Optogenetics is a technology that implements millisecond-precision control of activity patterns in defined cell types within freely behaving mammals.
As the first step toward making this technology possible, we developed a panel of functionally distinct microbial opsins (ChR2, NpHR, and VChR1, all light-activated regulators of transmembrane ion conductance) for application to neurobiology, and engineered them for novel capabilities. We first found that ChR2 (channelrhodopsin-2)-expressing neurons can fire blue light–triggered action potentials with high temporal precision, without addition of chemical cofactors; this approach has proved versatile across a variety of preparations, including worms, flies, fish, and mammals. We next found that neurons targeted to express the light-activated halorhodopsin (NpHR, a chloride pump from Natronomonas pharaonis) can be inhibited from firing action potentials when exposed to yellow light, even in intact tissue and behaving animals; because of the excitation wavelength difference, the two optical gates can be simultaneously used in the same cells even in vivo. We then employed genomic strategies to discover and adapt for neuroscience a cation channel (VChR1) with action spectrum red-shifted relative to ChR2 to allow tests of the combinatorial interaction of cell types in circuit computation or behavior.
Next, to enhance the power of these tools, we engaged in extensive molecular engineering of the opsins. For example, we have generated bistable (step-function) mutants of ChR2 that allow cells to be switched into and out of stable excitable states with only brief single flashes of light. We also have generated membrane-trafficking modifications of NpHR with much greater light-activated currents and tolerability in vivo. Finally, we are engineering opsins for enhanced speed, expression, and red-shifted action spectra; this will enhance combinatorial optogenetics and allow for better integration with blue light–activated Ca2+ indicators and deeper penetration of safer light with low-energy photons.
Also, as a key technological step to allow light delivery to even deep brain structures in vivo, we developed and published fiberoptic and solid-state laser diode tools to translate optogenetics to freely moving mammals and to allow depth targeting of optogenetic control to any brain region or tissue.
To allow simultaneous readouts of circuit dynamics, as well as animal behavior, we developed methods for achieving simultaneous millisecond-scale quantitative measures of circuit activity during optogenetic control, with voltage-sensitive dye imaging and novel integrated fiber-optic-electrode assemblies (optrodes).
To implement truly versatile targeting of optical control, we developed methods for targeting these light-sensitive proteins to subtypes of cells in vivo that go beyond simple promoter fragment-based methods. We have developed a double-floxed, inverse-open reading frame AAV strategy that provides for high specificity of expression, liberating optogenetics from the challenges of fitting promoter fragments into viruses, as this strategy decouples promoter strength from promoter specificity. This method opens the door to targeting virtually any cell type that is defineable genetically or by topological connectivity.
To broaden the reach of optogenetics to nonelectrical events (in neurons or in nonneuronal cells and tissues) we developed a family of light-activated G protein-coupled receptors (optoXRs) for fast control of biochemical events in freely behaving mammals. Optical control of Gs- and Gq-triggered pathways is now possible with green light, both in vitro and in vivo within freely behaving mammals.
As a physician I am particularly interested in technology application to pressing problems in biology and medicine. Therefore, in addition to our technology development work, we also have worked on applications of optogenetics. In one early effort, we and our collaborators engineered a lentivirus-based system for targeting ChR2 to the hypocretin (Hcrt, also known as orexin) neurons in the lateral hypothalamus that are important in narcolepsy, and found that specific types of electrical activity in the Hcrt neurons suffice to trigger sleep-state transitions. In this way we established a causal relationship between frequency-dependent activity of genetically defined neurons important in clinical neuropsychiatric disease and a complex mammalian behavior.
We also have employed optogenetics to determine the precise causal role of dopamine neurons in reward learning. In this work we drove defined dopamine neurons in the mouse VTA (ventral tegmental area) in different temporal patterns during free behavior, and found phasic spike parameters that were sufficient to subserve reward learning. This work is relevant to hedonic symptoms involved in depression and substance abuse, as well as in physiological reward processes.
We, along with our collaborators, also have used optogenetics to define a causal role for cortical parvalbumin neurons in specific kinds of brain rhythmicity (gamma oscillations) relevant to schizophrenia and autism, and to implicate these gamma waves in enhancing the flow of quantifiable information within neocortical microcircuits.
Finally, in preclinical work, we have used optogenetic approaches to depth target the subthalamic nucleus (STN) in animal models of Parkinson's disease, in an effort to determine the circuit and cellular mechanisms of deep brain stimulation (DBS). We were able to demonstrate that the likely direct target of DBS electrodes in the STN in Parkinson's disease is not a local cellular element in the STN, but afferent axons. This work may help us understand DBS, and assist in the design of more effective electrode-based treatments for Parkinson's disease and other disorders treatable with brain stimulation (such as depression).
In our future work, we will be engaged in further engineering and technology development work in the areas of optics, genomics, and combinatorial control. As a practicing psychiatrist as well as a neuroscientist, I am particularly interested in questions relating to depression, and we will work to extend optogenetics to this and other psychiatric and neurological diseases. We will continue to distribute optogenetic reagents to hundreds of laboratories via our lab Web site, which serves as a worldwide community resource, and to run optogenetics teaching and training courses and modules at Cold Spring Harbor Laboratory, Stanford University, and the Woods Hole Marine Biological Laboratory.