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Channelrhodopsin-2 and Optical Control of Excitable Cells

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Channelrhodopsin-2 and Optical Control of Excitable Cells PERSPECTIVE Channelrhodopsin-2 and optical control of excitable cells methods Feng Zhang1,3, Li-Ping Wang1,3, Edward S Boyden1 & Karl Deisseroth1,2 Electrically excitable cells are important in the normal functioning and in the .com/nature e pathophysiology of many biological processes. These cells are typically embedded .natur in dense, heterogeneous tissues, rendering them difficult to target selectively with w conventional electrical stimulation methods. The algal protein Channelrhodopsin-2 offers a new and promising solution by permitting minimally invasive, genetically targeted and http://ww temporally precise photostimulation. Here we explore technological issues relevant to the temporal precision, spatial targeting and physiological implementation of ChR2, in the oup r G context of other photostimulation approaches to optical control of excitable cells. lishing Electrically excitable cells include skeletal, cardiac and Photostimulation provides a versatile alternative to b smooth muscle cells, pancreatic beta cells, and neurons. electrode stimulation. Light beams can be easily and Pu Malfunction of these cells can lead to heart failure, mus- quickly manipulated to target one or many neurons, and cular dystrophies, diabetes, pain syndromes, cerebral could help relax the requirement for mechanical stability. Nature palsy, paralysis, depression and schizophrenia, among Richard Fork at Bell Laboratories introduced the semi- 6 many other diseases. Notably, unlike cells such as those nal idea in 1971 when he used an intense beam of blue 1 200 in the immune and gastrointestinal systems that respond light to evoke action potentials in Aplysia ganglion cells . © well to slow chemical modulation, electrically excitable Although Fork’s method was never widely adopted (pre- cells signal and react on timescales as short as millisec- sumably because it disrupted membranes, as later dem- onds. To date, stimulating electrodes have been a valu- onstrated for two-photon laser-based excitation2), several able tool for the study of excitable cells because they can groups have since developed improved photostimulation accurately simulate natural cellular signals by control- techniques to address different aspects of what has been ling electrical activity on the millisecond time scale. Two considered a ‘holy grail’ of neuroscience: minimally inva- experimental challenges, however, render investigation of sive, genetically targeted and temporally precise control excitable cells with electrodes difficult. First, it is generally of neural activity (for review see refs. 3,4). Here we will infeasible or impractical to target multiple cells of a spe- focus on physiological issues governing one such new cific class simultaneously. Extracellular electrodes have approach—application of an algal light-gated ion chan- limited spatial resolution for heterogeneous tissue, and nel Channelrhodopsin-2 for photostimulation5–8—and although intracellular electrodes do target specific neu- compare the technological capabilities of this approach rons, they are impractical for simultaneous targeting of with other optical methods. many cells of a particular class. Second, electrode meth- odologies typically rely on mechanical stability because Photostimulation techniques and physiological of the need to register electrodes with cells, and therefore considerations can be cumbersome to use in awake, behaving animals. Photostimulation techniques can be divided into three Solutions to these problems, which have challenged sci- approaches: light-mediated ‘uncaging’ of chemically entists and physicians alike, have begun to emerge from modified signaling molecules9–11, chemical modifi- the neuroengineering and neuroscience fields. cation of ion channels and receptors to render them 1Department of Bioengineering and 2Department of Psychiatry and Behavioral Sciences, Clark Center, Stanford University, 318 Campus Drive West, Stanford, California 94305, USA. 3These authors contributed equally to this work. Correspondence should be addressed to K.D. ([email protected]). PUBLISHED ONLINE 21 SEPTEMBER 2006; DOI:10.1038/NMETH936 NATURE METHODS | VOL.3 NO.10 | OCTOBER 2006 | 785 PERSPECTIVE light-responsive12,13 and introduction of light-sensitive proteins activating G protein–coupled receptors and downstream signal- into otherwise light-insensitive cells5–8,11,14–16. These techniques can ing pathways17. modulate membrane potential and activate downstream signaling Uncaging can be used to stimulate individual parts of a neuron, cascades with different levels of temporal and spatial control (sum- single neurons or larger networks of neurons. In particular, gluta- marized in Table 1). mate uncaging has become an important and useful tool for study- ing dendritic integration18,19, and mapping neural connectivity in Light-mediated uncaging of signaling molecules. In this intact tissues20,21 (for a detailed review of the pros and cons of glu- approach, membrane potential or cellular signaling can be mod- tamate uncaging, see ref. 22). Recently, researchers used glutamate ulated when light releases a blocking moiety covalently attached uncaging to probe the cellular connectivity of cortical networks by to a biochemically active compound (for example, glutamate, exciting neurons at several hundred sites20 and recording the result- GABA or second messengers such as Ca2+). Although glutamate ing synaptic transmission in postsynaptic cells. Populations of neu- is the most commonly used molecule, other types of caged com- rons could be stimulated with a temporal precision on the order methods pounds have been synthesized for controlling cellular activity by of several tens of milliseconds and spatial resolution down to fifty Table 1 | Temporal, spatial, and technical properties of different photostimulation techniques. .com/nature e Typical Sustained membrane Typical precisely Need for .natur Optical voltage single-photon Temporal timed spike exogeneous Spatial w method Mechanism actuation excitation spike jittera trainsb chemicals resolution References Glutamate Glutamate molecules are Depolarizing 355 nm 1–3 ms NM Caged glutamate 5 µmc 18,20,41 uncaging released from their caged ultraviolet (for example, http://ww form upon illumination light nitrobenzyl ester by uncaging light. Free form9) oup r glutamate can activate both G ionotropic and G protein– coupled metabotropic receptors. lishing b ChARGe Three-part Drosophila sp. Depolarizing 400-600 nm Seconds to NM Requires retinal16 Genetically 16 Pu G-protein phototransduction light minutes targetable cascade is introduced into neurons, driving downstream Nature endogenous ion channels. 6 ChR2 Naturally occurring light- Depolarizing 480 nm blue 1–3 ms 10 Hz routine, Not needed for Genetically 5–8,15,38 200 ≥ © activated cation channel is light 30 Hz mammalian cells; targetable opened upon exposure to readily non-mammalian blue light. achievable systems may in fast- require added all- spiking cells trans-retinal4 Modified Engineered iGluR receptor Depolarizing 380 nm for 100 ms NM Azobenzene- Genetically 13 glutamate binds to a synthetic ‘switch’ activation and tethered targetable receptor molecule. 580 nm for glutamate inactivation receptor agonist13 Ion channel ATP and capsaicin are Depolarizing 355 nm ≤1 s NM Caged ATP, Genetically 10,23 ligand uncaged to activate cells ultraviolet capsaicin, etc. targetable uncaging expressing exogenous light (e.g. nitrobenzyl ionotropic purinoceptors and ester form10) capsaicin receptors. Modified Engineered potassium Hyperpolarizing 380 nm for 1–3 s NA Azobenzene- Genetically 12 potassium channel binds to a synthetic activation and tethered targetable channel “switch” molecule. 580 nm for potassium channel inactivation blocker12 Vertebrate Vertebrate rat rhodopsin Hyperpolarizing 475 nm blue 1–3 s NA Requires retinal2 Genetically 6 rhodopsin 4 is introduced into light- light targetable insensitive neurons, driving downstream endogenous ion channels. Single-photon excitation wavelengths are indicated; two-photon excitation methods are typically suitable as well. The modified potassium channel has been recently modified to give depolarizing currents (R. Kramer, personal communication). GABA uncaging is also possible, which could give rise to either depolarizing or hyperpolarizing responses, depending on membrane voltage. aTemporal spike jitter is determined by the timing precision with which single spikes may be reliably evoked. bSustainability of precise firing refers to the ability of the technique to control repeated precisely timed spike trains (NM, not measured; NA, not applicable). cThe 5 µm spatial resolution for glutamate uncaging is based on reference 18. 786 | VOL.3 NO.10 | OCTOBER 2006 | NATURE METHODS PERSPECTIVE micrometers. In very recent work, acousto-optical deflectors were uncaging methods), the azobenzene compound must be supple- used to stimulate several different locations on the dendritic tree mented during intact-tissue studies. of a single cerebellar Purkinje cell in succession18; with this custom optical setup, rapid elicitation of uncaging responses may be more Naturally occurring photosensitive proteins. A potentially more readily achieved than with what is commercially available (for a direct approach for bestowing light-sensitivity is to use light-sens- similar study of dendritic integration, see ref. 19). The authors also ing rhodopsins. The first attempt at this strategy exploited the mul- presented data demonstrating precise timing of individual evoked tiple-component
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