Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral ...cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.
Over the last 10 years, optogenetics has become widespread in neuroscience for the study of how specific cell types contribute to brain functions and brain disorder states. The full impact of ...optogenetics will emerge only when other toolsets mature, including neural connectivity and cell phenotyping tools and neural recording and imaging tools. The latter tools are rapidly improving, in part because optogenetics has helped galvanize broad interest in neurotechnology development.
Expansion microscopy Chen, Fei; Tillberg, Paul W.; Boyden, Edward S.
Science,
01/2015, Letnik:
347, Številka:
6221
Journal Article
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In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it ...can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ∼70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ∼107 cubic micrometers of the mouse hippocampus with a conventional confocal microscope.
Many biological investigations require 3D imaging of cells or tissues with nanoscale spatial resolution. We recently discovered that preserved biological specimens can be physically expanded in an ...isotropic fashion through a chemical process. Expansion microscopy (ExM) allows nanoscale imaging of biological specimens with conventional microscopes, decrowds biomolecules in support of signal amplification and multiplexed readout chemistries, and makes specimens transparent. We review the principles of how ExM works, advances in the technology made by our group and others, and its applications throughout biology and medicine.
The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically ...targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells.
Optogenetics is currently the state-of-the-art method for causal-oriented brain research. Despite an increasingly large number of invertebrate and rodent studies showing profound electrophysiological ...and behavioral effects induced by optogenetics 1, 2, only two primate studies have reported modulation of local single-cell activity but with no behavioral effects 3, 4. Here, we show that optogenetic stimulation of cortical neurons within rhesus monkey arcuate sulcus, during the execution of a visually guided saccade task, evoked significant and reproducible changes in saccade latencies as a function of target position. Moreover, using concurrent optogenetic stimulation and opto-fMRI 5, 6), we observed optogenetically induced changes in fMRI activity in specific functional cortical networks throughout the monkey brain. This is critical information for the advancement of optogenetic primate research models and for initiating the development of optogenetically based cell-specific therapies with which to treat neurological diseases in humans.
► Optogenetics induces behavioral changes in monkeys ► Optogenetics evokes fMRI signal changes in functional networks of macaques ► Saccade latencies decrease by optogenetic stimulation of the arcuate sulcus
Optogenetic studies in mice have revealed new relationships between well-defined neurons and brain functions. However, there are currently no means to achieve the same cell-type specificity in ...monkeys, which possess an expanded behavioral repertoire and closer anatomical homology to humans. Here, we present a resource for cell-type-specific channelrhodopsin expression in Rhesus monkeys and apply this technique to modulate dopamine activity and monkey choice behavior. These data show that two viral vectors label dopamine neurons with greater than 95% specificity. Infected neurons were activated by light pulses, indicating functional expression. The addition of optical stimulation to reward outcomes promoted the learning of reward-predicting stimuli at the neuronal and behavioral level. Together, these results demonstrate the feasibility of effective and selective stimulation of dopamine neurons in non-human primates and a resource that could be applied to other cell types in the monkey brain.
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•Cell-type-specific promoter drives Cre-dependent ChR2 expression in monkey•Optogenetically activated neurons had dopamine-like features and reward responses•Dopamine neurons respond strongly to cues predicting optical stimulation•Monkeys choose predicted optogenetic stimulation over no predicted stimulation
Cell-type specific optogenetic stimulation in Rhesus monkeys allows manipulation of dopamine neurons to demonstrate their role in reward-based learning
An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still ...limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.
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•Evaluated multiple mouse transgenic intersectional strategies•Established TIGRE locus as a novel permissive docking site for transgene expression•Developed 21 new intersectional driver and reporter lines expressing novel tools•Demonstrated functionality of new voltage or calcium sensing or optogenetic lines
Madisen et al. developed multiple intersectional transgenic mouse lines and AAVs for gene expression control with high specificity, in particular TIGRE locus-based high-level expression of calcium sensors GCaMP6 and YCX2.60, voltage sensor VSFP Butterfly 1.2, and optogenetic silencer Jaws.
Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct ...spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.
In order to understand how the brain generates behaviors, it is important to be able to determine how neural circuits work together to perform computations. Because neural circuits are made of a ...great diversity of cell types, it is critical to be able to analyze how these different kinds of cell work together. In recent years, a toolbox of fully genetically encoded molecules has emerged that, when expressed in specific neurons, enables the electrical activity of the targeted neurons to be controlled in a temporally precise fashion by pulses of light. We describe this optogenetic toolbox, how it can be used to analyze neural circuits in the brain and how optogenetics is impacting the study of cognition.
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