Optogenetics: 10 years of microbial opsins in neuroscience

Abstract

Over the past 10 years, the development and convergence of microbial opsin engineering, modular genetic methods for cell-type targeting and optical strategies for guiding light through tissue have enabled versatile optical control of defined cells in living systems, defining modern optogenetics. Despite widespread recognition of the importance of spatiotemporally precise causal control over cellular signaling, for nearly the first half (2005-2009) of this 10-year period, as optogenetics was being created, there were difficulties in implementation, few publications and limited biological findings. In contrast, the ensuing years have witnessed a substantial acceleration in the application domain, with the publication of thousands of discoveries and insights into the function of nervous systems and beyond. This Historical Commentary reflects on the scientific landscape of this decade-long transition.

Figure 1

The biochemical foundations of the study of microbial light-activated proteins. (a) The three major classes of microbial proteins used for single-component optogenetics (adapted from ref. , Elsevier). (b) Light-activated transmembrane current mechanism of the proton pump bacteriorhodopsin (BR). Photon (hv) absorption initiates a conformational switch, leading to discontinuous proton transfers involving Asp85, Asp96, Asp212, Arg82 and the proton release complex (PRC), and net charge movement across the membrane. The core concept of single-component light-activated transmembrane ion conductance had become textbook material by the 1980s (reproduced from ref. , Elsevier). (c) Elucidation of channel-type conductance. The channelrhodopsin crystal structure revealed positioning of transmembrane helices (green), the binding pocket of all-trans retina (purple), and angstrom-scale positioning of residues lining the pore (left). In the course of testing the pore model, structure-guided mutagenesis of the residues in orange (left) shifted expected pore electrostatics from largely negative (red, center) to largely positive (blue, right) and switched ion selectivity from cation to anion (chloride) conductance. (d) All three classes of microbia opsin-derived proteins suffer to some degree from formation of aggregations within metazoan host cells^,,, but in all cases this can be addressed with membrane trafficking motifs borrowed from mammalian channels^,,,. Shown: original BR fused to enhanced yellow fluorescent protein (EYFP); upper left depicts accumulations seen with wild-type BR expression in mammalian neurons, upper right shows the effect on surface membrane expression of adding a neurite targeting motif (TS), and the lower row shows the effect of combined TS and ER (endoplasmic reticulum export) motif provision (reproduced from ref. , Elsevier).

Figure 2

Putting the pieces together. (a) Top, original notebook page from my laboratory’s first microbial opsin experiment. Several channel clones were tested in parallel to build strategies for neuronal control, including the highest-risk opsin expression; constructs and plasmid concentrations that I used to transduce the neurons on 1 July 2004 are recorded at top left, including a channelrhodopsin (clone K43) and wild-type (WT) or dominant-negative (DN) TASK1/TASK3 potassium channels (K44-47). Host neurons were differentiated from adult-derived mammalian CNS progenitors. Center: neuronal expression, localization and light activation; green, subcellular distribution of fluorescent protein fused to channelrhodopsin in the ~10-µ.m-diameter neuronal somata and proxima dendrites. After carrying out illumination for optogenetic photostimulation and the CREB Ser-133 phosphorylation (red) assay for reporting activation (14 July 2004), activation was noted as a neuron-by-neuron tally (bottom; z-test, χ² = 9.0634, d.f. = 3; P = 0.028) and neurons were transduced for the next steps. (b) The next steps included design of electrophysiological, imaging and behavioral readouts; design and introduction of high-titer opsin virus; and focal illumination of transduced brain regions (workflow of planned steps shown). (c) Stable, well-tolerated, reproducible control across experiments, crucia for optogenetics, was enabled with high-titer opsin viruses. Electrophysiological readout shows spikes from two cultured neurons receiving the same light pattern (adapted from ref. , Springer Nature). (d) Initial engineering sketch of the fiber-optic neural interface for spatially registering viral transduction with focal high-intensity illumination (drawings courtesy F. Zhang, Stanford). (e) Instantiation of the interface that ultimately allowed depth-targeted control and observation^4,111 of population-level activity in cells and projections of freely moving mammals (photo courtesy I. Goshen, Stanford). Supplementary Video 1 shows initial mammalian behavioral control.

Figure 3

Progress in genetically guided intervention for optogenetics. (a) Top, initial cell-type targeting for optogenetics in behaving mammals, based on a 3.1-kb hypocretin (Hcrt) promoter fragment in lentivirus; control vector without opsin gene at right. LTR, long terminal repeats; RRE, Rev-responsive element; WPRE, woodchuck post-transcriptional regulatory element; ChR2, channelrhodopsin-2; cPPT, central polypurine tract; Psi+, cis-acting packaging sequence; mCherry, a red fluorescent protein. Middle, specificity, penetrance and efficacy of expression in Hcrt neurons (green); ChR2–mCherry fusion (red) shown in mouse lateral hypothalamus (scale bar, 20 µm); right, photocurrent in hypothalamic slice. Bottom, neurons firing action potentials upon illumination; two sweeps superimposed. Error bars, s.e.m. (b) Dose-response of light flash effects; in experiment corresponding to a, latencies of wake transitions are shown from rapid eye motion (REM) sleep after a single 10-s photostimulation bout at different frequencies (15-ms light pulses; a,b adapted from ref. , Springer Nature). Error bars, s.e.m. (c) Mammalian genetic targeting for optogenetics: AND or NOT opsin-expression logic conditional on multiple recombinase-expression-defined genetic features using single adeno-associated viral vectors. Top left, schematics representing selection of target populations of cells expressing (or not) the recombinases Cre or Flp, which are often used to create animal lines with cell-type-targeted recombinase expression patterns. Top right, mechanism of targeting based on recombination followed by removal of recombination sites in introns. (d) Neurons transfected with combinations of Cre (blue), Flp (red) and the vectors that implement Cre AND NOT Flp (C_on/F_off) or Flp AND NOT Cre (C_off/F_on). ChR2-YFP is only expressed in cells marked by one or the other, but not both, recombinases (c,d adapted from ref. , Springer Nature).

Figure 4

Progress in activity-guided intervention for optogenetics. (a–d) Closed-loop targeting of thalamocortical neurons in epileptic cortex (reproduced from ref. , Springer Nature). Yellow light terminates seizures defined by EEG and behavior, detected and interrupted in real time with closed-loop optogenetic inhibition using eNpHR3.0, an engineered inhibitory halorhodopsin (a,c). Without yellow light, native epileptic events follow an unmodified time course (b,d). Thalamocortical activity was thus, surprisingly, identified as necessary for poststroke epileptic events in this context. (e) Optogenetic closed-loop control in 2009. Optogenetic stimulation of ChR2-expressing inhibitory (FS) neurons was made conditional on spike detection in pyramidal (PY) neurons, implementing feed-forward inhibition under experimenter control (adapted from ref. , Springer Nature). Black and red traces show action potentials in PY cells without or with, respectively, use of closed-loop optogenetic excitation of FS cells. (f) Closed-loop, all-optical control could be implemented using deep brain fluorescence (for example, GCaMP) detection of genetically specified activity signals, via techniques such as fiber photometry (shown here detecting in real time the activity of ventral tegmental area (VTA) dopamine neuron projections to the nucleus accumbens (NAc) during appetitive social interaction). Red bars indicate interaction episodes (social or object). (g) With considerable potential for closed-loop control of projection dynamics, the same fiber-optic interface^, can be used not only to observe activity in defined deep-brain projections, but also to control activity in deep-brain projections, as shown here modulating social behavior (f,g adapted from ref. , Elsevier). These specified methods for activity-guided opsin expression.

Figure 5

Progress in spatially guided intervention for optogenetics: beyond cell subpopulation or projection targeting. (a–d) Initial in vivo two-photon, single-cell-resolution optogenetics with guided light: optogenetic control of spiking in adult mice (adapted from ref. , Springer Nature). (a) Experimental setup targeting superficial layer 2/3 somatosensory neurons with C1V1 (ref. 175). (b,c) Transduced neurons in somatosensory cortex shown at low (b) and high (c) magnification with cell-filling fluorophore version of the opsin virus used to facilitate cell identification, imaging and control. (d) Left, layer 2/3 pyramidal cells transduced with C1V1 under loose patch conditions (note red dye-filled patch electrode). Lower left, trace showing 5-Hz control of spiking with 1,040-nm raster-scanning illumination. Right, axial (upper) and lateral (lower) single-cell resolution of two-photon optogenetic spiking control in vivo. Blue triangles indicate pyramidal neurons and red boxes illustrate region-of-interest raster-scan positioning; traces show spiking occurring only while scanning within the cell. (e) An example of single-cell targeting with optogenetics using temporally focused two-photon control of C1V1, with single-cell resolution optical feedback, in a virtual reality environment in behaving mice (adapted from ref. , Springer Nature); for spatial light modulator use together with C1V1, see refs. ,,. Target neurons are illuminated in turn via temporally focused soma-sized spots (<15 µm). Left, light path; center, lasers used; right, behavioral preparation. PMTs, photomultiplier tubes; ex., excitation; em., emission; H P, headplate.

Figure 6

Variations on the theme. (a) Optogenetic control of histone acetylation and gene expression. The LITE system uses light-responsive cryptochromes, not microbial opsins, and has been adapted to create histone effectors targeted to specific genomic sequences using TALE reagents (left). TALE, transcription activator–like effector; CRY2PHR, cryptochrome-2 photolyase homology region; CIB1, CRY2 interacting partner. The same laser diode/fiber-optic optogenetic interface developed for microbial opsins (middle) can be used to control this optogenetic system in vivo (right) (adapted from ref. , Springer Nature). ILC, infralimbic cortex; SID4X, four concatenated mSin3 domains for histone effector control. (b) Left, multicomponent transduction of magnetic signals into neural activity (left), via introduced nanoparticles (MNP) and TRPV1 temperature-sensitive ion channel genes. Middle, nanoparticles with polyacrylic acid coating (blue) and polyethylene glycol chains (orange). Right, the magnetic field exposure system. (Adapted from ref. , AAAS). (c) Multicomponent transduction of optical signals into neural activity via nanoparticles directly targeted to cells (which, as in b, must be distributed through tissue); light intensities required are many orders of magnitude greater than those used in microbial opsin optogenetics (adapted from ref. , Elsevier). (d) Single-component control of defined biochemical pathways in neurons by the optoXR method. IP₃, inositol trisphosphate; DAG, diacylglycerol. (e) Biochemical validation of signaling specificity; optoβ₂AR recruits the cAMP pathway in response to light (green bars) to a level comparable to that of direct pharmacological agonism of the original G protein–coupled receptor (blue bars), while not recruiting separate pathways; in contrast, other optoXRs recruit distinct signaling mechanisms^,–. (f) Optrode methodology for activity recording. As with the LITE system shown in a, biochemical systems continue to exploit the neural interfaces for electrophysiology and for behavior that were developed for microbial opsin optogenetics^,, (d–f adapted from ref. , Springer Nature).

Figure 7

Publication timeline for microbial opsins and optogenetics over 45 years. Trajectory of the number of papers per year searchable in PubMed by bacteriorhodopsin (triangles); the second trajectory (squares) shows papers searchable by keywords encompassing all other related efforts: halorhodopsin, channelrhodopsin or variations of optogenetics. Note steady progress of the groundbreaking bacteriorhodopsin literature, not surpassed by the rest of the field until 2010. Key pioneering papers relevant to bacteriorhodopsin, halorhodopsin^, and channelrhodopsin^,, are indicated. Publication counts: PubMed search on 1 July 2015. The first 5 years of single-component optogenetics are shown in orange, during which time few papers were published, and the second 5 years (to the present) shown in blue. Circular symbols for 2015 represent linear extrapolation based on the first 6 months.