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Optogenetics: shining a light on the brain

Optogenetics: shining a light on the brain Bioscience BioscienceHoriz Horizons ons Volume 5 2012 10.1093/biohorizons/hzr020 Research article James Butler* Imperial College London, London, UK. * Corresponding author: Email: james.butler08@imperial.ac.uk Supervisor: Stephen Brickley, Imperial College London, South Kensington Campus, London SW7 2AZ. In 2005, Boyden et al. used the protein channelrhodopsin-2 to generate the first ever light-induced action potential, involving a single component device. Since then, an explosion of so-called ‘optogenetic’ research has occurred. This abundance of new discoveries is reviewed here in depth. First, methods of targeting optogenetic techniques are discussed in brief. Next, both optogenetic sensors, used for observing neural circuits, and single-component optogenetic effectors, used for manipulating neural circuits, are assessed. The discoveries that these new technologies have led to is presented, current limitations of the respective technologies are examined and directions of future research discussed. Keywords: neural, neuron, brain, review, halorhodopsin, channelrhodopsin Submitted on 11 July 2011; accepted on 13 October 2011 Introduction and halorhodopsin (NpHR), are described, their influence on neural research is highlighted and their potential discussed. Optogenetics involves the combination of optic and genetic Lastly, the different optogenetic sensors are presented and techniques for the study of neural circuits. The term was first their respective benefits and drawbacks are discussed. coined by Deisseroth et al. (2006), the team that was quickest to realize the full potential of channelrhodopsin-2. Since then Targeting of optogenetic tools the neuroscience community has witnessed an explosion of optogenetic research. The brain is a complex (ordered) tangle of heterogeneous The area of optogenetics can be subdivided into optogenetic neurons that makes studying it extremely difficult. One of the sensors and effectors. The former is used to monitor neural main strengths of optogenetics is the impressive resolution it circuits and the latter is used to directly manipulate neural cir- allows, enabling the targeting of (and therefore study of) neu- cuits. The green fluorescent protein (GFP), discovered in 1962, ral subsets. The different techniques of optogenetic targeting revolutionized large areas of scientific research (Shimomura, are discussed below and the various advantages and disad- Johnson and Saiga, 1962). Current day commonly used GFP vantages of each technique highlighted. techniques fall under the umbrella term of optogenetics. Likewise, multi-component devices involving multiple genes Transgenic animals have been used to create light-sensitive action potentials (Lima Transgenic animals have been used for decades in scientific and Miesenbock, 2005) and would also be categorized as research (Jaenisch, 1988). Short transgene cassettes made optogenetic. However, in the case of optogenetic effectors, this from either recombinant promoters or bacterial artificial review will use a more concise definition. Here, only single- chromosomes (BAC) are inserted into the embryo of a mouse component optogenetic effectors are considered, due to the or rat and the strain is then bred until a stable line is pro- explosion of interest in this field over recent years. duced (Adamantidis et al., 2007). So far two such lines have This review first presents an overview of genetic targeting been produced, one expressing ChR2 (Arenkiel et al., 2007) methods used for both effectors and sensors. Next, the two and the other NpHR (Zhao et al., 2008), with no most commonly used effectors, channelrhodopsin-2 (ChR2) abnormalities detected. © The Author 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons 1 1 Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Review article Bioscience Horizons • Volume 5 2012 Transgenic animals exhibit a uniform level of expression activate the opsin gene by irreversibly inverting it. It does not and distribution, and a high level of specificity can be reached matter if the virus infects other cells as these will not be by using a BAC to introduce a large cell-type specific pro- expressing Cre-recombinase, hence the opsin gene will remain moter (Zhao et al., 2011). However, production of a trans- inactive (Chakravarthy et al., 2008). genic line is labour intensive, taking up to a year to produce A problem when using viral vectors is that some cell-type a stable line. Furthermore, levels of transgene expression can specific promoters have weak expression levels. This can be be lower than with other methods, leading to inadequate overcome in a Cre-dependant expression system through the optical control (Zhang et al., 2010). use of a strong Cre-dependant promoter (Petreanu et al., 2009). Viral vectors Viral vectors are the current mainstay of optogenetic research. Discussion This method takes advantage of the fact that some viruses Importantly, AAVs have been used to insert transgenes into (for example lentiviruses and adeno-associated viruses human neurones in a clinical setting (Kaplitt et al., 2007). (AAVs)) incorporate their DNA into the host cell genome as Therefore, if optogenetic effectors are of use in clinical treat- a part of their natural life cycle (Buchschacher, 2003). The ment of human disease, a viral vector targeting system can be opsin gene is again fused with a cell-type specific promoter used for administration. and a recombinant virus is then made. The virus is used as a vector to introduce the transgene into the cells of interest Genetic targeting is a crucial aspect of optogenetics which (Boyden et al., 2005). An example of a lentivirus vector can allows us to investigate neurons in the brain at a higher reso- be seen in Fig. 1. lution than ever before. Indeed, Gradinaru et al. (2010), using trans-synaptic trafficking, have developed a novel tech- Unlike transgenic animals, which are restricted to rats and nique with the potential to target specific axonal branches. mice, viral vectors have successfully been used in a range of Thus, the precision of optogenetic targeting is still increasing higher organisms (Kaplitt and During, 2006). A further and with it this should bring new discoveries. advantage of viral vectors is the short time (4–5 weeks) needed to achieve transgene expression. In contrast to trans- Further information on this topic can be found in the com- genic animals, high levels of transgene expression can be prehensive reviews of Luo, Callaway and Svoboda (2008) achieved by increasing the transgene copy number. Due to the and Yizhar et al. (2011). Next, this review looks at the bat- limited maximum size of a virus genome, viral vectors suffer tery of optogenetic tools that can be targeted using these from a limited packaging capacity (LaLumiere, 2011). This techniques, and the insights into neuroscience that they have can also cause lower specificity as it limits the length of the granted. promoter sequence used. Optogenetic effectors Cre-dependant expression system This method combines both viral and transgenic methods. A A variety of different opsins (see Fig. 2) have recently transgenic line of mouse is produced expressing Cre- been discovered and utilized which, when stimulated by light, recombinase in the cells of interest. Then a viral vector is used have the ability either to induce or inhibit synaptic activity. to deliver a doubly floxed inverted opsin gene (depicted in These have had a significant impact in neuroscience and are Fig. 1). Any cells expressing Cre-recombinase will then outlined below. Figure 1. Top: a lentiviral virus vector containing a cell-type specific Figure 2. Left: channelrhodopsin (in yellow) that, upon stimulation by promoter and an opsin. Bottom: a Cre-dependant AAV vector 470 nm blue light, opens and allows an Na influx into the cell. Right: containing a doubly floxed inverted opsin fusion gene (Zhang et al., NpHR that, upon stimulation by 580 nm yellow light, hyperpolarizes 2010). the membrane by allowing a Cl influx into the cell. 2 Bioscience Horizons • Volume 5 2012 Review article Recent studies have investigated the ability of light-sensi- neural activity by pumping Cl ions into the cell (Zhang tive chimeric G-protein-coupled receptors to induce intracel- et al., 2007). lular signalling pathways (Massack et al., 2011). The Since its discovery, upgraded versions of NpHR have been potential this technology carries is huge, however as this field produced with advantages over the original protein. The first, is still in its infancy, it will not be discussed within this review. eNpHR2.0, had a mammalian signal peptide grafted onto it, improving its membrane targeting and ER export. More Channelrhodopsin-2 recently, Gradinaru et al. (2010) added a C-terminal traffick- ChR2 was the first opsin to be discovered by Nagel et al. ing signal from the potassium ion channel Kir2.1, creating (2003). ChR2 is a light-gated ion channel isolated from the eNpHR3.0. eNpHR3.0 shows improved localization to the alga Chlamydomonas reinhardtii. Nagel et al. (2003) showed plasma membrane and has a significantly enhanced inhibi- that it is a cation selective ion channel that permits a Na influx tory capacity. Most importantly, eNpHR3.0 is stimulated by when illuminated by 470 nm blue light. Upon discovering red/far-red light, which penetrates deeper into biological tis- ChR2′s unique ability, the authors postulated that it could be sue, thereby increasing the ease with which the effector is used as a powerful research tool in other species, not just algae. activated (Mancuso et al., 2011). Subsequently, Boyden et al. (2005) used a lentiviral vector The opsins Archaerhodopsin-3 (Arch) and Mac, isolated to express ChR2 in mammalian neurons. They showed that, from Halorubrum sodomense and Leptosphaeria maculans, when stimulated with 470 nm light, ChR2 generated a large respectively, provide an alternative method of silencing neu- photocurrent capable of stimulating an action potential. rons. They are proton pumps that, when activated by the cor- Furthermore, ChR2 was so sensitive that millisecond-scale rect wavelength of light, pump protons out of the neuron, timing was possible, enabling sensitive temporal studies to thus collapsing the current normally generated by an action take place. potential. Arch and Mac are activated by different wave- lengths of light. Hence, it is possible to use them simultane- ChR2-based research ously and independently on two neural populations. Both have advantages and disadvantages when compared with Synaptic plasticity and long-term potentiation (LTP), the NpHR. For example, Arch is able to generate much larger hypothesized molecular mechanism behind memory in photocurrents than NpHR, but requires higher light levels to humans, are areas of intensive research in which there is still do so (Chow et al., 2010). much to learn (Blundon and Zakharenko, 2008). Zhang and Oertner (2007) showed that it was possible to induce LTP NpHR-based research using ChR2, and Oertner’s group went on to use this tech- nique to reveal input-specific accumulation of αCaMKII after As with ChR2, NpHR has also led to a plethora of research. LTP (Zhang et al., 2008). Thus, it is likely that optogenetic One of the earliest studies was by Gradinaru et al. (2009), effectors will help us unravel the mystery of LTP in the future. who used NpHR to identify the specific cell type responsible for the therapeutic effect of deep brain stimulation when used For many years it has been suggested that dopaminergic to treat neurological disorders, such as Parkinson’s disease. (DA) neurons are important in appetitive conditioning (Koob NpHR has also been used for research in swim circuitry in and Le Moal, 1997). Previous techniques, however, lacked zebrafish (Arrenberg, Del Bene and Baier, 2009), gamma both the temporal and spatial precision required to investi- oscillations (Sohal et al., 2009) and neurogenesis (Yang, gate this theory further. Tsai et al. (2009) used ChR2 to Ming and Song, 2011). manipulate selectively DA neurons of the ventral tegmental area to show that phasic (50 Hz), but not tonic (1 Hz), action Discussion potential firing of DA neurons is a driver of behavioural con- ditioning. This is a lovely example of the extra precision that The discovery of ChR2 and NpHR, and their potential in ChR2 is able to provide. neuroscience, has created an explosion of research. As the effectors are light-induced, this allows for millisecond-scale Therefore, the increased resolution that ChR2 provides control of neural circuits. Thus, this extremely high temporal enables many previously inaccessible avenues to be explored. and spatial resolution of NpHR and ChR2 makes them very The opsin has also already been used to uncover new infor- powerful research tools. mation on wakefulness (Adamantidis et al., 2007), depres- sion (Covington et al., 2010) and fear (Ciocchi et al., 2010). NpHR and ChR2 are stimulated by different wavelengths These are only but a few of the discoveries generated by of light. This allows NpHR to be used in conjunction with ChR2-based research. ChR2 to induce or inhibit neural activity, depending on the researcher’s purpose. This further increases the power of Halorhodopsin these effectors as research tools. NpHR is a light-sensitive chloride pump that can hyperpolar- ChR2-induced currents have different kinetics from native ize a neuron when stimulated by 580 nm light. When membrane channel-induced currents (Ritter et al., 2008). 2+ expressed in the cell membrane, NpHR can be used to inhibit Additionally, ChR2 is Ca permeable (Nagel et al., 2003). 3 Review article Bioscience Horizons • Volume 5 2012 As neurotransmitter release is intrinsically linked with cal- Optogenetic sensors cium influx (Kasparov, 2011), light-induced action potentials have a very high chance of neurotransmitter release. Although Optogenetic methods for monitoring synaptic activity have this problem is reduced in certain ChR2 variants (Gunaydin been invaluable in neural research. Outlined below are the et al., 2010), great care must be taken when interpreting the different methods, and their respective advantages and disad- results of studies using ChR2. vantages are discussed. Improved opsins have been produced with features such as Voltage-sensitive fluorescent proteins faster kinetics (Lin et al., 2009), increased membrane expres- Voltage-sensitive fluorescent proteins (VSFPs) are able to sion (Zhao et al., 2008) and increased light sensitivity (Berndt record changes in voltages across membranes, allowing the et al., 2009). These will further increase the effectiveness of visualization of action potentials. They consist of a voltage- opsins in neural circuitry research. sensitive domain, taken from a voltage-sensitive phosphatase Long-term expression of ChR2 in the primate brain is from the organism Ciona intestinalis, coupled to either one stable (Han et al., 2009) and ChR2 has already been shown or two fluorophores (Dimitrov et al., 2007). An example of a to be of use in Parkinson’s treatment (Gradinaru et al., VSFP can be seen in Fig. 3. 2009). Therefore, ChR2 may have a future role in human When a cell expressing VSFP becomes depolarized, a con- disease treatment. However, much work will have to be formational change in the voltage sensor domain brings the done as to the safety of long-term ChR2 expression in two fluorophores closer together. This results in a change in humans. Other work has shown the potential of optogenetic the ratio of fluorescence emitted by the two fluorophores, effectors in a clinical setting [for example, light-induced res- known as a fluorescence resonance energy transfer (FRET) cue of breathing after spinal cord injury (Alilain et al. 2008)] signal. The FRET signal can be measured and used to observe and it is, unfortunately, a topic too broad to cover within current changes in cells (Mutoh et al., 2011). this review. As with optogenetic effectors, a variety of VSFP variants Blood-oxygen-level dependence (BOLD) signals measured, have been produced with improved responsiveness (Lundby using functional magnetic resonance imaging, have been used et al., 2008), different fluorescent wavelengths (Mutoh since the early 1990s to measure neural activity in the brain et al., 2009) and even different fluorophores [Tsutsui et al. (Ogawa et al., 1990). Despite the many discoveries made (2008) used a fluorophore isolated from coral]. This has with this technology, until recently it was not clear what further increased the diagnostic capability of VSFPs as exactly caused BOLD signals. Lee et al. (2010a) used a ChR2- it allows the investigator to choose the right VSFP for based approach to show that stimulation of local CaMKIIα- their experiment. More information on VSFPs can be found expressing excitatory neurons (in the neocortex or thalamus) in the comprehensive review by Peterka, Takahashi and produce positive BOLD signals. This illustrates the ability of Yuste (2011). optogenetics to resolve previously unanswerable long-stand- ing questions. Genetically encoded calcium indicators Within the brain, light scattering limits the depth of light 2+ An intracellular calcium (Ca ) influx is associated with an to a few hundred micrometres. Therefore, this makes it hard action potential. Thus, detection of calcium can be used as a to activate opsins over a large area in a non-invasive fashion (Mancuso et al., 2011). Despite the improved version of NpHR, eNpHR3.0 (see above), there is still a need for fur- ther development of non-invasive techniques of delivering light to the opsins. Lewis et al. (2009) fused a myosin-binding domain to ChR2, which successfully targeted the opsin to the somato- dendritic compartment of neurons in mice. This ability to target opsins to specific regions of the cell, or to intracel- lular organelles, is largely unexplored. As we further the tar- geting precision of the optogenetic effectors, this will boost our understanding of the specific molecular mechanisms occurring in neurons underlying neural activity. Optogenetic effectors have exploded onto the scene over the past 6 years and the rate of new discoveries that this has brought has yet to slow down. It is still a young technology Figure 3. VSFP2.3 which consists of four transmembrane domains and it will be exciting to see the future developments that fused to the fluorophores mCerulean and citrine in tandem ( Akemann optogenetic effectors will bring. et al., 2010). 4 Bioscience Horizons • Volume 5 2012 Review article method of detecting neural activity (Spruston et al., 1995). were used to visualize changes in voltage but localised 2+ Consequently, different methods of detecting Ca levels in expression was hard to achieve (Kee et al., 2008). Thus, neurons have been explored. VSFPs have a higher level of precision compared with their predecessors. D3cpVenus is one such genetically encoded calcium indi- cators (GECI), which fluoresces in response to an increase in Despite VSFP development coming a long way over the 2+ Ca and has been successfully used, both in vitro and in vivo, past few years, there is little published data to show for it. to detect single action potential spiking. Likewise, Nakai, This is so especially when compared with optogenetic effec- Ohkura and Imoto (2001) developed G-CaMP, a GFP-based tors; a technology only 2 years older. Last year, for the first probe that also showed fluorescent changes in response to time, Akemann et al. (2010) successfully used VSFPs in vivo 2+ changes in Ca levels. This has since been improved upon to report cortical electrical responses to stimuli. Hopefully, and the latest version of G-CaMP, G-CaMP3, has an increased VSFPs are coming to a point where their potential is unlocked, baseline flourescence and dynamic range and a higher affinity and questions that were previously unanswerable with classi- 2+ for Ca (Tian et al., 2009). cal methods are answered. A fundamental problem with using calcium levels as a Chloride sensors measure of action potentials is that other cellular processes 2+ As a Cl influx is normally the mechanism of synaptic inhibi- involve the alteration of Ca levels (Mancuso et al., 2011). tion, it is possible to monitor synaptic inhibition by monitor- Therefore, no matter how sensitive GECIs become, they are − − ing Cl levels. Clomeleon is a Cl sensor that works in a still at risk of overestimating action potential rates. similar way to VSFPs. An increase in Cl concentration causes However, GECIs have been used successfully to study both a decrease in FRET and thereby causes the protein to fluo- spiking activity and synaptic activity across multiple neurons resce (Kuner and Augustine, 2000). simultaneously (Dreosti and Lagnado, 2011). It will be inter- esting to follow their development and the possible informa- pH sensors tion they could uncover about synaptic circuits. During an action potential, release of neurotransmitter causes Clomeleon suffers from a low-binding affinity for Cl and a loss of protons for the cell, thereby reducing the cells’ pH. interference from background Cl concentrations which hin- The optogenetic pH sensor, pHluorin, is fused to the walls of ders Clomeleon-based studies. An improved protein, vesicles within the cell and is quenched in the acidic environ- Superclomeleon, has now been produced which is free from ment of the vesicle. Upon neurotransmitter release the pH these drawbacks. Already, Clomeleon has proved to be a use- of the vesicle increases, causing the pH sensor to fluoresce ful tool in the investigation of inhibitory neural responses which can then be recorded (Miesenbock, De Angelis and (Berglund et al., 2008; Lee et al., 2010b) and it will be inter- Rothman, 1998). esting to see if Superclomeleon is able to expand the potential Optogenetic pH sensors have been used successfully in the of Cl sensing in neural activity studies (Mancuso et al., study of synaptic vesicle exocytosis and endocytosis (Dittman 2011). and Kaplan, 2006). Recent improvements have enabled Optogenetic sensors are a diverse group which are able to researchers to detect the release or retrieval of individual monitor synaptic transmission at a variety of different stages. vesicles (Kim and Ryan, 2009). By using them in concert with one another, it would be pos- sible to compare the sensitivity and accuracy of each tech- Neurotransmitter release sensors nique at measuring neural activity. GluSnFR and FLIPEO are two FRET-based optogenetic sen- sors that are anchored to the cell membrane and fluoresce in Concluding remarks response to glutamate, the most common excitatory neu- rotransmitter (Hires, Zhu and Tsien, 2008; Okumoto et al., Optogenetics is a fast growing field; more than 800 laborato- 2005) These sensors are sensitive enough to detect a single ries around the world are now engaged in optogenetic spike (Dreosti and Lagnado, 2011). research (Deisseroth, 2011). Despite copious amounts of research over the past 5 years, a consistent flow of new dis- Discussion coveries is being produced, both in neural circuitry research Neurotransmitter release sensors are yet to be used to uncover and in improving current optogenetic methods. any novel information. Despite the impressive ingenuity Optogenetic effectors have increased our ability to manip- behind neurotransmitter release sensors, it is difficult to ulate neural circuits while optogenetic sensors are increasing imagine their use in neural circuit research reaching levels our ability to observe such circuits. It will be exciting to see if comparable to other optogenetic methods. further developments will increase their ability to comple- As VSFPs are expressed by cells, this allows them to ben- ment one another, which would cause further acceleration to efit from genetic targeting. Previously, voltage-sensitive dyes the already fast pace of neural research. 5 Review article Bioscience Horizons • Volume 5 2012 Deisseroth, K. (2011) Optogenetics, Nature Methods, 8, 26–29. Author biography Deisseroth, K., Feng, G., Majewska, A. K. et  al. (2006) Next-generation James Butler has just finished a biology degree at Imperial optical technologies for illuminating genetically targeted brain cir- College London. While there, he developed a fascination cuits, The Journal of Neuroscience, 26 (41), 10380–10386. with neuroscience that has resulted in him pursuing a research Dimitrov, D., He, Y., Mutoh, H. et al. (2007) Engineering and characteriza- career in this field. In 2011, he is due to start a masters in tion of an enhanced fluorescent protein voltage sensor, PLoS One, 2, neuroscience at University College of London, Europe’s lead- e440. ing neuroscience institution. Dittman, J. S. and Kaplan, J. M. (2006) Factors regulating the abundance References and localization of synaptobrevin in the plasma membrane, Proceedings of the National Academy of Sciences USA, 103, 11399– Adamantidis, A. R., Zhang, F., Aravanis, A. M. et  al. 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Optogenetics: shining a light on the brain

Bioscience Horizons , Volume 5 – Dec 26, 2012

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10.1093/biohorizons/hzr020
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Bioscience BioscienceHoriz Horizons ons Volume 5 2012 10.1093/biohorizons/hzr020 Research article James Butler* Imperial College London, London, UK. * Corresponding author: Email: james.butler08@imperial.ac.uk Supervisor: Stephen Brickley, Imperial College London, South Kensington Campus, London SW7 2AZ. In 2005, Boyden et al. used the protein channelrhodopsin-2 to generate the first ever light-induced action potential, involving a single component device. Since then, an explosion of so-called ‘optogenetic’ research has occurred. This abundance of new discoveries is reviewed here in depth. First, methods of targeting optogenetic techniques are discussed in brief. Next, both optogenetic sensors, used for observing neural circuits, and single-component optogenetic effectors, used for manipulating neural circuits, are assessed. The discoveries that these new technologies have led to is presented, current limitations of the respective technologies are examined and directions of future research discussed. Keywords: neural, neuron, brain, review, halorhodopsin, channelrhodopsin Submitted on 11 July 2011; accepted on 13 October 2011 Introduction and halorhodopsin (NpHR), are described, their influence on neural research is highlighted and their potential discussed. Optogenetics involves the combination of optic and genetic Lastly, the different optogenetic sensors are presented and techniques for the study of neural circuits. The term was first their respective benefits and drawbacks are discussed. coined by Deisseroth et al. (2006), the team that was quickest to realize the full potential of channelrhodopsin-2. Since then Targeting of optogenetic tools the neuroscience community has witnessed an explosion of optogenetic research. The brain is a complex (ordered) tangle of heterogeneous The area of optogenetics can be subdivided into optogenetic neurons that makes studying it extremely difficult. One of the sensors and effectors. The former is used to monitor neural main strengths of optogenetics is the impressive resolution it circuits and the latter is used to directly manipulate neural cir- allows, enabling the targeting of (and therefore study of) neu- cuits. The green fluorescent protein (GFP), discovered in 1962, ral subsets. The different techniques of optogenetic targeting revolutionized large areas of scientific research (Shimomura, are discussed below and the various advantages and disad- Johnson and Saiga, 1962). Current day commonly used GFP vantages of each technique highlighted. techniques fall under the umbrella term of optogenetics. Likewise, multi-component devices involving multiple genes Transgenic animals have been used to create light-sensitive action potentials (Lima Transgenic animals have been used for decades in scientific and Miesenbock, 2005) and would also be categorized as research (Jaenisch, 1988). Short transgene cassettes made optogenetic. However, in the case of optogenetic effectors, this from either recombinant promoters or bacterial artificial review will use a more concise definition. Here, only single- chromosomes (BAC) are inserted into the embryo of a mouse component optogenetic effectors are considered, due to the or rat and the strain is then bred until a stable line is pro- explosion of interest in this field over recent years. duced (Adamantidis et al., 2007). So far two such lines have This review first presents an overview of genetic targeting been produced, one expressing ChR2 (Arenkiel et al., 2007) methods used for both effectors and sensors. Next, the two and the other NpHR (Zhao et al., 2008), with no most commonly used effectors, channelrhodopsin-2 (ChR2) abnormalities detected. © The Author 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons 1 1 Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Review article Bioscience Horizons • Volume 5 2012 Transgenic animals exhibit a uniform level of expression activate the opsin gene by irreversibly inverting it. It does not and distribution, and a high level of specificity can be reached matter if the virus infects other cells as these will not be by using a BAC to introduce a large cell-type specific pro- expressing Cre-recombinase, hence the opsin gene will remain moter (Zhao et al., 2011). However, production of a trans- inactive (Chakravarthy et al., 2008). genic line is labour intensive, taking up to a year to produce A problem when using viral vectors is that some cell-type a stable line. Furthermore, levels of transgene expression can specific promoters have weak expression levels. This can be be lower than with other methods, leading to inadequate overcome in a Cre-dependant expression system through the optical control (Zhang et al., 2010). use of a strong Cre-dependant promoter (Petreanu et al., 2009). Viral vectors Viral vectors are the current mainstay of optogenetic research. Discussion This method takes advantage of the fact that some viruses Importantly, AAVs have been used to insert transgenes into (for example lentiviruses and adeno-associated viruses human neurones in a clinical setting (Kaplitt et al., 2007). (AAVs)) incorporate their DNA into the host cell genome as Therefore, if optogenetic effectors are of use in clinical treat- a part of their natural life cycle (Buchschacher, 2003). The ment of human disease, a viral vector targeting system can be opsin gene is again fused with a cell-type specific promoter used for administration. and a recombinant virus is then made. The virus is used as a vector to introduce the transgene into the cells of interest Genetic targeting is a crucial aspect of optogenetics which (Boyden et al., 2005). An example of a lentivirus vector can allows us to investigate neurons in the brain at a higher reso- be seen in Fig. 1. lution than ever before. Indeed, Gradinaru et al. (2010), using trans-synaptic trafficking, have developed a novel tech- Unlike transgenic animals, which are restricted to rats and nique with the potential to target specific axonal branches. mice, viral vectors have successfully been used in a range of Thus, the precision of optogenetic targeting is still increasing higher organisms (Kaplitt and During, 2006). A further and with it this should bring new discoveries. advantage of viral vectors is the short time (4–5 weeks) needed to achieve transgene expression. In contrast to trans- Further information on this topic can be found in the com- genic animals, high levels of transgene expression can be prehensive reviews of Luo, Callaway and Svoboda (2008) achieved by increasing the transgene copy number. Due to the and Yizhar et al. (2011). Next, this review looks at the bat- limited maximum size of a virus genome, viral vectors suffer tery of optogenetic tools that can be targeted using these from a limited packaging capacity (LaLumiere, 2011). This techniques, and the insights into neuroscience that they have can also cause lower specificity as it limits the length of the granted. promoter sequence used. Optogenetic effectors Cre-dependant expression system This method combines both viral and transgenic methods. A A variety of different opsins (see Fig. 2) have recently transgenic line of mouse is produced expressing Cre- been discovered and utilized which, when stimulated by light, recombinase in the cells of interest. Then a viral vector is used have the ability either to induce or inhibit synaptic activity. to deliver a doubly floxed inverted opsin gene (depicted in These have had a significant impact in neuroscience and are Fig. 1). Any cells expressing Cre-recombinase will then outlined below. Figure 1. Top: a lentiviral virus vector containing a cell-type specific Figure 2. Left: channelrhodopsin (in yellow) that, upon stimulation by promoter and an opsin. Bottom: a Cre-dependant AAV vector 470 nm blue light, opens and allows an Na influx into the cell. Right: containing a doubly floxed inverted opsin fusion gene (Zhang et al., NpHR that, upon stimulation by 580 nm yellow light, hyperpolarizes 2010). the membrane by allowing a Cl influx into the cell. 2 Bioscience Horizons • Volume 5 2012 Review article Recent studies have investigated the ability of light-sensi- neural activity by pumping Cl ions into the cell (Zhang tive chimeric G-protein-coupled receptors to induce intracel- et al., 2007). lular signalling pathways (Massack et al., 2011). The Since its discovery, upgraded versions of NpHR have been potential this technology carries is huge, however as this field produced with advantages over the original protein. The first, is still in its infancy, it will not be discussed within this review. eNpHR2.0, had a mammalian signal peptide grafted onto it, improving its membrane targeting and ER export. More Channelrhodopsin-2 recently, Gradinaru et al. (2010) added a C-terminal traffick- ChR2 was the first opsin to be discovered by Nagel et al. ing signal from the potassium ion channel Kir2.1, creating (2003). ChR2 is a light-gated ion channel isolated from the eNpHR3.0. eNpHR3.0 shows improved localization to the alga Chlamydomonas reinhardtii. Nagel et al. (2003) showed plasma membrane and has a significantly enhanced inhibi- that it is a cation selective ion channel that permits a Na influx tory capacity. Most importantly, eNpHR3.0 is stimulated by when illuminated by 470 nm blue light. Upon discovering red/far-red light, which penetrates deeper into biological tis- ChR2′s unique ability, the authors postulated that it could be sue, thereby increasing the ease with which the effector is used as a powerful research tool in other species, not just algae. activated (Mancuso et al., 2011). Subsequently, Boyden et al. (2005) used a lentiviral vector The opsins Archaerhodopsin-3 (Arch) and Mac, isolated to express ChR2 in mammalian neurons. They showed that, from Halorubrum sodomense and Leptosphaeria maculans, when stimulated with 470 nm light, ChR2 generated a large respectively, provide an alternative method of silencing neu- photocurrent capable of stimulating an action potential. rons. They are proton pumps that, when activated by the cor- Furthermore, ChR2 was so sensitive that millisecond-scale rect wavelength of light, pump protons out of the neuron, timing was possible, enabling sensitive temporal studies to thus collapsing the current normally generated by an action take place. potential. Arch and Mac are activated by different wave- lengths of light. Hence, it is possible to use them simultane- ChR2-based research ously and independently on two neural populations. Both have advantages and disadvantages when compared with Synaptic plasticity and long-term potentiation (LTP), the NpHR. For example, Arch is able to generate much larger hypothesized molecular mechanism behind memory in photocurrents than NpHR, but requires higher light levels to humans, are areas of intensive research in which there is still do so (Chow et al., 2010). much to learn (Blundon and Zakharenko, 2008). Zhang and Oertner (2007) showed that it was possible to induce LTP NpHR-based research using ChR2, and Oertner’s group went on to use this tech- nique to reveal input-specific accumulation of αCaMKII after As with ChR2, NpHR has also led to a plethora of research. LTP (Zhang et al., 2008). Thus, it is likely that optogenetic One of the earliest studies was by Gradinaru et al. (2009), effectors will help us unravel the mystery of LTP in the future. who used NpHR to identify the specific cell type responsible for the therapeutic effect of deep brain stimulation when used For many years it has been suggested that dopaminergic to treat neurological disorders, such as Parkinson’s disease. (DA) neurons are important in appetitive conditioning (Koob NpHR has also been used for research in swim circuitry in and Le Moal, 1997). Previous techniques, however, lacked zebrafish (Arrenberg, Del Bene and Baier, 2009), gamma both the temporal and spatial precision required to investi- oscillations (Sohal et al., 2009) and neurogenesis (Yang, gate this theory further. Tsai et al. (2009) used ChR2 to Ming and Song, 2011). manipulate selectively DA neurons of the ventral tegmental area to show that phasic (50 Hz), but not tonic (1 Hz), action Discussion potential firing of DA neurons is a driver of behavioural con- ditioning. This is a lovely example of the extra precision that The discovery of ChR2 and NpHR, and their potential in ChR2 is able to provide. neuroscience, has created an explosion of research. As the effectors are light-induced, this allows for millisecond-scale Therefore, the increased resolution that ChR2 provides control of neural circuits. Thus, this extremely high temporal enables many previously inaccessible avenues to be explored. and spatial resolution of NpHR and ChR2 makes them very The opsin has also already been used to uncover new infor- powerful research tools. mation on wakefulness (Adamantidis et al., 2007), depres- sion (Covington et al., 2010) and fear (Ciocchi et al., 2010). NpHR and ChR2 are stimulated by different wavelengths These are only but a few of the discoveries generated by of light. This allows NpHR to be used in conjunction with ChR2-based research. ChR2 to induce or inhibit neural activity, depending on the researcher’s purpose. This further increases the power of Halorhodopsin these effectors as research tools. NpHR is a light-sensitive chloride pump that can hyperpolar- ChR2-induced currents have different kinetics from native ize a neuron when stimulated by 580 nm light. When membrane channel-induced currents (Ritter et al., 2008). 2+ expressed in the cell membrane, NpHR can be used to inhibit Additionally, ChR2 is Ca permeable (Nagel et al., 2003). 3 Review article Bioscience Horizons • Volume 5 2012 As neurotransmitter release is intrinsically linked with cal- Optogenetic sensors cium influx (Kasparov, 2011), light-induced action potentials have a very high chance of neurotransmitter release. Although Optogenetic methods for monitoring synaptic activity have this problem is reduced in certain ChR2 variants (Gunaydin been invaluable in neural research. Outlined below are the et al., 2010), great care must be taken when interpreting the different methods, and their respective advantages and disad- results of studies using ChR2. vantages are discussed. Improved opsins have been produced with features such as Voltage-sensitive fluorescent proteins faster kinetics (Lin et al., 2009), increased membrane expres- Voltage-sensitive fluorescent proteins (VSFPs) are able to sion (Zhao et al., 2008) and increased light sensitivity (Berndt record changes in voltages across membranes, allowing the et al., 2009). These will further increase the effectiveness of visualization of action potentials. They consist of a voltage- opsins in neural circuitry research. sensitive domain, taken from a voltage-sensitive phosphatase Long-term expression of ChR2 in the primate brain is from the organism Ciona intestinalis, coupled to either one stable (Han et al., 2009) and ChR2 has already been shown or two fluorophores (Dimitrov et al., 2007). An example of a to be of use in Parkinson’s treatment (Gradinaru et al., VSFP can be seen in Fig. 3. 2009). Therefore, ChR2 may have a future role in human When a cell expressing VSFP becomes depolarized, a con- disease treatment. However, much work will have to be formational change in the voltage sensor domain brings the done as to the safety of long-term ChR2 expression in two fluorophores closer together. This results in a change in humans. Other work has shown the potential of optogenetic the ratio of fluorescence emitted by the two fluorophores, effectors in a clinical setting [for example, light-induced res- known as a fluorescence resonance energy transfer (FRET) cue of breathing after spinal cord injury (Alilain et al. 2008)] signal. The FRET signal can be measured and used to observe and it is, unfortunately, a topic too broad to cover within current changes in cells (Mutoh et al., 2011). this review. As with optogenetic effectors, a variety of VSFP variants Blood-oxygen-level dependence (BOLD) signals measured, have been produced with improved responsiveness (Lundby using functional magnetic resonance imaging, have been used et al., 2008), different fluorescent wavelengths (Mutoh since the early 1990s to measure neural activity in the brain et al., 2009) and even different fluorophores [Tsutsui et al. (Ogawa et al., 1990). Despite the many discoveries made (2008) used a fluorophore isolated from coral]. This has with this technology, until recently it was not clear what further increased the diagnostic capability of VSFPs as exactly caused BOLD signals. Lee et al. (2010a) used a ChR2- it allows the investigator to choose the right VSFP for based approach to show that stimulation of local CaMKIIα- their experiment. More information on VSFPs can be found expressing excitatory neurons (in the neocortex or thalamus) in the comprehensive review by Peterka, Takahashi and produce positive BOLD signals. This illustrates the ability of Yuste (2011). optogenetics to resolve previously unanswerable long-stand- ing questions. Genetically encoded calcium indicators Within the brain, light scattering limits the depth of light 2+ An intracellular calcium (Ca ) influx is associated with an to a few hundred micrometres. Therefore, this makes it hard action potential. Thus, detection of calcium can be used as a to activate opsins over a large area in a non-invasive fashion (Mancuso et al., 2011). Despite the improved version of NpHR, eNpHR3.0 (see above), there is still a need for fur- ther development of non-invasive techniques of delivering light to the opsins. Lewis et al. (2009) fused a myosin-binding domain to ChR2, which successfully targeted the opsin to the somato- dendritic compartment of neurons in mice. This ability to target opsins to specific regions of the cell, or to intracel- lular organelles, is largely unexplored. As we further the tar- geting precision of the optogenetic effectors, this will boost our understanding of the specific molecular mechanisms occurring in neurons underlying neural activity. Optogenetic effectors have exploded onto the scene over the past 6 years and the rate of new discoveries that this has brought has yet to slow down. It is still a young technology Figure 3. VSFP2.3 which consists of four transmembrane domains and it will be exciting to see the future developments that fused to the fluorophores mCerulean and citrine in tandem ( Akemann optogenetic effectors will bring. et al., 2010). 4 Bioscience Horizons • Volume 5 2012 Review article method of detecting neural activity (Spruston et al., 1995). were used to visualize changes in voltage but localised 2+ Consequently, different methods of detecting Ca levels in expression was hard to achieve (Kee et al., 2008). Thus, neurons have been explored. VSFPs have a higher level of precision compared with their predecessors. D3cpVenus is one such genetically encoded calcium indi- cators (GECI), which fluoresces in response to an increase in Despite VSFP development coming a long way over the 2+ Ca and has been successfully used, both in vitro and in vivo, past few years, there is little published data to show for it. to detect single action potential spiking. Likewise, Nakai, This is so especially when compared with optogenetic effec- Ohkura and Imoto (2001) developed G-CaMP, a GFP-based tors; a technology only 2 years older. Last year, for the first probe that also showed fluorescent changes in response to time, Akemann et al. (2010) successfully used VSFPs in vivo 2+ changes in Ca levels. This has since been improved upon to report cortical electrical responses to stimuli. Hopefully, and the latest version of G-CaMP, G-CaMP3, has an increased VSFPs are coming to a point where their potential is unlocked, baseline flourescence and dynamic range and a higher affinity and questions that were previously unanswerable with classi- 2+ for Ca (Tian et al., 2009). cal methods are answered. A fundamental problem with using calcium levels as a Chloride sensors measure of action potentials is that other cellular processes 2+ As a Cl influx is normally the mechanism of synaptic inhibi- involve the alteration of Ca levels (Mancuso et al., 2011). tion, it is possible to monitor synaptic inhibition by monitor- Therefore, no matter how sensitive GECIs become, they are − − ing Cl levels. Clomeleon is a Cl sensor that works in a still at risk of overestimating action potential rates. similar way to VSFPs. An increase in Cl concentration causes However, GECIs have been used successfully to study both a decrease in FRET and thereby causes the protein to fluo- spiking activity and synaptic activity across multiple neurons resce (Kuner and Augustine, 2000). simultaneously (Dreosti and Lagnado, 2011). It will be inter- esting to follow their development and the possible informa- pH sensors tion they could uncover about synaptic circuits. During an action potential, release of neurotransmitter causes Clomeleon suffers from a low-binding affinity for Cl and a loss of protons for the cell, thereby reducing the cells’ pH. interference from background Cl concentrations which hin- The optogenetic pH sensor, pHluorin, is fused to the walls of ders Clomeleon-based studies. An improved protein, vesicles within the cell and is quenched in the acidic environ- Superclomeleon, has now been produced which is free from ment of the vesicle. Upon neurotransmitter release the pH these drawbacks. Already, Clomeleon has proved to be a use- of the vesicle increases, causing the pH sensor to fluoresce ful tool in the investigation of inhibitory neural responses which can then be recorded (Miesenbock, De Angelis and (Berglund et al., 2008; Lee et al., 2010b) and it will be inter- Rothman, 1998). esting to see if Superclomeleon is able to expand the potential Optogenetic pH sensors have been used successfully in the of Cl sensing in neural activity studies (Mancuso et al., study of synaptic vesicle exocytosis and endocytosis (Dittman 2011). and Kaplan, 2006). Recent improvements have enabled Optogenetic sensors are a diverse group which are able to researchers to detect the release or retrieval of individual monitor synaptic transmission at a variety of different stages. vesicles (Kim and Ryan, 2009). By using them in concert with one another, it would be pos- sible to compare the sensitivity and accuracy of each tech- Neurotransmitter release sensors nique at measuring neural activity. GluSnFR and FLIPEO are two FRET-based optogenetic sen- sors that are anchored to the cell membrane and fluoresce in Concluding remarks response to glutamate, the most common excitatory neu- rotransmitter (Hires, Zhu and Tsien, 2008; Okumoto et al., Optogenetics is a fast growing field; more than 800 laborato- 2005) These sensors are sensitive enough to detect a single ries around the world are now engaged in optogenetic spike (Dreosti and Lagnado, 2011). research (Deisseroth, 2011). Despite copious amounts of research over the past 5 years, a consistent flow of new dis- Discussion coveries is being produced, both in neural circuitry research Neurotransmitter release sensors are yet to be used to uncover and in improving current optogenetic methods. any novel information. Despite the impressive ingenuity Optogenetic effectors have increased our ability to manip- behind neurotransmitter release sensors, it is difficult to ulate neural circuits while optogenetic sensors are increasing imagine their use in neural circuit research reaching levels our ability to observe such circuits. It will be exciting to see if comparable to other optogenetic methods. further developments will increase their ability to comple- As VSFPs are expressed by cells, this allows them to ben- ment one another, which would cause further acceleration to efit from genetic targeting. Previously, voltage-sensitive dyes the already fast pace of neural research. 5 Review article Bioscience Horizons • Volume 5 2012 Deisseroth, K. (2011) Optogenetics, Nature Methods, 8, 26–29. Author biography Deisseroth, K., Feng, G., Majewska, A. K. et  al. (2006) Next-generation James Butler has just finished a biology degree at Imperial optical technologies for illuminating genetically targeted brain cir- College London. While there, he developed a fascination cuits, The Journal of Neuroscience, 26 (41), 10380–10386. with neuroscience that has resulted in him pursuing a research Dimitrov, D., He, Y., Mutoh, H. et al. (2007) Engineering and characteriza- career in this field. In 2011, he is due to start a masters in tion of an enhanced fluorescent protein voltage sensor, PLoS One, 2, neuroscience at University College of London, Europe’s lead- e440. ing neuroscience institution. Dittman, J. S. and Kaplan, J. M. (2006) Factors regulating the abundance References and localization of synaptobrevin in the plasma membrane, Proceedings of the National Academy of Sciences USA, 103, 11399– Adamantidis, A. R., Zhang, F., Aravanis, A. M. et  al. 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Bioscience HorizonsOxford University Press

Published: Dec 26, 2012

Keywords: Neural neuron brain review halorhodopsin channelrhodopsin

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