane (1.5 g/kg, i.p.) and placed in astereotaxic frame. It should be noted that urethane anesthesia does not alter dopamine uptakedynamics, although it has been reported to diminish total DA release (Garris et al. 2003;Sabeti et al. 2003). The cement cap was removed and a hole was drilled above the striatum(from bregma: anterior-posterior, 1.3 mm; lateral, 2 mm). The hole that was prepared earlierabove the SN was carefully redrilled and cleaned. An Ag/AgCl reference electrode wasimplanted in the contralateral hemisphere and a carbon fiber electrode (~100 μm in length, 6μm in diameter) was positioned in the striatum (dorsal-ventral, 4.5 – 7.5 mm). An opticalfiber (200 μm in diameter), used for optically stimulating ChR2 expressing dopamineneurons (delivering light) was positioned at the SN (from bregma: anterior-posterior, 5.6mm; lateral, 2.0 mm dorsal-ventral, 7.6 mm). In a separate set of experiments, a bipolarstimulating electrode was lowered to the SN using the same coordinates as for the opticalfiber. The reference and carbon fiber electrodes were connected to a voltammetric amplifier(UNC Electronics Design Facility, Chapel Hill, NC) and voltammetric recordings weremade at the carbon fiber electrode every 100 ms by applying a triangular waveform (-0.4 to+1.3 V, 400 V/s). Data were digitized (National Instruments, Austin, TX) and stored on acomputer. Light evoked dopamine release was identified by the background-subtractedcyclic voltammogram, which was compared to voltammograms from detection of dopaminein vitro and after electrical stimulation. Carbon fiber microelectrodes were calibrated in vitrowith known concentrations of dopamine (2-5 μM). Calibrations were done in triplicate andthe average value for the current at the peak oxidation potential was used to normalize invivo signals to dopamine concentration. Dopamine uptake was determined from theclearance rate of dopamine and was assumed to follow Michaelis-Menten kinetics. Thechanges in dopamine during and after optical or electrical stimulation were fit using theequation:

where f is the stimulation frequency (Hz), [DA]p is the concentration of dopamine releasedper stimulus pulse, and Vmax is the maximal rate of dopamine uptake, which is proportionalto the number of available DAT proteins. The baseline value of Km was calculated to bebetween 0.16 -0.2 μM, a value determined in rat brain synaptosomes and commonly used inthe analysis of voltammetric data (Near et al. 1988; Garris and Rebec 2002). The derivative

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form of the above equation was used to simulate the dopamine response (Garris et al. 1997;Budygin et al. 1999; Wu et al. 2001; Garris and Rebec 2002; Garris et al. 2003; Oleson et al.2009a; Oleson et al. 2009b).

Optical deliveryThe optical setup consisted of a laser at wavelength 473 nm (Viasho) with a maximumoutput of 100 mW. The laser was fiber pigtailed into a glass fiber of core 200 μm (Thorlabs– BFL37200 – Custom) and 75 cm in length. The laser was modulated using the TTL inputcontrol port on the laser power supply via a USB Data Acquisition unit (NationalInstruments 6221-USB). The data acquisition unit was controlled by a desktop computerusing LabVIEW software (National Instruments). The software allowed us to control thefrequency of the square pulses, the total number of pulses in one data stream, and the widthof each pulse as a fraction of the period between pulses. The laser power output wasmeasured using a commercial power meter (Newport Model 1815C). We measured the laseroutput pulses that resulted from the electrical pulses that were used to drive the TTL input ofthe laser, and the laser pulses faithfully replicated the electrical pulses driving the TTL inputof the laser. To understand how the dopamine stimulation depended on pulse width, weselected three pulse widths: 4, 10 and 20 ms, a range that reflects common pulse widths forelectrical stimulation (4 ms) and optical stimulation (20 ms). For each of these pulse widths,we collected the dopamine response as a function of frequency. To study the effects offrequency on the dopamine response, we varied the duty cycle as frequency changed inorder to hold the pulse width at the fixed value for that set of experiments. Thus for a set ofexperiments or trials at one fixed pulse width (say 4 ms), we would vary the frequency andduty cycle, but keep the total time of the pulse train fixed at 1 or 0.5 second. Keeping thetime of the pulse train fixed meant that for each different frequency, we had to adjust thenumber of pulses being produced to match the frequency.

A single desktop computer controlled both the voltammetric electrodes (and amplifier) aswell as the USB Data Acquisition unit (National Instruments 6221-USB) that controlled thelaser. Two separate LabVIEW programs were used to achieve synchronization between thelaser pulse train and the recording of the voltammetric dopamine current by the electrode.One program controlled the voltammetric parameters and readings, while the otherLabVIEW program controlled the USB Data Acquisition unit. The USB Data Acquisitionunit, which has both inputs and outputs, interfaced to the laser. Synchronization of the laserto the voltammetric recording was achieved through an electronic controller instrument thatwas built by the Electronics Design Facility at the University of North Carolina at ChapelHill. The first LabVIEW program controlled the voltammetric electrode via this instrument.Once the instrument received a trigger signal from the LabVIEW program, it triggered theData Acquisition unit input to begin a sequence controlled in software by the secondLabVIEW program that then triggered the laser and sent the laser the appropriate set ofpulses (with a preselected width for each pulse, a preselected delay to the beginning of thepulse train, and a preselected number of pulses per triggering event).

Statistical analysisData were analyzed in GraphPad Prism (GraphPad Software, San Diego, CA). A t-test, one-way repeated measures and two-way ANOVAs with a Bonferroni post test were used todetermine statistical significance. The data are presented as mean ± SEM and the criterion ofsignificance was set at p < 0.05.

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ResultsOptical stimulation of SN induces dopamine release in the dorsal striatum

ChR2-AAV was injected into the substantia nigra and rats were assessed three weeks later.FSCV was used to monitor extracellular dopamine changes in the rat striatum followingexposure of the substantia nigra to blue light. Optical stimulation produced a rapid increasein the current amplitude, which was used to calculate the dopamine concentration based oncalibration of the electrode immediately after the experiment (Fig. 1A). The rising phase ofdopamine efflux was restricted in time with blue light stimulation, while the falling phasebegan immediately after stimulation stopped. Therefore, the peak amplitude ([DA]max) ofthe light-induced dopamine signal can be considered an index of the balance between releaseand uptake of this neurotransmitter. To remove the background and charging currents, thecyclic voltammograms were subtracted from the average of the first 10 scans. Subtractedvoltammograms were then encoded with the current in false color, which demonstrates all ofthe electrochemical changes that occur in the detection time frame (Fig. 1B).

During optical stimulation, an increase in current at 0.65 V and decrease at -0.2 V wereobserved, which correspond to the potential at which dopamine oxidizes and reduces,respectively. No other electrochemical activity changes were detected. This is in starkcontrast to electrically evoked dopamine release, which is often accompanied by pH changesindicated by peaks around -0.25 V and 0.20 V on the positive-going scan and at -0.10 V onthe negative-going scan. A marked blue trace representing a basic pH shift can often be seenafter the electrical stimulation (Heien et al. 2005). These changes easily contaminate themeasurements of nearby dopamine spikes, particularly during multiple stimulations. In thecase of optically induced dopamine release, even multiple stimulations can be performedwithout any interference from pH changes (Fig. 1, last panel). The background – subtractedcyclic voltammograms recorded at the end of the optical stimulations (Fig. 1C) additionallyconfirm that the signal measured is dopamine on the basis of comparison to voltammogramsof dopamine obtained in vitro.

Dopamine release was evoked using different stimulating parameters, including altering thenumber of pulses, frequency and single pulse duration (Fig. 1), which results in phasicpatterns of dopamine release. The peak amplitude ([DA]max) was varied in range from 20nM (near the detection limit of FSCV) to several μM depending on the parameters of opticalstimulation. Most importantly, evident dopamine release was clearly detected following asingle light pulse, which is rarely achieved with one pulse of electrical stimulation. It isimportant to note that the magnitudes and frequencies we were able to induce with light arecomparable to changes in dopamine release observed under different behavioral conditionsand drug treatments (Robinson et al. 2001;Phillips et al. 2003;Wightman et al.2007;Anstrom et al. 2009).

Dopamine release and uptake parameters from light and electrically induced dopamineefflux are similar

To assess dopamine release and uptake parameters, evoked levels of extracellular dopaminewere modeled as a balance between release and uptake (Wightman et al. 1988b; Wightmanet al. 1988a; Wu et al. 2001). Overall, there was good agreement between the collected dataand modeled dopamine curves (r2 at least 0.93). Representative light evoked dopamineresponses with overlaid fitted curves, based on calculated parameters, are shown in Fig. 2.The magnitudes of the [DA]p, Vmax and Km were quite similar to those previously reportedfor the rat dorsal striatum using electrically evoked dopamine signals (Wightman et al.1988b; Garris et al. 1997; Budygin et al. 1999; Wu et al. 2001; Garris et al. 2003). However,to more precisely compare dopamine parameters obtained from optical and electrical

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stimulation conditions, we performed additional experiments where electrically induceddopamine efflux was measured under the same conditions as optically evoked dopamine.The analysis revealed no significant difference between uptake rates (Vmax) calculated fromoptically and electrically evoked dopamine effluxes (3.49 ± 0.23 and 3.20 ± 0.30 μM/s,respectively, P = 0.46; n=6). In both cases, dopamine responses were well fitted when Kmwas set between 0.16 and 0.2 μM (0.187 ± 0.008 vs 0.193 ± 0.007 μM, P = 0.55; n = 6).Therefore, uptake parameters were identical between electrically and optically evokeddopamine release. In contrast, there was a non-significant trend towards a decrease in the[DA]p, dopamine release parameter during optical stimulation in comparison with electricalstimulation (0.072 ± 0.007 and 0.097 ± 0.011 μM, respectively, P = 0.08; n = 6).

Dopamine release dynamics can be manipulated by different light parametersThe responsiveness of ChR2 expressing dopamine neurons to different parameters of lightstimulation was evaluated. In the first experiment we stimulated SN neurons with light pulsetrains over a range of pulse widths (4-40 ms) and frequencies (from 10-200 Hz), and theamount of dopamine release ([DA]max) was determined. Both parameters proved efficaciousunder various conditions with some overlap. The most relevant frequency response curvesare presented in Fig. 3A. Two-way ANOVA analysis revealed significant effect of pulsewidth on dopamine release (F[2,108] = 9.935, P < 0.001). Interestingly, stimulation withlower pulse durations, which better mimic physiological conditions, more effectivelyincreased extracellular dopamine concentration than stimulation with higher pulse widths.The effect of frequency on dopamine release was also statistically significant (F[8,108] =2.496, P < 0.05; two-way ANOVA). The maximal dopamine efflux for stimulation with 4,10 and 20 ms pulse widths was observed with 40, 30 and 20 Hz frequency, respectively. Inthe second experiment, we tested whether light induced dopamine release is sensitive to thetotal duration of stimulus using the same pulse width and frequency (Fig. 3B). When theduration of stimulation was decreased from 1 second to 0.5 second (pulse width of 4 ms,frequency of 10-120 Hz), the amplitude of dopamine release was markedly decreased by 2-4fold (F[1,72] = 29.86, P < 0.0001). As expected, dopamine release under these conditionswas frequency dependent (F[11,72] = 7.034, P < 0.0001). Importantly, the maximal increasein dopamine release was observed at the same frequency (40 Hz) for both durations, thoughthe dopamine release was 4-fold higher following 1 s versus 0.5 s of optical stimulation.Finally, we evaluated the intensity of the laser power (from 0 to 5.7 mW) delivered throughthe fiber optic to the SN on the efficacy of dopamine release in striatum (Fig. 4). There wasa significant main effect of laser power on dopamine release (F[6,18] = 9.700, P < 0.0001; n= 4; one-way ANOVA) (Fig. 5). The minimum laser power which was required to induceChR2 positive neurons in the SN to release dopamine in the terminal field was around 1mW.With maximal laser power (5.7 mW) we could reliably induce dopamine release in thestriatum by lowering the fiber optic and stimulating the SN over 1 mm dorsoventral. Theoverall range of induced dopamine release at this location (AP - 5.6, L - 2.0) correspondedwell to the shape of the SN, which extends approximately 1 mm dorsoventrally. Our rangeof light induced release agrees well with previously published results on blue light (473 nm)penetration and tissue geometry for this brain region (Gradinaru et al. 2009).

Selective stimulation of striatal subregions by optical stimulation of SNThe optogenetic approach allows us to selectively stimulate dopamine release specifically inthe dorsal striatum by stimulating a small region of the SN and without affecting the VTA(Fig. 5). This precision is achieved both by a small injection of virus and using a smalldiameter fiber optic (200 μm). As the recording electrode was lowered into the striatum,robust dopamine effluxes were detected in the dorsal part of the region. The dopaminesignals decreased when the electrode was lowered more ventrally and completelydisappeared in the nucleus accumbens core. Cyclic voltammograms confirmed that all the

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signals were dopamine. It is important to note that the stimulation of the SN with blue lightwas often, but not always, accompanied by whisker twitch behaviors that were perfectlytimed with the light stimulus (see supplementary video). We occasionally failed to observewhisker twitches even when optical stimulation was producing a prominent dopamine signalin the dorsal striatum. Given the smaller area of stimulation with the blue light compared toelectrical stimulation, it seems probable that a small region of the SN is responsible fordopamine associated whisker twitch behaviors.

DiscussionIn order to characterize optically induced phasic dopamine release we took advantage of thesubsecond resolution of FSCV, which allowed us to precisely pair dopamine release withlight application, and FSCV can measure small, physiologically relevant dopamineconcentrations with precise spatial resolution at the micron scale. We have shown thatoptogenetics provides unprecedented control over extracellular dopamine levels by allowingus to induce physiologically relevant concentrations of dopamine, trains of stimulationwithout confounding electrochemical alterations, and extremely precise restriction ofdopamine release to relevant subregions of the striatum.

Our results indicate that optical stimulation is not accompanied by several confounds thatoften occur with electrical stimulation. For example, alterations in pH are very common withelectrical stimulation, apparently due to increases in blood flow that rapidly removeextracellular CO2, which helps maintain pH levels in the brain. Removal of the CO2 leads toalkaline pH shifts, which are more pronounced when electrical stimulation occurs at thedopamine cell bodies (Venton et al. 2003). However, in our hands, pH changes were notdetected in fast-scan cyclic voltammograms recorded following optical stimulation, evenwith repeated stimulations. This finding is relatively important since basic shifts in pH canlead to alterations in receptor affinities (Vyklicky et al. 1990; Huang and Dillon 1999)andion channel kinetics (Tombaugh and Somjen 1996). Furthermore, pH shifts seen on cyclicvoltammograms can often lead to misinterpretations due to their location near the dopaminesignal. It also suggests that electrically induced alterations in blood flow are minimal afteroptical stimulation, leading to two important implications that may only be addressed usinga genetic or optical approach. First, are alterations in blood flow due to electrical stimulationaltering our neurotransmitter release, reuptake or signaling, thereby obscuring the efficacy ofvarious interventions? If so, does this imply that some of the effects resulting from DBS aredue to alterations in blood flow as well as increased neuronal firing?

It is well established both in anesthetized and freely moving rats that the concentration ofextracellular dopamine during a series of action potentials increases as a consequence ofeach impulse and decreases as a consequence of reuptake (Wightman et al. 1988a; Garris etal. 1997). The peak concentration of electrically evoked extracellular dopamine increaseswith stimulation frequency. Dopamine concentration changes approach steady state at 10 Hzfrequency with electrical stimulation, whereas at higher frequencies (20-60 Hz) extracellulardopamine increases linearly during the stimulus train (Garris et al. 1997). In contrast to theseresults, light stimulation resulted in inverted U-shaped frequency dependence curves. Themaximal peaks for dopamine release were observed at 20, 30 and 40 Hz frequency with 4,10 and 20 ms light pulse widths respectively. After reaching these peaks, the concentrationof optically evoked dopamine declined and then reached a steady state at relatively lowlevels. This unique frequency dependence is likely based on the biological properties of theChR2 proteins.

Electrophysiological studies demonstrated that many cells cannot follow ChR2-drivenspiking above the 40 Hz range in sustained trains (Gunaydin et al.; Boyden et al. 2005; Lin

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et al. 2009). The inability of single cells to respond at the higher light pulse rates was linkedto ChR2 photocycle kinetics (Gunaydin et al.; Ernst et al. 2008) and to host cell-specificproperties of potassium and sodium channel activation/inactivation kinetics (Mainen et al.1995). The 40 Hz limitation was mirrored in our experiments in vivo, where maximaldopamine release has been observed in the 40-50 Hz range at 4 ms pulse width.

Another common artifact observed in several electrophysiological studies with ChR2 is thepresence of extra spikes (Gunaydin et al.; Boyden et al. 2005). FSCV measures anintegrative dopamine response, which originates from numerous synapses at the terminallevel. This response is not always proportional to changes of single neuron activity at thecell body region. Therefore, it is not surprising that artifactual dopamine spikes were notvoltammetrically detected in the striatum during light stimulation of the SN. It is possiblethat many artifacts detected at the single cell level are not relevant to the integrativeresponse, or on the level of neurotransmitter release. Finally, it is important to emphasizethat the current limitations on frequency dependence exhibited in our study are well withinthe boundaries of natural, behavioral and drug induced dopamine concentrations. In otherwords, even though we cannot successfully drive release at higher frequencies using thecurrent ChR2 system, the amount of release we observe is well within the range found orinduced in most experimental conditions.

Perhaps the most attractive application of optically induced dopamine release is the ability tospatially separate the release events according to the anatomical distribution of the dopaminecell bodies in the SN. With our experimental approach, we were able to anatomically restrictthe dopamine release exclusively to the dorsal striatum. As the detecting electrode waslowered throughout the striatum, the resulting signal decreased with no detectable release inthe nucleus accumbens. It is extremely difficult, if not impossible, to electrically stimulaterat SN alone, without also recruiting the VTA and other nearby regions, as well as fibers ofpassage. In recent years, it has been shown that various projections from the VTA aresensitive to different behaviors and drugs of abuse which may help explain various naturaland pathological states (Ikemoto 2007). Since we can constrain optical stimulation to verysmall subregions of the VTA or SN, this approach will undoubtedly increase ourunderstanding of the neural microcircuitry underlying dopamine transmission.

The promise of optogenetics is that the resolution of the effect also occurs on the geneticlevel where various components of the transgene cassette can be used to regulate theexpression of the opsin, either temporally, spatially, or in various neuronal subtypes. Wehave elected to proceed with generalized expression to adequately compare and contrast theparameters of electrical and optical induced dopamine release and to validate an approachthat can be easily and readily applied to other brain regions, neurotransmitter systems, andanimal models, particularly those involving rats. However, in order to leverage the ability torestrict expression, particularly with regards to FSCV, new viral vectors that utilize subtypespecific promoters, such as tyrosine hydroxylase, should be used.

In summary, by combining real time measurements of dopamine dynamics using FSCV withoptical control over nigral activation, we have demonstrated that optogenetics can be used tomirror natural patterns of dopamine release in the striatum, with extremely precise temporaland spatial resolution, and absent any chemical artifacts. Our data establish optogenetics as aviable method to establish cause and effect relationships and to dissect the neural circuitryunderlying striatal based phenomena.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

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AcknowledgmentsWe thank Drs. David Roberts, Jeffery Weiner and Jack Strandhoy for helpful comments and Dr. Karl Deisseroth forproviding the EF1α-ChR2-EYFP AAV plasmid. This work was supported by WFU Cross-Campus CollaborativeFund Award (EAB and KDB) and NIH grants DA021634 (EAB) and DA024763 (CEB).

Abbreviations used

VTA ventral tegmental area

SN substantia nigra

DBS deep brain stimulation

ChR2 channelrhodopsin-2

FSCV fast-scan cyclic voltammetry

AAV adeno-associated virus

WPRE woodchuck postregulatory element

DAT dopamine transporter

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Figure 1.Light stimulation of SN dopamine neurons produce transient dopamine release in thestriatum detected by FSCV. (A) Representative traces of dopamine signals detected in dorsalstriatum of anesthetized rats in response to light stimulation. The pulse widths, which wereused to induce these 3 signals, were 10, 20 and 4 ms, respectively. (B) Representative colorplots, which topographically depict the voltammetric data before, during and after lightstimulation, with time on the x-axis, applied scan potential on the y-axis and background-subtracted faradaic current shown on the z-axis in pseudo-color. The color scales for the firstand third panels begin with +4.0 nA and ends with -2.7 nA. The second panel scale beginswith +10 nA and ends with -5 nA. (C) Background-subtracted cyclic voltammograms takenfrom the peak of light stimulation. Every signal has an oxidation peak at +0.65 V andreduction peak at -0.2 V vs. Ag/AgCl reference, identifying the released species asdopamine.

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Figure 2.Curve fits of light evoked dopamine release in rat striatum. Data from one representativesubject (black lines) were fit to a Michaelis-Menten kinetic model to determine theparameters for dopamine release and uptake. Simulation lines (red) were calculated frombest-fit parameters. The magnitudes of the [DA]p, Vmax and Km for the dopamine effluxesinduced by 20- and 40-pulse stimulation (4 ms pulse width) were 45 nM, 3500 nM/s, 200nM and 95 nM, 3500 nM/s, 200 nM, respectively. The total length of light stimulation (1 s)is indicated by the red line under the efflux curves.

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Figure 3.Frequency, pulse width, and train duration dependency of light induced dopamine release.(A) The total duration of the stimulation for every frequency and pulse width combinationwas kept the same (1 s). (B) Data from one representative rat with 4 ms pulse widthstimulation at various frequencies (20, 30 or 40 Hz), maintained for either 0.5 s (solid line)or 1 s (dashed line) train duration. An optical fiber was positioned in the SN at the followingcoordinates: anterior-posterior, 5.6 mm; lateral, 2.0 mm; dorsal-ventral, 7.6 mm.Voltammetric recordings were performed in the striatum at the following coordinates:anterior-posterior, 2.0 mm; lateral, 1.3 mm; dorsal-ventral, 5.0 mm. The data are presentedas mean ± SEM (n =5).

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Figure 4.Evaluation of laser power intensity on the efficacy of dopamine release. Laser power from 0to 5.7 mW intensities were delivered through the fiber optic to the SN and dopamine releasewas voltammetrically detected in the striatum. (A) Dopamine signals recorded in 1 minintervals in the striatum of a representative subject during stimulation with different laserintensity values (0.0, 0.1, 0.6, 1.3, 2.3, 3.4, 4.5, 5.7 mW from bottom). (B) Effect of laserpower on dopamine release in the striatum. The data are presented as mean ± SEM andsignificance versus minimal triggered dopamine release (≈ 1mW) is indicated (*P < 0.05,**P < 0.01, *** P < 0.001, n =4).

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Figure 5.Optical stimulation of dopamine release in the striatum is spatially restricted to the dorsalregion. Dopamine traces from one representative animal are presented. The fiber optic wasfixed in the SN (anterior-posterior, 5.6 mm; lateral, 2.0 mm; dorsal-ventral, 7.6 mm) whilethe carbon fiber recording electrode was lowered to various depths throughout the striatumto determine level of dopamine release (anterior-posterior, 1.3 mm; lateral, 2.0 mm anddorsal-ventral varied with 1: 5.0 mm; 2: 5.5 mm; 3: 6.4 mm; 4: 6.8 mm; 5: 7.4 mm).

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Optogenetic control of striatal dopamine release in rats

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