Chapter 16 Optogenetics

To truly understand biological systems, one must possess the ability to selectively manipulate their parts and observe the outcome. (For purposes of this review, we refer mostly to targets of neuroscience; however, the principles covered here largely extend to myriad samples from microbes to plants to the intestine, etc.). Drugs are the most commonly employed way of introducing such perturbations, but they act on endogenous proteins that frequently exist inmultiple cell types, complicating the interpretation of experiments. Whatever the applied stimulus, it is best to introduce optimized exogenous reagents into the systems under studydenabling manipulations to be targeted to specific cells and pathways. (It is also possible to target manipulations through other means, such as drugs that acquire cell-type specificity through targeting via antibodies and/or cell surface receptor ligands, but as far as we are aware, existing reagents fall short in terms of necessary specificity.) Many types of perturbations are useful in living systems and can be divided into rough categories such as the following: depolarize or hyperpolarize cells, induce or repress the activity of a specific pathway, induce or inhibit expression of a particular gene, activate or repress a specific protein, degrade a specific protein, etc. User-supplied triggers for such manipulations to occur include the following: addition of a small molecule (“chemogenetics”dideally inert on endogenous proteins) [1], sound waves (“sonogenetics”) [2], alteration of temperature (“thermogenetics”d almost exclusively used for small invertebrates) [3], and light (“optogenetics”). There are reports of using magnetic fields (“magnetogenetics”) [4], but there is no evidence that such effects are reproducible or even physically possible [5,6]. Of these, themost commonly used, for multiple reasons, is light. Many factors make light an ideal user-controlled stimulus for the manipulation of samples. Light is quickly delivered, and most light-sensitive proteins and other molecules respond quickly to light stimuli, making many optogenetic systems relatively rapid in comparison to, for instance, drug-modulated systems. Light is also quite easy to deliver in localized patterns, allowing for targeted stimulation. Multiple wavelengths can be delivered separately to distinct (or overlapping) regions, potentially allowing combinatorial control of diverse components. Finally, light can be delivered to shallow brain regions (and peripheral sites) relatively noninvasively, and to deeper brain regions with some effort. However, there are also a number of shortcomingsof using light for control. Robust and uniform penetration of light into the sample is the most significant concern. For systems requiring modulation of many cells, particularly at depth, the use of systems controlled by small molecule drugs would generally be recommended instead of optogenetic approaches. When light is delivered through the use of fibers, lenses, or other optical devices, such interventions can produce significant cellular death, scar formation, and biofouling. The foreign-body response of tissue to objects triggers substantial molecular alterations, the implications of which are incompletely defined, but can involve reactive astrogliosis, oxidative stress, and perturbed vascularization. Head-mounted lightdelivery devices can be heavy and/or restrictive, and thus perturb behavior, particularly for small animals (e.g., mouse behavior is much more disrupted than rat behavior). More generally, all light causes tissue heating, which can have dramatic effects on cell health, physiology, and animal behavior. This is most concerning for tiny animals such as flies. Light itself also damages tissue, most obviously through photochemistry (e.g., oxidation and radicalization) and photobleaching of critical endogenousmolecules. Furthermore, of course, light is ubiquitous, meaning that the sample is never completely unstimulated, despite precautions. Light passes through the eyes into the brain with surprising ease, and even through the skull withmodest efficacy [7]dwhich can disrupt animal behavior (as can the converse: stimulating light in the brain perceived as a visual stimulus through the back of the eyes.) Light-responsive proteins exist in all samples, particularly in the eyes but to some extent in all tissuesdnotably, deep-brain photoreceptors [8].