A history of optogenetics: the development of tools for controlling brain circuits with light

Understanding how different kinds of neuron in the brain work together to implement sensations, feelings, thoughts, and movements, and how deficits in specific kinds of neuron result in brain diseases, has long been a priority in basic and clinical neuroscience. “Optogenetic” tools are genetically encoded molecules that, when targeted to specific neurons in the brain, enable their activity to be driven or silenced by light. These molecules are microbial opsins, seven-transmembrane proteins adapted from organisms found throughout the world, which react to light by transporting ions across the lipid membranes of cells in which they are genetically expressed. These tools are enabling the causal assessment of the roles that different sets of neurons play within neural circuits, and are accordingly being used to reveal how different sets of neurons contribute to the emergent computational and behavioral functions of the brain. These tools are also being explored as components of prototype neural control prosthetics capable of correcting neural circuit computations that have gone awry in brain disorders. This review gives an account of the birth of optogenetics and discusses the technology and its applications.National Institutes of Health (U.S.) (New Innovator Award (DP2OD002002))National Science Foundation (U.S.) (EFRI 0835878)National Science Foundation (U.S.) (DMS 0848804)National Science Foundation (U.S.) (DMS 1042134)National Institutes of Health (U.S.) (grant 1R01DA029639)National Institutes of Health (U.S.) (grant 1RC1MH088182)National Institutes of Health (U.S.) (grant 1RC2DE020919)National Institutes of Health (U.S.) (grant 1R01NS067199)National Institutes of Health (U.S.) (grant 1R43NS070453)United States. Dept. of Defense (CDMRP PTSD Program

Q&A: Expansion microscopy

Expansion microscopy (ExM) is a recently invented technology that uses swellable charged polymers, synthesized densely and with appropriate topology throughout a preserved biological specimen, to physically magnify the specimen 100-fold in volume, or more, in an isotropic fashion. ExM enables nanoscale resolution imaging of preserved samples on inexpensive, fast, conventional microscopes. How does ExM work? How good is its performance? How do you get going on using it? In this Q & A, we provide the answers to these and other questions about this new and rapidly spreading toolbox

Optogenetic and pharmacological suppression of spatial clusters of face neurons reveal their causal role in face gender discrimination

Neurons that respond more to images of faces over nonface objects were identified in the inferior temporal (IT) cortex of primates three decades ago. Although it is hypothesized that perceptual discrimination between faces depends on the neural activity of IT subregions enriched with “face neurons,” such a causal link has not been directly established. Here, using optogenetic and pharmacological methods, we reversibly suppressed the neural activity in small subregions of IT cortex of macaque monkeys performing a facial gender-discrimination task. Each type of intervention independently demonstrated that suppression of IT subregions enriched in face neurons induced a contralateral deficit in face gender-discrimination behavior. The same neural suppression of other IT subregions produced no detectable change in behavior. These results establish a causal link between the neural activity in IT face neuron subregions and face gender-discrimination behavior. Also, the demonstration that brief neural suppression of specific spatial subregions of IT induces behavioral effects opens the door for applying the technical advantages of optogenetics to a systematic attack on the causal relationship between IT cortex and high-level visual perception.National Institutes of Health (U.S.) (Grant K99 EY022924)National Institutes of Health (U.S.) (Grant R21 EY023053)National Institutes of Health (U.S.) (Grant R01 EY14970

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (<0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.National Institutes of Health (U.S.) (NIH Director's New Innovator Award (DP2 OD002002-01)National Institutes of Health (U.S.) (NIH Challenge Grant 1RC1MH088182-01)National Institutes of Health (U.S.) (NIH Grand Opportunities Grant 1RC2DE020919-01)National Institutes of Health (U.S.) (NIH Grand Opportunities Grant NIH 1R01NS067199-01)National Science Foundation (U.S.) (NSF 0848804)National Science Foundation (U.S.) (NSF 0835878)McGovern Institute for Brain Research at MIT (Neurotechnology Award Program)National Alliance for Research on Schizophrenia and Depression (U.S.)Alfred P. Sloan FoundationDr. Gerald Burnett and Marjorie BurnettUnited States. Dept. of DefenseSociety for Neuroscience (SFN Research Award for Innovation in Neuroscience)Massachusetts Institute of Technology. Media LaboratoryBenesse FoundationWallace H. Coulter Foundatio

Programmable RNA-binding protein composed of repeats of a single modular unit

The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby–RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.United States. National Institutes of Health (1R01NS075421)National Science Foundation (U.S.) (1344219)United States. National Institutes of Health (1U01MH106011)United States. National Institutes of Health (1R01MH103910)United States. National Institutes of Health (1DP1NS087724

Optogenetic astrocyte activation modulates response selectivity of visual cortex neurons in vivo

Astrocytes play important roles in synaptic transmission and plasticity. Despite in vitro evidence, their causal contribution to cortical network activity and sensory information processing in vivo remains unresolved. Here we report that selective photostimulation of astrocytes with channelrhodopsin-2 in primary visual cortex enhances both excitatory and inhibitory synaptic transmission, through the activation of type 1a metabotropic glutamate receptors. Photostimulation of astrocytes in vivo increases the spontaneous firing of parvalbumin-positive (PV[superscript +]) inhibitory neurons, while excitatory and somatostatin-positive (SOM[superscript +]) neurons show either an increase or decrease in their activity. Moreover, PV[superscript +] neurons show increased baseline visual responses and reduced orientation selectivity to visual stimuli, whereas excitatory and SOM[superscript +] neurons show either increased or decreased baseline visual responses together with complementary changes in orientation selectivity. Therefore, astrocyte activation, through the dual control of excitatory and inhibitory drive, influences neuronal integrative features critical for sensory information processing.National Institutes of Health (U.S.)National Science Foundation (U.S.)Picower Institute for Learning and Memory (Innovation Fund)Simons FoundationMarie Curie International Outgoing Fellowship (FP7-253635)Consolider (CDS2010-00045)Ramon y Cajal Program (RYC-2012-12014

A fully genetically encoded protein architecture for optical control of peptide ligand concentration

Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin’s local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology.National Institutes of Health (U.S.) (grant NIH 1DP2OD002002)National Institutes of Health (U.S.) (grant NIH 1R01DA029639)National Institutes of Health (U.S.) (grant NIH 1R01NS075421)National Institutes of Health (U.S.) (grant NIH 1RC1MH088182)National Science Foundation (U.S.) (NSF CAREER Award CBET 1053233)United States. Defense Advanced Research Projects Agency (DARPA Living Foundries, Contract HR0011-12-C-0068)New York Stem Cell Foundation (Robertson Investigator Award)Damon Runyon Cancer Research Foundation (DRG 2095-11)Fannie and John Hertz FoundationNational Science Foundation (U.S.) (Graduate Research Fellowship under grant no. 1122374)Massachusetts Institute of Technology. Synthetic Intelligence Laboratory (project