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For exceptions, permission may be sought for such use through Elsevier’s permissions site at: http://www.elsevier.com/locate/permissionusematerial New optical tools for controlling neuronal activity Stefan Herlitze and Lynn T Landmesser Corresponding author: Herlitze, Stefan ([email protected]) This review comes from a themed issue on Development Edited by Ben Barres and Mu-Ming Poo Available online 15th December 2006 or 's 0959-4388/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2006.12.002 Introduction Au th In cells, light is used for two main purposes: first, to produce energy through photosynthesis; and second, to couple extracellular stimuli to intracellular signaling pathways, such as enzymes, ion transporters or ion channels. The second use is particularly interesting to us because switching on and off ion transporters, ion channels or second messenger pathways by light provides an opportunity to control non-invasively the membrane potentials and second messenger cascades in a living animal. Recently, it has been demonstrated that neuronal circuits can be manipulated through the exogenous expression of mutated ion channels and G-protein-coupled receptors (GPCRs). Here we review recent progress made in this area and discuss the specific shortcomings of some of the methods proposed. www.sciencedirect.com y co p Invertebrate rhodopsins During activation of the invertebrate visual system, the excited GPCR (rhodopsin) stimulates a Gq/11 protein, which then activates phospholipase C, leading to the production of inositol (1,4,5)-trisphosphate and diacylglyercol. These second messengers activate non-specific cation channels, which depolarize the cell. The G protein signal is turned off when arrestin binds to the GPCR to initiate regeneration of 11-cis retinal, the ligand of the photoreceptor. al pe Current Opinion in Neurobiology 2007, 17:87–94 In the visual system of invertebrates and vertebrates, light of different frequencies can activate a molecular cascade leading to the depolarization of a nerve cell in invertebrates or the hyperpolarization of a nerve cell in vertebrates. The potential for the invertebrate light cascade to depolarize cells outside their normal environment was first recognized by Miesenbock and colleagues [1]. By expressing ten different proteins of the invertebrate light cascade, they identified the three minimal structural components of an invertebrate GPCR light-induced cationic current. The three proteins that were necessary and sufficient were the GPCR (NinaE), the G protein aq subunit and arrestin-2. Exogenous expression of these three proteins, collectively called chARGe, in hippocampal neurons was found to induce action potential firing (spiking) after light activation. The technique, however, had some limitations. rs Addresses Department of Neurosciences, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4975, USA Visual system GPCRs for slow control of nerve cell activity on A major challenge in understanding the relationship between neural activity and development, and ultimately behavior, is to control simultaneously the activity of either many neurons belonging to specific subsets or specific regions within individual neurons. Optimally, such a technique should be capable of both switching nerve cells on and off within milliseconds in a non-invasive manner, and inducing depolarizations or hyperpolarizations for periods lasting from milliseconds to many seconds. Specific ion conductances in subcellular compartments must also be controlled to bypass signaling cascades in order to regulate precisely cellular events such as synaptic transmission. Light-activated G-protein-coupled receptors and ion channels, which can be genetically manipulated and targeted to neuronal circuits, have the greatest potential to fulfill these requirements. First, the slow activation and deactivation of neuronal firing made it difficult to control precisely the firing pattern of the neuron. In addition, the precise cellular mechanism by which these constructs regulated neuronal firing was not investigated. The induction of neuronal firing might be mediated through a decrease in K+ conductance, for example, by KCNQ channels, which are inhibited by activation of Gq-coupled receptors. If this is the case, then chARGe would have to be expressed in neurons with sufficient M-currents to be able to affect neuronal firing. Second, the construct is very complex, making it difficult to express as a transgene in other animals [1]. As an alternative to invertebrate rhodopsin, melanopsin, a photopigment of retinal ganglion cells, could be used. Three independent studies have recently shown that vertebrate melanopsins couple to the Gq pathway and can, for example, activate transient receptor potential (TRP) channels [2–4]. Whether or not this approach will work in neurons remains to be demonstrated. Current Opinion in Neurobiology 2007, 17:87–94 88 Development y co p Indirect photoactivation: ligands and channel blockers In the first approach, chemical compounds were synthesized for the light activation of two ligand-gated ion channels: the capsaicin receptor TRPV1 and the purinergic receptor P2X2 [12]. Mammalian TRPV1 had previously been used to confer capsaicin avoidance responses in Caenorhabditis elegans, which is normally not responsive to capsaicin [13]. Ester linkage of an inactive, photosensitive molecule to the active ligand facilitated activation of the ligand and channel by 1 s pulses of short wavelength (<400 nm) light in neurons to trigger action potential firing with some delay. According to the dose–response relationship of the current response, this method enabled the firing frequencies to be adjusted by the application of different concentrations of ligands. This approach would thus be very useful for inducing different firing patterns during constant application of light and/or ligand. rs Indeed, other Gi/o-coupled receptors have been recently used to silence neuronal firing. The silencing of cortical and thalamic neurons from various species, including rats, ferrets and monkeys, was achieved by exogenously expressing the insect GPCR AlstR (the Drosophila allatostatin receptor). Application of its peptide ligand allatostatin in neurons expressing the AlstR was found to inactivate the neurons reversibly within minutes [8,9!]. the tool of choice for rapid control of nerve cell activity. The regional expression of a genetically modified K+ channel in Drosophila provided the first example of using an ion channel to control the excitability of the targeted cell (i.e. muscle, neurons, photoreceptors). Unfortunately, at higher transgene dosages the expression of these channels led to the loss of neuronal function; thus, the extent of excitability of the targeted cell could not be controlled during the experiment [11]. This problem has been overcome by the development of photoactivated (caged) chemicals, which are used as ligands or blockers to increase or to decrease ion conductances once activated by light. al In vertebrates, light activation of the GPCR rhodopsin leads to dissociation of the G protein and subsequent activation of a phosphodiesterase, which hydrolyzes cyclic guanosine monophosphate (cGMP) to 50 GMP. The reduction in cGMP induces the closure of cGMP-gated cation channels, resulting in hyperpolarization owing to a reduction in Na+ and Ca2+ influx [5,6]. Vertebrate rhodopsin was found to couple to the G protein transducin. Because the a subunit of transducin belongs to the Gi/o subfamily [7], it seemed possible that the mammalian rhodopsins might couple to other members of this subfamily. Activation of the Gi/o pathway in neurons usually leads to a reduction in the firing rate through activation of a G-protein-coupled inward rectifier K+ channel (GIRK) and a reduction in synaptic transmitter release through inhibition of presynaptic Ca2+ channels. on Vertebrate rhodpsins Au th or 's pe We and our co-workers [10!!] also used this approach when we expressed the vertebrate rhodopsin RO4 together with GIRK or Ca2+ channels in HEK293 cells, which enabled us to demonstrate that the GPCR can activate K+ currents and inhibit Ca2+ currents expressed through recombinant constructs. Expression and light activation of RO4 in cultured hippocampal neurons hyperpolarized the cell membrane within a second, leading to a reduction in the firing rate of the neurons and suggesting that, in neurons, RO4 activates GIRK currents on somatodendritic areas. Analysis of the presynaptic transmission parameter ‘paired pulse facilitation’ revealed that, once RO4 was activated, an increase in paired pulse facilitation in hippocampal autapses was induced, suggesting that RO4 was also transported and was active at presynaptic sites. This result has interesting implications for controlling Gi/o pathways in defined subcellular structures by targeting the light-activated rhodopsin in the cell area of choice. Thus, light-activated GPCRs of the rhodopsin family can be used to increase neuronal activity when coupled to the Gq pathway and to decrease activity when coupled to the Gi/o pathway. Ion channels for rapid control of nerve cell activity Activation and deactivation of GIRK channels by pertussis toxin (PTX)-sensitive G proteins in neurons occur within one to several seconds. By contrast, ligand-gated and voltage-gated ion channels can be activated within microseconds, making the direct gating of ion channels Current Opinion in Neurobiology 2007, 17:87–94 The second approach was based on the photoconversion of a K+ channel blocker [14]. This blocker is in the stretched trans configuration at longer wavelengths (500 nm) and is converted to a more bulky cis form at shorter wavelength (380 nm). The stretched form can enter the open pore and reduce the K+ conductance, whereas the bulky form cannot. The experiments were done with the shaker K+ channel, a voltage-gated channel, and were based on the idea that molecules containing a quaternary ammonium, such as tetraethylammonium, block the channel when entering from the extracellular site. A glutamate to cysteine substitution in proximity of the tetraethylammonium-binding site facilitated tethering of a photoactivated compound containing the quaternary ammonium ion MAL-AZO-QA (MAL is malemide for attaching the molecule to the cysteine near the channel pore, AZO is a photoisomerizable group, and QA is the quaternary ammonium). By expressing the modified shaker K+ channel in hippocampal pyramidal neurons and loading the neurons with MAL-AZO-QA 15 min before recording, it was shown that switching the wavelength between 500 and 390 nm could control action potential firing in these neurons. At 500 nm, neuronal activity was induced owing to blockage of the shaker K+ www.sciencedirect.com Controlling neuronal activity Herlitze and Landmesser 89 channel, whereas at 390 nm neurons were silenced because the blocker was released from the channel. acids, has weak sequence similarity to bacteriorhodopsin and halorhodopsin, and comprises seven putative transmembrane segments. Most importantly, expression of ChR1 or carboxy (C)-terminally truncated ChR1 mRNAs in the Xenopus oocyte expression system results in a robust photocurrent with fast activation and deactivation kinetics during light pulses. The current carrier is H+ and current activation is wavelength dependent. The inward photocurrent in Xenopus oocytes is maximal at "500 nm, and there is no detectable activation of ChR1 at either 400 nm or 600 nm, raising the possibility of using blue- and red-shifted variants of the photoreceptor as probes for coactivation and/or colocalization. Furthermore, during light activation of the channel, the H+ current does not attenuate. Whether H+ conductances can be used to depolarize neurons sufficiently to trigger action potential firing remains to be demonstrated but ChR1, with its fast activation and deactivation kinetics, set the stage for using a light-activated channel to control membrane potential. co p al on A breakthrough came with the cloning and electrophysiological characterization of channelrhodopsin-2 (ChR2) [21]. ChR2 is also a proton channel, but it is non-selective and permeable to various cations including Na+, K+ and Ca2+. The ChR2 current has two components: a large inactivating or desensitizing current, followed by a steady-state current. The activation and deactivation kinetics, the current size, and the ratio between the inactivation current and the steady-state current are dependent on the duration and intensity of the light [10!!,22!]. This channel therefore provides the potential to control precisely the amount of depolarization by adjusting the light stimulation protocol. The ChR2 current is maximally activated around 480 nm and shows no activation at wavelengths >575 nm [21]. or 's pe rs In a similar approach, the ligand-binding domain of the ionotropic glutamate receptor (iGluR-L439C mutation) was modified to bind a photoactivated-glutamate-containing compound called MAG (M is the cysteine-reactive maleimide, A is the azobenzene photoswitch, and G is the glutamate head group) [16!]. This mutated channel, termed LiGluR, can be gated by both light and ligand. The covalent attachment of MAG to the cysteine located at the lip of the clam shell binding pocket of the GluR facilitates stabilization of the closed and open state of the channel, depending on whether the compound is in the trans (closed channel) or cis (open channel) configuration. Short wavelengths of light (380 nm) stabilize the cis conformation, whereas 500 nm light stabilizes the trans form. Although the biophysical designs of these experiments are very elegant, the modified channels must be loaded with the light-activated blocker before the experiment for a lengthy amount of time. This drawback might limit the application of this approach in terms of neurons, but it has potential to be very useful for controlling cell activity in other organs such as heart, where the compound can be applied through the blood stream at sufficient concentration. y Introduction of the point mutation V443Q in the selectivity filter converted the K+-selective shaker channel into a non-selective cation channel [15!]. When expressed in primary neuronal cultures, this channel could conduct Na+ and thus depolarize the cell membrane. Action potential firing was elicited once QA-mediated channel block was released at 390 nm. These light-controllable K+ channels are now called H-SPARK and D-SPARK for ‘synthetic photoisomerizable asobenzene-regulated K+ channels and will, respectively, hyperpolarize and depolarize the cell membrane. Au th In general, for ion channels and GPCRs activated by chemical stimuli, the need for ligand application and especially the time for wash out will limit the use of these constructs in large tissues such as brain slices or whole brain. The above examples demonstrate, however, that expression of foreign genes in neurons or other cells is a suitable method to non-invasively modulate the state of a cell and thus to elucidate mechanisms underlying neuronal development, neuronal circuit function, and behavior. Direct photoactivation: channelrhodopsin In phototactic green algae, photoreceptor currents induced by light are carried by Ca2+ or H+ [17] and are activated within 30 ms [18,19]. One of the rhodopsinmediated photoresponses from Chlamydomonas reinhardtii is mediated by a rhodopsin-like protein, the channelrhodopsin-1 (ChR1) [20]. This protein consists of 712 amino www.sciencedirect.com Three groups have independently tested the potential of ChR2 to control nerve cell firing [10!!,22!,23!] and demonstrated that, in both cultured hippocampal neurons and hippocampal slices, short light pulses (5–10 ms) are sufficient to trigger action potentials; in addition, neurons can follow light stimulation protocols with action potential firing up to 30 Hz. These observations correlate well with the deactivation of ChR2 within 20–40 ms [10!!,22!]. Thus, ChR2 is currently the molecular probe of choice to control action potential firing in neurons. One problem might be the small single channel conductance of ChR2, which has been estimated at "50 fS. As a result, high expression of ChR2 in the target cell might be necessary to activate sufficient inward current to depolarize the cell membrane. Chloride conductance As mentioned above, hyperpolarization within a cell can be caused by increasing not only the K+ conductance but also the Cl# conductance. Thus, another way to silence Current Opinion in Neurobiology 2007, 17:87–94 90 Development y co p RO4 and ChR2 in chicken spinal cord al We and our co-workers [10!!] have tested two constructs for their capability to silence or to activate neuronal circuits. Embryonic chicken spinal cord was electroporated with either vertebrate rhodopsin (RO4) or ChR2 to try and control precisely the spontaneous firing of the spinal cord motor neurons [10!!]. Embryonic chicken spinal cords show rhythmic episodes of spontaneous bursting activity, the frequency of which is important for motor axon pathfinding. Spinal cord preparations were incubated with all-trans retinal 2 min before the recording to supply a sufficient amount of chromophore for either RO4 or ChR2. rs The concept that controlling the Cl# conductance in cultured hippocampal neurons could silence neuronal activity was first demonstrated with an invertebrate ligand-gated Cl# channel [30]. Coexpression of a and b subunits of the glutamate-gated Cl# channel from C. elegans in neurons was necessary to activate the channels by application of the chemical compound ivermectin. Light-activated Cl# transport also could be achieved with halorhodopsin, which was first detected in the purple membranes of Halobacterium salinarum. Halorhodopsin is activated by light and is a Cl# transporter that carries Cl# ions inward, thereby resulting in hyperpolarization of the cell membrane. When halorhodopsin is excited, Cl# binds to the extracellular site of the receptor [31]. Light activation leads to several conformational changes in the protein, accompanied by an alteration in the accessibility and affinity of the binding site, resulting in release of Cl# at the intracellular surface of the cell and subsequent restoration of the unexcited state of the receptor [32]. Thus, light-activated Cl# transport provides another mechanism for hyperpolarizing cells and could be of particular interest for developmental neuroscientists. In the second application [33!!], P2X2 receptors were expressed in Drosophila dopaminergic neurons to elucidate the modulatory function of dopamine on movement. Four 150 ms UV light pulses, separated by 1.5 s, increased locomotor activity in flies, as evaluated by travel speed and pause duration. Interestingly, light-induced dopamine release also seemed to influence the flight routes, suggesting that dopamine might be involved in controlling motivational behavior in flies. This study shows that caged compounds, which activate ion channels, can be used to control neuronal circuits in invertebrate systems. on neurons would be through an increase in Cl# conductance. The equilibrium potential of Cl# changes, however, during development. Although Cl#-based g-amino butyric acid (GABA)-mediated currents in the hippocampus, neocortex, hypothalamus and spinal cord are depolarizing early in development, they are hyperpolarizing in the adult animal [24–29]. Thus, an increase in Cl# conductance in adult mice will cause hyperpolarization rather than depolarization. pe Light application increased the intervals between bursting episodes and decreased asynchronous firing of motor units between episodes in spinal cords expressing RO4, as would be expected if RO4 were causing hyperpolarization of the cell membrane. Notably, switching off the light often induced immediate bursting of the neuronal network, which suggests that the RO4 induced hyperpolarization acts to synchronize motor neurons most probably by relieving the Na+ channels from inactivation. By contrast, exposing cords expressing ChR2 to continuous light (485 nm) increased the frequency of bursting of the neuronal circuit and decreased the intervals between episodes. The precise pattern of frequency bursts could be controlled by brief, 3 s applications of light, each of which elicited immediate bursts. 's Light-activated switches in physiologically relevant neuronal networks or So far three different light-activated proteins have been used to control neuronal circuits in intact tissue and live animals. P2X2 receptors in Drosophila Au th The first approach, involving the expression of the ATPgated P2X2 receptors in the Drosophila nervous system, has been used to trigger action potentials for controlling several escape-related behaviors and to investigate the role of dopaminergic input for motor control [33!!]. Specific expression of the P2X2 receptors in the Drosophila giant fiber system — a well-defined and well-characterized neuronal circuit responsible for the escape movements of jumping and initiation of flight — revealed that short (150–250 ms) UV laser pulses were sufficient to trigger flight behavior. Flight and jumping could be induced in both blind and decapitated flies, supporting the notion that the light-induced behavior was due to the exogenous receptors. Flies had to be microinjected with caged-ATP 10 min before the experiments and the efficacy of the response behavior had a half-life of "75 min. Current Opinion in Neurobiology 2007, 17:87–94 To examine the possibility that ChR2 could be used to control neuronal activity in intact animals, we also assessed whether light could induce axial movement of intact embryos in ovo. Depending on the developmental stage, bursts of cord activity cause axial movements. Light application through an egg shell window was sufficient to activate ChR2 and to induce movement of the embryo. This observation indicates that a retinal derivative sufficient to act as a light-activated ligand is supplied by the embryo or egg. This approach has potential applications. First, expression of RO4 and ChR2 in specific subsets of spinal cord motor neurons and interneurons could be used to understand the circuitry in the spinal cord and the influence that rhythmic activity has on spinal cord development. Second, in cases of spinal cord injury, www.sciencedirect.com Controlling neuronal activity Herlitze and Landmesser 91 light could be used either to non-invasively activate regenerating descending input to determine the effect of activity on regeneration, or to directly activate denervated neurons to prevent inactivity-induced changes. co p al In addition, ChR2(H134R) was expressed in mechanosensitive neurons in C. elegans, with the result that light activation of these neurons led to withdrawal responses that are normally observed when the worms are tapped. Interestingly, the Na+ and Ca2+ influx through ChR2(H134R) could substitute for mechanosensitive ion channels: worms carrying mutations in MEC-4 and MEC-10 mechanosensitive ion channels are insensitive to touch, but touch responses could be restored by light activation of ChR2. Ultimately, the withdrawal response habituated during the light stimulation as do touch responses. The mechanism underlying habituation has not been addressed in detail. In summary, this study shows that ChR2 can be used not only to induce action potentials but also to trigger signaling events, which depend on both depolarization and ion specificity such as Ca2+. Lastly, the transparency of C. elegans makes it the optimal biological system for using light-activated switches to understand neuronal circuitry underlying behavior. Au th or 's pe rs Expression of ChR2 was found to be stable for up to 16 months, suggesting that viral gene transfer might be useful for long-term expression of ChR2 not only in the eye but also in other tissues [34!!]. Electrophysiological recordings from ChR2-expressing retinal neurons indicated that light induced cell-type-specific spiking in these neurons. Spike frequency was dependent on the light intensity, suggesting that third-order neurons in the retina (which are normally not responsive to light) can translate light intensities into spike firing rates. Lightinduced responses could be observed for several hours without loss of activity, which suggests that the lightsensitive chromophore (the retinal compound) is constantly available and can be restored for ChR2 within the retinal ganglion cells without external application. By measuring visually evoked potentials from the visual cortex — the brain area into which retinal ganglion cells project — 460 nm light but not 580 nm light evoked potentials in blind rd1/rd1 mice that expressed ChR2 in the ganglion cells. Whether these mice were now able to see or to react to light stimuli in a meaningful, behavioral context was not assessed; however, the study shows that ChR2 has the potential to restore light sensitivity in the vertebrate eye. on Indeed, ChR2 has been recently used to restore visual responses in mice by converting inner retinal neurons, which are normally light insensitive, into light-sensitive neurons [34!!]. In a mouse model deficient in functional photoreceptor cascades, ChR2 was expressed throughout the retina, and prominently in retinal ganglion cells, by an adeno-associated virus vector. Maximal ChR2 currents were measured, without the addition of all-trans retinal, with 460 nm light in acutely dissociated inner retinal neurons from wild-type mice. ChR2 was also expressed in homozygote rd1 mice through injection of ChR2 into the eye of newborn or adult mice. rd1 mice carry a mutation in cGMP phosphodiesterase (PDE6) and therefore their photoreceptor signaling is non-functional. Because similar mutations are found in humans in some forms of retinitis pigmentosa, it was proposed that ChR2 might be used as a therapeutic approach to cure blindness. y ChR2 in mouse retina osensory neurons of C. elegans. They used ChR2(H134R), a mutant with a larger stationary photocurrent in comparison to wild-type protein, for these studies. The transgenic worms were raised in the presence of all-trans retinal and ChR2 was activated by light of 450–490 nm. ChR2(H134R) was expressed in the body wall and egglaying muscles and was localized in the muscles in the endoplasmic reticulum and the cell membranes, as expected for a transmembrane protein and as previously observed in hippocampal neurons and HEK293 cells [10!!,23!]. Light activation of ChR2(H134R) induced muscle contraction, often accompanied with egg-laying as a result of the contraction of the vulva muscles. Switching off the light led to muscle relaxation within a second. Interestingly, ChR2(H134R) might be sufficient to induce contraction directly by means of Na+ and Ca2+ influx through its own pore, bypassing the nicotinic nAChR and L-type Ca2+ channels (egl-19). Note that muscle contraction in C. elegans is mediated by Na+ influx through nAChR and the depolarization-induced opening of voltage-gated Ca2+ channels (egl-19), followed by Ca2+-induced Ca2+ release from the sarcoplasmic reticulum through activating ryanodine receptors. ChR2 in C. elegans A limitation of light-activated constructs in, for example, deep brain areas is that light might not be able to penetrate to the relevant cellular layers. This problem should not exist for transparent or thin cell layers, or for transparent animals such as C. elegans. Nagel et al. [35!] have therefore expressed ChR2 in muscle and mechanwww.sciencedirect.com Application of light-activated ligands: the cell helps itself Light-activated receptors or channels require the application and regeneration of a light-sensitive ligand to activate the receptor, which can be a problem for approaches that use synthesized compounds. The receptors have to be loaded before the experiment with a compound that has the potential to induce toxicity depending on the length of application and the compound itself. Fortunately, by using retinal-derived ligands, the cellular environment seems to help itself by providing the receptor with its own active molecule. The importance of this issue warrants that we discuss it in more detail. Current Opinion in Neurobiology 2007, 17:87–94 92 Development Conclusions co p y In any case, whether one uses vertebrate, invertebrate, bacterial or plant-derived light-sensitive proteins, application of all-trans retinal seem to be sufficient to achieve light responses in mammalian cells. At least in the vertebrate eye and the chicken spinal cord, sufficient retinal compounds are available in the tissue to drive lightactivated currents without external supply of the chromophore. Whether this is true for every tissue remains to be investigated. al Controlling cellular signaling by light using light-activated switches has great potential for understanding basic cellular mechanisms and to elucidate how these mechanisms influence and determine system function and animal behavior. Several studies in Drosophila, C. elegans, chicken and mice have revealed the feasibility of the approach. In addition, development of light-activated molecular machines will have important implications for curing disease. For example, controlling ion conductance in the heart through light could be used to regulate the frequency of the heartbeat in disease conditions such as atrial fibrillation. In addition, locomotor circuits capable of producing walking behavior are contained in the lower spinal cord after cord injury, but the level of excitability is below the threshold for their activation. Light-controlled ion conductances could here be used to activate these circuits after spinal cord injury or to raise the level of excitation so that spared fibers of the brain are capable of causing locomotion. Moreover, neuronal function in the basal ganglia is severely impaired in Parkinson’s disease. Deep brain stimulation is currently used in individuals with Parkinson’s to overcome tremor, rigidity, akinesia and gait. Light-activated channels could be used for precise activation or silencing of neuronal circuits of hyper- or hypoactive neuronal circuits involved in Parkinson’s disease such as the internal segment of the globus pallidus and subthalamic nucleus. pe rs In contrast to invertebrates, the vertebrate system possesses numerous enzymes and retinoid-binding proteins in its visual cycle [37]. In vertebrates, the cis–trans and trans–cis isomerization of retinal are locally separated and do not occur in the same photoreceptor; thus, the isomerization products must move between different cell types and distinct subcellular regions within a cell of the retina by means of retinoid-binding proteins. The photoconversion from trans to cis is mediated by an isomerase. to all-trans is driven by the energy conversion produced by light and by conformational changes in the protein during ion translocation. on Phototransduction in many systems involves the isomerization of a photosensitive pigment — namely, an aldehyde of vitamin A retinal. This light-sensitive pigment binds to a lysine through a protonated Schiff base in all rhodopsin-type proteins. Vertebrates and invertebrates use derivatives of 11-cis retinal as chromophores. Light responses are initially mediated by the photoisomerization of this 11-cis retinal to all-trans retinal in all systems described so far, but differences exist in the regeneration of 11-cis chromophores. In invertebrates, the all-trans isomer remains bound in the receptor pocket in the thermally stable metarhodopsin state. The advantage here is that the active photoproduct can be reformed without deprotonation of the Schiff base by the absorption of a second photon [36]. Thus, the equilibrium between rhodopsin and metarhodopsin is sufficient to drive the photocycle and additional retinoid-binding proteins are not necessary. th or 's In contrast to the complicated mechanism in the vertebrate eye, in heterologous expression systems such as HEK293 cells, activation of human rhodopsin (when expressed in these cells) can be achieved for longer than 4 h once the cells are loaded with 11-cis retinal. No further application of 11-cis retinal is necessary, suggesting that at least HEK293 cells contain the intrinsic capability to regenerate 11-cis retinal or other analogs such as 9-cis or 13-cis retinal to activate their photoreceptors [38]. Thus, single application and/or long-time perfusion of 11-cis retinal or its analogs 9-cis or 13-cis or even all-trans retinal (see [38]) is sufficient to regenerate the active compounds necessary for repetitive light activation of vertebrate photoreceptors. Au Bacteria and plants use the all-trans isomer of retinal as the chromomer of choice for the active receptor state. In the dark, retinal in its receptor-binding pocket is in an equilibrium of the all-trans, 15-anti and 13-cis, 15-syn configurations [39,40]. Only the all-trans configuration can induce the ion transport mechanism, at least in bacteriorhodopsins and halorhodopsins [41]. During illumination, all-trans retinal is converted to 13-cis retinal, accompanied by several conformational changes in the protein, resulting in net ion transport. Reisomerization to all-trans retinal after the ion is released restores halorhodopsin in the unexcited state [31,32,42]. Thus, the isomerization of the retinal from all-trans to 13-cis and back Current Opinion in Neurobiology 2007, 17:87–94 Whatever new applications and designs for light-activated switches arise, improvement in light sources are needed for precise triggering of signaling events, such as activation of channels or GPCRs at defined subcellular regions such as presynaptic terminals. In addition, light sources must be developed that can be used as implants in living animals to control cellular activity in vivo and to deliver light deep into tissue — an issue that will be particularly important for controlling neuronal activity in the brain. Chemically controlled methods using compounds that can cross the blood–brain barrier might provide an alternative to light activation. For example, the chemical induction of homo- or heterodimerization of modified synaptic proteins has been recently applied to reversibly www.sciencedirect.com Controlling neuronal activity Herlitze and Landmesser 93 4. Melyan Z, Tarttelin EE, Bellingham J, Lucas RJ, Hankins MW: Addition of human melanopsin renders mammalian cells photoresponsive. Nature 2005, 433:741-745. 5. Ebrey T, Koutalos Y: Vertebrate photoreceptors. Prog Retin Eye Res 2001, 20:49-94. 6. Montell C: Visual transduction in Drosophila. Annu Rev Cell Dev Biol 1999, 15:231-268. Among the different light switches that have been used, ChR2 seems to have the greatest potential for fast and continuous control of membrane depolarization; however, further improvements of ChR2 regarding its ion selectivity, ion conductance and its spectral sensitivity are needed. Combination of light-activated GPCRs and ion channels that differ in their spectral sensitivity would provide the means for simultaneous control of depolarization and hyperpolarization. We think that we are looking at a bright future. Let the light shine! 7. Downes GB, Gautam N: The G protein subunit gene families. Genomics 1999, 62:544-552. 8. Lechner HA, Lein ES, Callaway EM: A genetic method for selective and quickly reversible silencing of Mammalian neurons. J Neurosci 2002, 22:5287-5290. co p 9. ! Tan EM, Yamaguchi Y, Horwitz GD, Gosgnach S, Lein ES, Goulding M, Albright TD, Callaway EM: Selective and quickly reversible inactivation of Mammalian neurons in vivo using the Drosophila allatostatin receptor. Neuron 2006, 51:157-170. This paper, together with [8], establishes use of the Drosophila allatostatin receptor, a Gi/o-coupled receptor, to silence neuronal function. Functionality in several tissues is demonstrated. 10. Li X, Gutierrez D, Hanson MG, Han J, Mark MD, Chiel H, !! Hegemann P, Landmesser LT, Herlitze S: Fast non-invasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc Natl Acad Sci USA 2005, 102:17816-17821. This study uses the vertebrate rhodopsin and the green algae channelrhodopsin-2 (ChR2) to either hyperpolarize or depolarize the cell membrane of HEK293 cells, hippocampal neurons and chicken spinal cord. It is the first paper to demonstrate the use of antagonistically acting light switches in an intact tissue. on al Update 11. White BH, Osterwalder TP, Yoon KS, Joiner WJ, Whim MD, Kaczmarek LK, Keshishian H: Targeted attenuation of electrical activity in Drosophila using a genetically modified K+ channel. Neuron 2001, 31:699-711. or 's pe rs Recent work has also described the application of ChR2 in Drosophila larvae to determine whether defined neuronal circuits can distinguish between appetitive or aversive learning [44]. Pairing of odor stimuli, which were either appetitive or aversive, with a second neutral stimuli (fructose) led to an odor specific behavior. Expression and activation of ChR2 either in octopaminergic or dopaminergic neurons during application of the odors could substitute for fructose, suggesting that octopaminergic pathways underlie appetitive learning while the dopaminergic pathway determines aversive learning. In another study, ChR2 was delivered stereotaxically using lentivirus into the hippocampus to control excitation of ChR2 negative cells by light [45,46]. In a very interesting study a photoacivated adenylyl cyclase (PAC) from the flagellate Euglena gracilis was used to control cAMP levels in Xenopus oocytes, HEK293 cells, and Drosophila. PAC is activated by 455 nm and does not need any cofactor. 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