Optogenetics and Clarity

This chapter includes an accompanying lecture presentation that has been prepared by the authors: .

Key Concepts

Optical techniques for manipulating and recording neural activity, as well as for high-resolution structural mapping, have emerged over the past 10 to 15 years to transform fundamental and translational neuroscience research.
Optogenetics uses genetically encoded, light-gated ion channels—originally discovered in microbial organisms but engineered in the laboratory—to confer the ability for live neurons to be excited or inhibited on a millisecond timescale by specific wavelengths of light.
By combining with (typically virus-based) genetic tools for targeting neurons based on their anatomic location, connectivity within the brain, and molecular signatures, optogenetics enables the direct activation or inhibition of a specifically defined subpopulation of neurons in live, freely moving animals.
Optogenetics has enabled a broad range of discoveries in neuroscience, ranging from fundamental circuits governing biologic processes and behaviors to numerous disease models including epilepsy, depression, and stroke, and has been established in many animals including nonhuman primates, and preliminary clinical studies.
Genetically encoded calcium indicators are hybrid protein sensors that emit fluorescence that is proportional to the intracellular calcium concentration, and is thus a reliable proxy for electrical activity within neurons.
Recording the fluorescent activity of genetically defined populations of neurons using, for example, microscopy, enables the recording of neural dynamics across hundreds to thousands of individual cells in a spatially resolved manner inaccessible to traditional electrical recording methods.
Tissue clearing methods, such as CLARITY, can be used to render a tissue specimen, such as a whole mouse brain or clinical biopsy, fully transparent while structurally intact and accessible by molecular probes.
Applications of tissue clearing include visualization of three-dimensional structures such as long-range neuronal projections, and volumetric histopathology of clinical specimens for identifying spatial features such as tumor margins.

Electricity was first demonstrated as the basis of neural activity through an unusual series of experiments conducted by Luigi Galvani in the late 18th century. In the preparation of a frog nerve from a dissected specimen, Galvani found that electrical stimulation of the nerve caused the intact muscles to contract violently and reproducibly. Grasping the novelty of finding that the electrical spark was conducted along the nerve, he produced a comprehensive documentation of his experiments that laid the foundation for electrophysiology and modern therapeutic stimulatory interventions used today, fulfilling his hopes that his results “might be able more surely to heal [neurological] diseases.”

Recordings of the nervous system were first made more than 100 years later when, in 1929, Hans Berger published the first electrical recordings of human CNS activity. The usefulness of this technique was quickly realized, and epileptiform and interictal abnormalities in electroencephalography were described soon thereafter. In clinical medicine today, activity in the brain is commonly assayed, or induced, to localize epileptogenic regions or to manipulate aberrant circuitry, but resolution in these interventions is limited to a similar degree as that of early experiments—that is, to a scale not useful for investigation of local network activity, let alone temporally refined information about the identity or activity of a single neuron.

To address these shortcomings, the development of new tools and techniques has allowed researchers unprecedented insight into the workings of the nervous system through the ability to control and assess neural activity with subcellular resolution, as well as to explore healthy and diseased specimens on the intact, whole-brain level with molecular resolution. This proliferation of neuroscience research has generated extraordinary interest in the field from clinical, governmental, and business organizations that wish to apply new results and further expand the fund of knowledge. It is hoped that some of these novel approaches will be put to therapeutic or diagnostic use.

This chapter serves as a general introduction to optical methods that allow neuroscientists to study the brain in three separate ways: first, by direct control of the membrane potential through the use of optically modulated, single-component ion channels and pumps (optogenetics); second, by optical readout of changes in calcium concentrations within neurons as a proxy for action potential generation, with the use of either dyes or genetically encoded calcium indicators (GECIs); and third, by rendering intact nervous system samples translucent, to allow molecular identification and imaging of entire circuits, nuclei, or whole brains in situ without sectioning (CLARITY and other tissue clearing methods). This chapter is not meant to be a comprehensive or detailed description of these techniques but rather a starting point; it is likely that they will have been improved or expanded on by the time of publication. With that in mind, the following sections describe the development and general principles of these three topics, detailed and referenced to allow interested readers to explore further. Clinical and human application that has already been completed is noted, and potential future translational opportunities are highlighted. Collectively, these approaches have revolutionized neuroscience research and have enabled experimental approaches and results that were only dreamed of beforehand. Their effect on neurological surgery has yet to be tested, but the potential for fundamental change is real.

Optogenetics

The ability to precisely manipulate the activity of defined populations of neurons is an acute challenge for neuroscience. Although electrical interventions enable precise, temporal neural modulation, neither the functional role, nor genetic identity, nor connectivity of the target population can be selected; all neurons within a given volume of parenchyma are modulated. Of the approaches developed to address this obstacle, optical techniques have proved highly successful in experimental models, in large part owing to the innate spatiotemporal precision, inherent multiplexing capacity across a broad spectrum of colors, and general safety of light as an actuator.

In initial approaches to modulating intracellular events with light, researchers used lasers to photoablate signaling pathway proteins or to stimulate action potentials via laser irradiation of axon initial segments. In work completed soon thereafter, investigators coupled light stimulation with genetically encoded effector proteins to directly depolarize the neuron membrane potential with light through targeted coexpression of Drosophila arrestin-2, rhodopsin, and a G-protein subunit (chARGe). The targeted expression of optically modulated proteins, combined with the controlled introduction of light, was a revolutionary approach to neural control, but was limited by the slow speed at which neuronal activity could be induced, by poor transduction of light, and by the difficulty inherent in working with a system with distinct molecular optical sensors and signal transducers.

The adaptation of an obscure class of light-sensitive ion channels and pumps known as microbial opsins removed these barriers. Lacking the complex sensory organs of higher order eukaryotes, some prokaryotes use protein sensors to detect visible light as a means to control various homeostatic functions, such as maintaining osmotic balance in seawater. The microbial opsins unite the distinct functions of light sensing and signal transduction into a single molecule, and the adaptation of these optically modulated, genetically encoded molecular tools to achieve control or readout of biologic system activity with high spatial and temporal precision is known as optogenetics. Optogenetic techniques are used in neuroscience to modulate neural circuits with specificity and precision that are not attainable with electrical or pharmacologic modulation. Although this approach has thus far been demonstrated only in experimental animal platforms, it holds significant promise in describing the origins of psychiatric and neurological disease and potential direct therapeutic application.

Opsins

Opsins are categorized as type I, or microbial, opsins (present in prokaryotes, algae, and fungi ) and type II, or eukaryotic, opsins (primarily used for visual sensation in eyes, including those in humans). Both types are seven-transmembrane proteins, although type I opsins are ion channels, whereas type II are G-protein–coupled receptors. Both make use of a vitamin A derivative (retinal) as the light-sensing chromophore. On absorption of a photon of the correct wavelength (color), a retinal molecule bound to an opsin (a “rhodopsin”) photoisomerizes and induces a conformational change in its partner opsin. This change in shape of the bound opsin activates it and, depending on the type of opsin, initiates transmembrane ion flux or a signaling cascade.

Opsins that function as ion channels or pumps, which are used in optogenetic approaches, are sensitive to light at characteristic wavelengths that induce large photocurrents of specified ions, causing either membrane depolarization or hyperpolarization when expressed in neurons. The discovery of combined prokaryotic sensor/effector proteins in 1971 ^, was unappreciated by neuroscientists until the sequencing and characterization of channelrhodopsin (ChR) from the green algae Chlamydomonas reinhardtii. With peak absorption in the visible blue light spectrum at approximately 460 nm and millisecond time constants of opening and closure with pulsed light, ChR has optimal characteristics for reliably driving action potentials in defined neuron populations.

Diverse Optogenetic Tools for Neural Control

In the context of manipulating neural activity, optogenetic tools may be divided broadly into excitatory and inhibitory opsins ( Fig. 62.1 ).

Excitatory opsins are generally true channels in that they allow ions to flow through a pore in the membrane when activated, whereas inhibitory opsins can be channels or pumps that move ions in a stepwise manner across the membrane. FLOAT NOT FOUND

Channelrhodopsin-2 (ChR2), the prototypical excitatory opsin, was the first to be demonstrated as a useful single-component optogenetic tool. As with all opsins, ChR2 uses retinal as its light-sensing partner; serendipitously, it was found that the concentration of retinol in vertebrate tissues is sufficient to enable opsin expression without exogenous supplementation. ^, Since its initial use in cultured rat neurons, ChR2 has been used in all standard neuroscience model organisms and has been highly engineered to alter its spectral sensitivity, ion conductance properties, ^, kinetics for high-frequency and low-frequency ^, stimulation, and potency ^, (for review, see Mattis et al. ). In addition, the conservation of protein structure across microbes has allowed for the development of mutants or chimeras ^, that combine useful functional features from multiple source opsins. Some of these, such as red light–activated C1V1 ¹⁴ or bReaChES, have been developed for use in conjunction with ChR2 to achieve independent, simultaneous control of distinct groups of neurons within the same parenchymal volume, or for simultaneous modulation and recording of neural activity when used in conjunction with fluorescent activity reporters (see “Optical Calcium Sensors” section, later). The development of optogenetic tools with red-shifted activation wavelengths (currently up to 630 nm) for neural excitation is especially useful in nonhuman primates or with other anatomic structures that require large-volume stimulation. Red light is less scattered by tissue and less absorbed by blood and blood vessels, which allows more uniform tissue penetration at greater distances from the light source, potentially even through the skull.

With respect to neuronal inhibition, the first and most commonly used opsin for neuron hyperpolarization, halorhodopsin (NpHR), is a chloride influx pump isolated from the halobacterium Natronomonas pharaonis and is maximally activated by yellow light at wavelengths of 573 to 613 nm. More recently, proton efflux pumps—including enhanced bacteriorhodopsin (eBR) from Halobacterium species, archaerhodopsin (Arch) from Halobacterium sodomense, and Mac from Leptosphaeria. maculans —have been developed as additional control tools for neural inhibition. These have been multiply engineered for improved eukaryotic cellular trafficking to increase functional expression. ^, ^, More recently, the crystal structures of both excitatory and inhibitory opsins, such as Gt ACR1 from Guillardia theta , have allowed for structure-guided engineering of chloride-conducting inhibitory opsins with large photocurrents and fast kinetics, key features of an effective inhibitory tool. ^,

Animal Models

In addition to the development and refinement of opsins, much work has been done to optimize their delivery into neural systems in vitro and in vivo. Viral expression systems based on lentivirus and adeno-associated virus (AAV) vectors have high infectivity and low toxicity and have been successfully used for stable and reliable expression of opsins in mammalian neural tissues. ^, Viral systems can allow cellular specificity by linking opsin payloads to specific promoters or by restricting viral injection or light delivery to specific anatomic subregions, ^, and have been engineered for advantageous properties such as retrograde transport or blood-brain barrier penetration for minimally invasive systemic delivery into the CNS.

Viral delivery of optogenetic tools has enabled researchers to model myriad psychiatric conditions, including obsessive-compulsive disorder, by targeting ChR2 to cortical glutamatergic neurons for specific modulation of corticostriatal circuitry ; to ameliorate the epileptogenic activity of thalamocortical neurons after cerebrovascular injury through the use of NpHR to inhibit their activity ; and to probe the dynamics of the hypocretin- and norepinephrine-mediated arousal pathways. Although this research is robust, the development of similar approaches in human patients is still in its infancy, with few viral delivery systems (gene therapies) presently approved by the US Food and Drug Administration (at the time of writing, Zolgensma for spinal muscular atrophy and Luxturna for a rare retinal dystrophy, as well as chimeric antigen T-cell therapies for oncology that are genetically engineered ex vivo ). However, numerous viral-based gene therapies are in clinical studies, including in the CNS (e.g., RESTORE trial in Parkinson disease ).

Of the greatest interest to researchers working toward the use of optogenetic technologies in the clinic and operating room is their application to nonhuman primate and human tissue. To this end, an “optogenetic toolbox” for primates has been developed that established and validated combinations of opsins and delivery methods tailored to the unique challenges of primate research, including large-volume stimulation and low toxicity over long time scales ( Fig. 62.2 ).

Figure 62.2, Optogenetic control of cortical activity in nonhuman primates.

After the first successful optogenetic study of nonhuman primates, several groups further applied these approaches, especially in the rhesus macaque, but also other species such as marmosets and squirrel monkeys. Work has included modulation of oculomotor behavior through the use of optogenetic stimulation of the macaque arcuate sulcus or primary visual cortex or optogenetic inhibition of the superior colliculus, which disrupts motor planning and thus modulates motor behavior through optogenetic stimulation of dorsal premotor cortex. FLOAT NOT FOUND

Future work will be facilitated by the development of a novel combined microelectrode–optical fiber apparatus for optogenetic modulation of deep subcortical primate brain structures. In this research, light stimulation of a target neuron population is delivered via optical fiber after stereotactic craniotomy. ^, Potentially less invasive alternatives for targeted, chronic stimulation, including implantable micron-scale light-emitting diodes for optogenetic stimulation in vivo, are being actively developed. Both excitatory and inhibitory opsins have been functionally expressed in human tissues, including embryonic stem cells and retinal explants, setting the stage for future experimentation aimed at eventual use of optogenetic technologies in the clinic. At the time of writing, the first human study with a ChR2-encoding AAV has been initiated as part of a phase 1/2a clinical trial for retinitis pigmentosa.

Outlook

Optogenetics has enabled exciting and diverse avenues of investigation into basic principles of neuroscience with experimental designs that were not previously possible. However, the true power in optogenetics lies in the potential it affords in investigating clinical phenomena. Indeed, optogenetics has been envisioned as a powerful method to treat neurological and psychiatric disease, potentially replacing techniques such as deep brain stimulation. Many researchers have sought to apply optogenetic technologies to modeling and modulating the symptoms of nervous system disorders, including depression, ^, anxiety, obsessive-compulsive disorder, ^, addiction, epilepsy, pain, movement disorders, and demyelinating disorders. In addition, optogenetic technologies applied to basic neuroscientific questions have helped reveal fundamental principles of neurobiology: for example, activity-induced differentiation of oligodendrocyte and neural precursors into oligodendrocytes. The neuron-glia interaction, however, when dysregulated, could underlie gliomagenesis in pediatric brain tumors.

Together, the development and application of optogenetics have substantially increased fundamental knowledge of nervous system function in health and disease. The technical, safety-related, and even ethical challenges inherent in the application of optogenetic tools to the nervous system in living human patients are numerous and will need to be overcome before the direct application of these tools in the clinic in the near future. However, the deep understanding of the mechanistic underpinnings of nervous system disorders afforded by optogenetics will certainly provide insight to psychiatrists, neurologists, and neurosurgeons seeking to refine—or redefine—current therapeutic strategies.

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