Optogenetics is not simply photoexcitation or photoinhibition of targeted cells; rather, optogenetics must deliver gain or loss of function of precise events—just as in genetics, where single-gene manipulations are the core currency of the field. This means that in neuroscience, millisecond-scale precision is essential to true optogenetics, to keep pace with the known dynamics of the targeted neural events such as action potentials and synaptic currents. Moreover, this level of precision must be operative within intact systems including freely moving mammals. All strategies to achieve optical control, including those involving microbial opsin genes, initially displayed serious limitations in meeting this goal. The multicomponent character, longer-timescale temporal properties, and/or requirement for high-intensity UV light characteristic of the earlier strategies () have limited adoption and application to mammalian and other systems, but single-component microbial opsin gene strategies also initially displayed problems as well ranging from inadequate control capability () to toxicity () to challenges linked to light delivery in vivo (). A long process of tool engineering and substantial development of enabling technologies was required over the next several years.

The key properties of these microbial optogenetic tools relate to the ecology of their original host organisms, which respond to the environment using seven-transmembrane proteins encoded by the type I class of opsin gene (). Type I opsins are protein products of microbial opsin genes and are termed rhodopsins when bound to retinal. However, in typical heterologous expression experiments the precise composition of retinoid-bound states is uncharacterized. Therefore in the setting of neuroscience application, the tools are conservatively referred to as opsins (a more accurate and convenient shorthand for common use, since only “opsin” correctly applies to the genes as well as to the protein products). These proteins are distinguished from their mammalian (type II) counterparts, in that they are single-component light-sensing systems; the two operations—light sensing and ion conductance—are carried out by the same protein.

The first identified, and still by far the best studied, type I protein is the haloarchaeal proton pump bacteriorhodopsin (BR; Figure 1 A ;). Under low-oxygen conditions, BR is highly expressed in haloarchaeal membranes and serves as part of an alternative energy-production system, pumping protons from the cytoplasm to the extracellular medium to generate a proton-motive force to drive ATP synthesis (). These light-gated proton pumps have since also been found in a wide range of marine proteobacteria as well as in other kingdoms of life, where they employ similar photocycles () and have been hypothesized to play diverse roles in cellular physiology ().

(B) Kinetic and spectral attributes of optogenetic tool variants for which both of these properties have been reported and for which minimal activity in the dark is observed. Visible spectrum shown; not venturing into the ultraviolet is preferred, for safety and light penetration reasons, although the 450–470 nm peak probes also can be excited very effectively with UV light (∼360–390 nm). Decay kinetics are plotted against peak activation wavelength only to demonstrate groupings and classes over the range of spectral and temporal characteristics and the feasibility of dual channel control using tools that are well separated in the spectral and temporal domains; see Table 1 for additional information and references. Kinetic data are not published for the proton pump Mac but the Mac action spectrum peak ∼565 nm is identical to that of Arch (). Opto-XR kinetics were obtained in vivo and should be taken only as an upper bound since the assay involved a downstream measure (spiking). Decay kinetics are temperature dependent; all other reported values except ChRGR are recorded at RT, with ∼50% decrease in τexpected at 37C.Since ChRGR has only been studied at elevated (34°C) T, we denote likely RT range for ChRGR shifted to the right. Values for channelrhodopsin/fast receiver and channelrhodopsin/wide receiver () can be estimated at 7 and 14 ms, respectively; these are not shown but respond at 470 nm and have not yet been functionally validated in neurons. L132C (CatCH) τvalue was not measured in neurons, and its properties may depend on other channels in the host cell as well as the host cell tolerance of, and response to, higher levels of elevated intracellular Ca).

A second class of microbial opsin genes encodes halorhodopsins ( Figure 1 A). Halorhodopsin (HR) is a light-activated chloride pump first discovered in archaebacteria (). The operating principles of halorhodopsin (HR) are similar to those of BR (), with the two main differences being that halorhodopsin pumps chloride ions and its direction of transport is from the extracellular to the intracellular space. Specific amino acid residues have been shown to underlie the differences between BR and HR in directionality and preferred cargo ion (). After initial identification of halorhodopsin, other members of this class soon followed; for example, Lanyi and colleagues expanded the family by identifying a halorhodopsin from Natronomonas pharaonis in 1982 (NpHR;).

Next, a third class of conductance-regulating microbial opsin gene (channelrhodopsin or ChR) was identified ( Figure 1 A). Nagel and Hegemann demonstrated light-activated ion-flux properties () for a protein encoded by one of the genomic sequences from the green algae Chlamydomonas reinhardtii, as Stoeckenius, Oesterhelt, Matsuno-Yagi, and Mukohata had earlier for the proteins halorhodopsin and bacteriorhodopsin. Subsequent papers from several groups described a second and third channelrhodopsin (), and many more will follow. While ChR is highly homologous to BR, especially within the transmembrane helices that constitute the retinal-binding pocket, in channelrhodopsins the ion-conducting activity is largely uncoupled from the photocycle (); an effective cation channel pore is opened, which implies that ion flux becomes independent of retinal isomerization and rather depends on the kinetics of channel closure. In neurons, net photocurrent due to ChR activation is dominated by cation flow down the electrochemical gradient (resulting in depolarization), rather than by the pumping of protons. Like the BRs and HRs, ChRs from various species () are functional in neurons with a range of distinct and useful intrinsic properties.

The single-component optogenetic palette available to neuroscientists now contains tools for four major categories of fast excitation, fast inhibition, bistable modulation, and control of intracellular biochemical signaling in neurons and other cell types ( Figure 1 B, Table 1 ). This array of optogenetic tools, the result of molecular engineering and genomic efforts, allows experimental manipulations tuned for (1) the desired physiologic effect; (2) the desired kinetic properties of the light-dependent modulation; and (3) the required wavelength, power, and spatial extent of the light signal to be deployed.

Decay kinetics are temperature dependent; values were taken from or estimated from published traces where available and necessary. Opto-XR kinetics were obtained in vivo and should be taken only as an upper bound since the assay involved a downstream measure (spiking). All other reported values except ChRGR are recorded at RT, with ∼50% decrease in τexpected at 37°C; since ChRGR has only been studied at elevated (34°C) T, we denote likely RT range for ChRGR in parentheses. Values for channelrhodopsin/fast receiver and channelrhodopsin/wide receiver (value was not measured in neurons, and its properties may depend on other channels in the host cell as well as the host cell tolerance of, and response to, higher levels of intracellular Ca

Decay kinetics are temperature dependent; values were taken from or estimated from published traces where available and necessary. Opto-XR kinetics were obtained in vivo and should be taken only as an upper bound since the assay involved a downstream measure (spiking). All other reported values except ChRGR are recorded at RT, with ∼50% decrease in τexpected at 37°C; since ChRGR has only been studied at elevated (34°C) T, we denote likely RT range for ChRGR in parentheses. Values for channelrhodopsin/fast receiver and channelrhodopsin/wide receiver (value was not measured in neurons, and its properties may depend on other channels in the host cell as well as the host cell tolerance of, and response to, higher levels of intracellular Ca

Decay kinetics are temperature dependent; values were taken from or estimated from published traces where available and necessary. Opto-XR kinetics were obtained in vivo and should be taken only as an upper bound since the assay involved a downstream measure (spiking). All other reported values except ChRGR are recorded at RT, with ∼50% decrease in τexpected at 37°C; since ChRGR has only been studied at elevated (34°C) T, we denote likely RT range for ChRGR in parentheses. Values for channelrhodopsin/fast receiver and channelrhodopsin/wide receiver (value was not measured in neurons, and its properties may depend on other channels in the host cell as well as the host cell tolerance of, and response to, higher levels of intracellular Ca

Decay kinetics are temperature dependent; values were taken from or estimated from published traces where available and necessary. Opto-XR kinetics were obtained in vivo and should be taken only as an upper bound since the assay involved a downstream measure (spiking). All other reported values except ChRGR are recorded at RT, with ∼50% decrease in τexpected at 37°C; since ChRGR has only been studied at elevated (34°C) T, we denote likely RT range for ChRGR in parentheses. Values for channelrhodopsin/fast receiver and channelrhodopsin/wide receiver (value was not measured in neurons, and its properties may depend on other channels in the host cell as well as the host cell tolerance of, and response to, higher levels of intracellular Ca

Fast Optogenetic Excitation for Neuroscience

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Bamberg E. Structural guidance of the photocycle of channelrhodopsin-2 by an interhelical hydrogen bond. Yizhar et al., 2011a Yizhar O.

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et al. Neocortical excitation/inhibition balance in social dysfunction and information processing. SFOs have recently been shown to deliver bistable optogenetic control in C. elegans neurons and muscle cells () and in the brains of awake, behaving primates (). Additional and combinatorial mutagenesis based on these initial principles has led to additional SFOs (), with time constants of deactivation up to 30 min (). With these stabilized SFOs, targeted neurons can in principle be “stepped” to a stable depolarized resting potential, which could be followed by removal of the light source and initiation of behavioral or physiological experimentation in the complete absence of light or other hardware. Moreover, the use of long low-intensity light pulses (in the setting of the steady photon-integration properties of cells expressing the stable SFOs) could allow elimination of variability of recruitment of cells in vivo attributable to variations in light intensity experienced, since the full population of opsin-expressing cells even in a large volume of tissue could be brought to saturating photocurrent levels over time.

While these tools afford experimental opportunities, an important caveat of this approach is that it must be validated in each system to quantify the effect on targeted cells. The published SFOs have slower activation kinetics that do not tend to directly elicit spikes or drive neurons into a state of depolarization block (the latter of which could give rise to a paradoxical inhibition rather than excitation of the targeted cells), but studies involving SFOs (indeed involving any optogenetic intervention) should still be accompanied by electrophysiological validation at the corresponding experimental time point (matching opsin expression levels) so that the effect on the targeted cell and tissue may be understood for proper interpretation of experimental results. Here, the SFOs, and indeed all optogenetic tools, offer a class of validation not typically possible with electrical stimulation, since with electrical stimulation it remains unclear precisely how the targeted region is responding due to the difficulties associated with electrical recording in the setting of electrical stimulation artifacts.

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Oertner T.G. High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels. Freund, 2003 Freund T.F. Interneuron Diversity series: Rhythm and mood in perisomatic inhibition. Gunaydin et al., 2010 Gunaydin L.A.

Yizhar O.

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Hegemann P. Ultrafast optogenetic control. None of the ChRs described above were initially shown to directly evoke reliable spiking above 40 Hz, while many neuronal cell types and physiological processes involve or require high-frequency spike trains (>40 Hz). Even the seemingly fast off-kinetics of wild-type ChR2 (τ ∼10 ms), and certainly those of H134R (τ ∼20 ms), are insufficient for precise control at high spike rates, a phenomenon that may be compounded by the further depolarization-dependent slowing of deactivation observed for most ChRs (). An important group of relevant neurons are the fast-spiking inhibitory parvalbumin-expressing interneurons, which in cortex are thought to be involved in generation of oscillatory rhythms and synchronization across brain regions (). Activation of these neurons with wild-type ChR2 is not sufficiently precise above 40 Hz, due to spike doublets, plateau potentials, and temporal nonstationarity in the form of missed spikes late in sustained high-frequency light pulse trains (which may result from the failure of full membrane repolarization and consequent insufficient voltage-dependent deinactivation of voltage-gated sodium channels;).

While the specificity of optogenetics presents an opportunity to understand precisely how cells and circuits give rise to nervous system function, experimental effects will depend on the type of neuron and cellular compartment targeted as well as the stimulation parameters employed (pulse frequency, duration, amplitude, and other factors, just as with electrical stimulation). Moreover, opsin choice (e.g., ChETA versus H134R or L132C) could affect the extent to which paired-pulse or plasticity effects are elicited in a manner distinct from electrical stimulation, especially in experiments where light is directly applied to the axons and the ChR therefore directly influences presynaptic terminal ion flux; in contrast, where light is delivered directly to the soma and propagating sodium action potentials are generated, the resulting presynaptic bouton (and downstream postsynaptic) spikes may look indistinguishable from those generated by native electrical spike generation mechanisms in terms of ion flux and kinetics.