optogenetics

History

In 1979, Francis Crick suggested that controlling all cells of one type in the brain, while leaving the others more or less unaltered, is a real challenge for neuroscience. Crick speculated that a technology using light might be useful to control neuronal activity with temporal and spatial precision but at the time there was no technique to make neurons responsive to light.

By the early 1990s LC Katz and E Callaway had shown that light could uncage glutamate. Heberle and Büldt in 1994 had already shown functional heterologous expression of a bacteriorhodopsin for light-activated ion flow in yeast. In 1995, Georg Nagel, Ernst Bamberg and their colleagues tried the heterologous expression of microbial rhodopsins (also bacteriorhodopsin and also in a non-neural system, Xenopus oocytes) and showed light-induced current.

The earliest genetically targeted method that used light to control rhodopsin-sensitized neurons was reported in January 2002, by Boris Zemelman and Gero Miesenböck, who employed Drosophila rhodopsin cultured mammalian neurons. In 2003, Zemelman and Miesenböck developed a second method for light-dependent activation of neurons in which single ionotropic channels TRPV1, TRPM8 and P2X2 were gated by photocaged ligands in response to light. Beginning in 2004, the Kramer and Isacoff groups developed organic photoswitches or "reversibly caged" compounds in collaboration with the Trauner group that could interact with genetically introduced ion channels. TRPV1 methodology, albeit without the illumination trigger, was subsequently used by several laboratories to alter feeding, locomotion and behavioral resilience in laboratory animals. However, light-based approaches for altering neuronal activity were not applied outside the original laboratories, likely because the easier to employ channelrhodopsin was cloned soon thereafter.

Peter Hegemann, studying the light response of green algae at the University of Regensburg, had discovered photocurrents that were too fast to be explained by the classic g-protein-coupled animal rhodopsins. Teaming up with the electrophysiologist Georg Nagel at the Max Planck Institute of Biophysics in Frankfurt, they could demonstrate that a single gene from the alga Chlamydomonas produced large photocurrents when expressed in the oocyte of a frog. To identify expressing cells, they replaced the cytoplasmic tail of the algal protein with a fluorescent protein YFP, generating the first generally applicable optogenetic tool. They stated in the 2003 paper that "expression of ChR2 in oocytes or mammalian cells may be used as a powerful tool to increase cytoplasmic Ca2+ concentration or to depolarize the cell membrane, simply by illumination".

Description

Optogenetics provides millisecond-scale temporal precision which allows the experimenter to keep pace with fast biological information processing. Optogenetics by definition must operate on the millisecond timescale to allow addition or deletion of precise activity patterns within specific cells in the brains of intact animals (see Figure 1) and keep pace with optical control. This can be done with electrical recordings ("optrodes") or with biosensing reporter proteins created through the fusion of fluorescent proteins to detector proteins. Beyond its scientific impact, optogenetics represents an important case study in the value of both ecological conservation and in the importance of pure basic science. These opsins were studied over decades for their own sake by biophysicists and microbiologists before considering their potential value in neuroscience and neuropsychiatric disease.

Light-activated proteins: channels, pumps and enzymes

The hallmark of optogenetics is the introduction of fast light-activated channels, pumps, and enzymes that allow temporally precise manipulation of electrical and biochemical events while maintaining cell-type resolution through the use of specific targeting mechanisms. Among the microbial opsins which can be used to investigate the function of neural systems are the channelrhodopsins (ChR2, ChR1, VChR1, and SFOs) to excite neurons and anion-conducting channelrhodopsins for light-induced inhibition. Indirectly light-controlled potassium channels have recently been engineered to prevent action potential generation in neurons during blue light illumination. Light-driven ion pumps are also used to inhibit neuronal activity, e.g. halorhodopsin (NpHR), enhanced halorhodopsins (eNpHR2.0 and eNpHR3.0, see Figure 2), archaerhodopsin (Arch), fungal opsins (Mac) and enhanced bacteriorhodopsin (eBR).

Optogenetic control of well-defined biochemical events within behaving mammals is also possible. Building on prior work fusing vertebrate opsins to specific G-protein coupled receptors a family of chimeric single-component optogenetic tools was created that allowed researchers to manipulate within behaving mammals the concentration of defined intracellular messengers such as cAMP and IP3 in targeted cells. Other biochemical approaches to optogenetics followed soon thereafter when optical control over small GTPases and adenylyl cyclase was achieved in cultured cells using novel strategies from several different laboratories. Photoactivated adenylyl cyclases have been discovered in fungi and successfully used to control cAMP levels in mammalian neurons. Gi/o-coupled opsins (eOPN3, PdCO) were used to inhibit the fusion of synaptic vesicles in neurons in response to light, silencing their output. This emerging repertoire of optogenetic actuators allows for cell-type-specific and temporally precise control of multiple axes of cellular function within intact animals.

Technique

The technique of using optogenetics is flexible and adaptable to the experimenter's needs. Cation-selective channelrhodopsins (e.g. ChR2) are used to excite neurons, anion-conducting channelrhodopsins (e.g. GtACR2) inhibit neuronal activity. Combining these tools into a single construct (e.g. BiPOLES) allows for both inhibition and excitation, depending on the wavelength of illumination.

Introducing the microbial opsin into a specific subset of cells is challenging. One popular approach is to introduce an engineered viral vector that contains the optogenetic actuator gene attached to a specific promoter, such as CAMKIIα, allowing for some level of specificity. A more specific approach based on transgenic "driver" mice express Cre recombinase, an enzyme that catalyzes recombination between two lox-P sites in a specific subset of cells. By introducing an engineered viral vector containing the optogenetic actuator gene in between two lox-P sites, only the cells producing Cre recombinase will express the microbial opsin. This technique has allowed for modified optogenetic actuators to be used without the need to create a whole line of transgenic animals every time a new microbial opsin is needed.

After the introduction and expression of the microbial opsin, a computer-controlled light source has to be optically coupled to the brain region in question. LEDs or fiber-coupled diode-pumped solid-state lasers (DPSS) are frequently used. Recent advances include the advent of wireless head-mounted devices that apply LEDs to the targeted areas and as a result, give the animals more freedom to move.

Fiber-based approaches can also be used to combine optical stimulation and calcium imaging. This enables researchers to visualize and manipulate the activity of single neurons in awake behaving animals. It is also possible to record from multiple deep brain regions at the same using GRIN lenses connected via optical fiber to an externally positioned photodetector and photostimulator.

Technical challenges and limitations

Selective expression

One of the main problems of optogenetics is that not all the cells in question may express the microbial opsin gene at the same level. Thus, even illumination with a defined light intensity will have variable effects on individual cells. Optogenetic stimulation of neurons in the brain is less controlled as the light intensity drops from the light source. Due to this, it remains difficult to target opsin to defined subcellular compartments. Restricting the opsin to specific regions of the plasma membrane such as dendrites, somata or axon terminals provides a more robust understanding of neuronal circuitry.

Mathematical modelling shows that selective expression of opsin in specific cell types can dramatically alter the dynamical behavior of the neural circuitry. In particular, optogenetic stimulation that preferentially targets inhibitory cells can transform the excitability of the neural tissue, affecting non-transfected neurons as well.

Kinetics and synchronization

The original channelrhodopsin-2 was slower closing than typical cation channels of cortical neurons, leading to prolonged depolarization and calcium influx. Furthermore, differences between natural spike and optogenetic activation patterns synchronously activate expressing neurons with pulsed light simulation, removing the possibility of sequential activity in the stimulated population. This not only makes it difficult to understand how the affected cells communicate with one another, but how their phasic properties of activation relate to circuit function.

To overcome these challenges, optogenetic activation has been combined with functional magnetic resonance imaging (fMRI) to elucidate the connectome, a thorough map of the brain's neural connections. Precisely timed optogenetic activation is used to calibrate the delayed hemodynamic signal (BOLD) fMRI is based on.

Light absorption spectrum

The opsin proteins currently in use have absorption peaks across the visual spectrum, but remain considerably sensitive to blue light. This spectral overlap makes it very difficult to combine opsin activation with genetically encoded indicators (GEVIs, GECIs, GluSnFR, synapto-pHluorin), most of which need blue light excitation. Opsins with infrared activation would, at a standard irradiance value, increase light penetration and augment resolution through reduction of light scattering.

Spatial response

Due to scattering, a narrow light beam to stimulate neurons in a patch of neural tissue can evoke a response profile that is much broader than the stimulation beam. In this case, neurons may be activated (or inhibited) unintentionally. Computational simulation tools are used to estimate the volume of stimulated tissue for different wavelengths of light.

Applications

The field of optogenetics has furthered the fundamental scientific understanding of how specific cell types contribute to the function of biological tissues such as neural circuits in vivo. On the clinical side, optogenetics-driven research has led to insights into restoring with light[1], Parkinson's disease and other neurological and psychiatric disorders such as autism, Schizophrenia, drug abuse, anxiety, and depression. An experimental treatment for blindness involves a channel rhodopsin expressed in ganglion cells, stimulated with light patterns from engineered goggles.

Identification of particular neurons and networks

Optogenetic approaches have been used to map neural circuits in the amygdala that contribute to fear conditioning. One such example of a neural circuit is the connection made from the basolateral amygdala to the dorsal-medial prefrontal cortex where neuronal oscillations of 4 Hz have been observed in correlation to fear induced freezing behaviors in mice. Transgenic mice were introduced with channelrhodoposin-2 attached with a parvalbumin-Cre promoter that selectively infected interneurons located both in the basolateral amygdala and the dorsal-medial prefrontal cortex responsible for the 4 Hz oscillations. The interneurons were optically stimulated generating a freezing behavior and as a result provided evidence that these 4 Hz oscillations may be responsible for the basic fear response produced by the neuronal populations along the dorsal-medial prefrontal cortex and basolateral amygdala.

Further optogenetic manipulations of the central amygdala (CeA) revealed the regions role in robust sensorimotor functions. Using mice models, in vivo calcium imaging revealed activation of neuronal populations expressing the transcription factor Isl1 at the onset of biting. Neuronal activity was directly proportional to the hardness of the object being bitten, suggesting the neurons' role in force modulation. Optogenetic activation of CeAIsl1 neurons reinforced and enhanced biting behaviors while inhibition impaired biting through reducing jaw-closing muscle activity. Furthermore, activation of CeAIsl1 projections to the parvocellular reticular formation (PCRt) and pedunculopontine tegmental nucleus (PPtg) resulted in increased biting frequency and duration, suggesting a link between motivational states and motor output.

Anterior Cingulate Cortex

Optogenetic techniques have also been utilized to investigate pain pathways involved in chronic and neuropathic pain. The anterior cingulate cortex (ACC) is a highly interconnected structure within the limbic system responsible for pain processing. Long-term potentiation of these signals characterizes neuropathic pain development. Simulation of inhibitory neurons in the ACC expressing channelrhodopsin-2 resulted in a reduction of reflexive acute pain responses in mice. Such studies show that modulation of inhibitory pathways involved in pain processing yield a viable method for controlling inflammatory and neuropathic pain.

Precise temporal control of interventions

The currently available optogenetic actuators allow for the accurate temporal control of the required intervention (i.e. inhibition or excitation of the target neurons) with precision routinely going down to the millisecond level. The temporal precision varies, however, across optogenetic actuators, and depends on the frequency and intensity of the stimulation.

Experiments can now be devised where the light used for the intervention is triggered by a particular element of behavior (to inhibit the behavior), a particular unconditioned stimulus (to associate something to that stimulus) or a particular oscillatory event in the brain (to inhibit the event). This kind of approach has already been used in several brain regions:

Sharp waves and ripple complexes (SWRs) are distinct high frequency oscillatory events in the hippocampus thought to play a role in memory formation and consolidation. These events can be readily detected by following the oscillatory cycles of the on-line recorded local field potential. In this way the onset of the event can be used as a trigger signal for a light flash that is guided back into the hippocampus to inhibit neurons specifically during the SWRs and also to optogenetically inhibit the oscillation itself. These kinds of "closed-loop" experiments are useful to study SWR complexes and their role in memory.

Cellular biology/cell signaling pathways

Analogously to how natural light-gated ion channels such as channelrhodopsin-2 allows optical control of ion flux, which is especially useful in neuroscience, natural light-controlled signal transduction proteins also allow optical control of biochemical pathways, including both second-messenger generation and protein-protein interactions, which is especially useful in studying cell and developmental biology. In 2002, the first example of using photoproteins from another organism for controlling a biochemical pathway was demonstrated using the light-induced interaction between plant phytochrome and phytochrome-interacting factor (PIF) to control gene transcription in yeast. By fusing phytochrome to a DNA-binding domain and PIF to a transcriptional activation domain, transcriptional activation of genes recognized by the DNA-binding domain could be induced by light. This study anticipated aspects of the later development of optogenetics in the brain, for example, by suggesting that "Directed light delivery by fiber optics has the potential to target selected cells or tissues, even within larger, more-opaque organisms." The literature has been inconsistent as to whether control of cellular biochemistry with photoproteins should be subsumed within the definition of optogenetics, as optogenetics in common usage refers specifically to the control of neuronal firing with opsins, and as control of neuronal firing with opsins postdates and uses distinct mechanisms from control of cellular biochemistry with photoproteins.

In addition to phytochromes, which are found in plants and cyanobacteria, LOV domains(Light-oxygen-voltage-sensing domain) from plants and yeast and cryptochrome domains from plants are other natural photosensory domains that have been used for optical control of biochemical pathways in cells. In addition, a synthetic photosensory domain has been engineered from the fluorescent protein Dronpa for optical control of biochemical pathways. In photosensory domains, light absorption is either coupled to a change in protein-protein interactions (in the case of phytochromes, some LOV domains, cryptochromes, and Dronpa mutants) or a conformational change that exposes a linked protein segment or alters the activity of a linked protein domain (in the case of phytochromes and some LOV domains). Light-regulated protein-protein interactions can then be used to recruit proteins to DNA, for example to induce gene transcription or DNA modifications, or to the plasma membrane, for example to activate resident signaling proteins. CRY2 also clusters when active, so has been fused with signaling domains and subsequently photoactivated to allow for clustering-based activation. The LOV2 domain of Avena sativa(common oat) has been used to expose short peptides or an active protein domain in a light-dependent manner. Introduction of this LOV domain into another protein can regulate function through light induced peptide disorder. The asLOV2 protein, which optogenetically exposes a peptide, has also been used as a scaffold for several synthetic light induced dimerization and light induced dissociation systems (iLID and LOVTRAP, respectively). The systems can be used to control proteins through a protein splitting strategy. Photodissociable Dronpa domains have also been used to cage a protein active site in the dark, uncage it after cyan light illumination, and recage it after violet light illumination.

Optogenetic noise-photostimulation

Professor Elias Manjarrez's research group introduced the Optogenetic noise-photostimulation. This is a technique that uses random noisy light to activate neurons expressing ChR2. An optimal level of optogenetic-noise photostimulation on the brain can increase the somatosensory evoked field potentials, the firing frequency response of pyramidal neurons to somatosensory stimulation, and the sodium current amplitude.

Awards

In 2010, optogenetics was chosen as the "Method of the Year" across all fields of science and engineering by the interdisciplinary research journal Nature Methods and was highlighted in the academic research journal Science on "Breakthroughs of the Decade". In the same year, Georg Nagel, Peter Hegemann and Ernst Bamberg were awarded the Wiley Prize in Biomedical Sciences and were among those awarded the Karl Heinz Beckurts Prize. Additionally, Karl Deisseroth was awarded the inaugural HFSP Nakasone Award for "his pioneering work on the development of optogenetic methods for studying the function of neuronal networks underlying behavior".

In 2012, Bamberg, Deisseroth, Hegemann and Nagel were awarded the Zülch Prize by the Max Planck Society, and Miesenböck was awarded the Baillet Latour Health Prize for "having pioneered optogenetic approaches to manipulate neuronal activity and to control animal behaviour."

In 2013, Georg Nagel and Hegemann were among those awarded the Louis-Jeantet Prize for Medicine. Also that year, year Bamberg, Boyden, Deisseroth, Hegemann, Miesenböck and Georg Nagel were jointly awarded The Brain Prize for "their invention and refinement of optogenetics."

In 2017, Deisseroth was awarded the Else Kröner Fresenius Research Prize for "his discoveries in optogenetics and hydrogel-tissue chemistry, as well as his research into the neural circuit basis of depression."

In 2018, the Inamori Foundation presented Deisseroth with the Kyoto Prize for "spearheading optogenetics" and "revolutionizing systems neuroscience research."

In 2019, Bamberg, Boyden, Deisseroth, Hegemann, Miesenböck and Georg Nagel were awarded the Rumford Prize by the American Academy of Arts and Sciences in recognition of "their extraordinary contributions related to the invention and refinement of optogenetics."

In 2020, Deisseroth was awarded the Heineken Prize for Medicine from the Royal Netherlands Academy of Arts and Sciences, for developing optogenetics and hydrogel-tissue chemistry. The same year, Miesenböck, Hegemann and Georg Nagel jointly received the Shaw Prize in Life Science and Medicine.