Long-term all-optical interrogation of cortical neurons in awake-behaving nonhuman primates

Whereas optogenetic techniques have proven successful in their ability to manipulate neuronal populations—with high spatial and temporal fidelity—in species ranging from insects to rodents, significant obstacles remain in their application to nonhuman primates (NHPs). Robust optogenetics-activated behavior and long-term monitoring of target neurons have been challenging in NHPs. Here, we present a method for all-optical interrogation (AOI), integrating optical stimulation and simultaneous two-photon (2P) imaging of neuronal populations in the primary visual cortex (V1) of awake rhesus macaques. A red-shifted channel-rhodopsin transgene (ChR1/VChR1 [C1V1]) and genetically encoded calcium indicators (genetically encoded calmodulin protein [GCaMP]5 or GCaMP6s) were delivered by adeno-associated viruses (AAVs) and subsequently expressed in V1 neuronal populations for months. We achieved optogenetic stimulation using both single-photon (1P) activation of neuronal populations and 2P activation of single cells, while simultaneously recording 2P calcium imaging in awake NHPs. Optogenetic manipulations of V1 neuronal populations produced reliable artificial visual percepts. Together, our advances show the feasibility of precise and stable AOI of cortical neurons in awake NHPs, which may lead to broad applications in high-level cognition and preclinical testing studies.

[1]  Jacob G. Bernstein,et al.  Millisecond-Timescale Optical Control of Neural Dynamics in the Nonhuman Primate Brain , 2009, Neuron.

[2]  Edward S Boyden,et al.  FEF inactivation with improved optogenetic methods , 2016, Proceedings of the National Academy of Sciences.

[3]  K. Svoboda,et al.  Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice , 2008, Nature.

[4]  Karl Deisseroth,et al.  Optogenetics enables functional analysis of human embryonic stem cell–derived grafts in a Parkinson's disease model , 2015, Nature Biotechnology.

[5]  M. Jazayeri,et al.  Saccadic eye movements evoked by optogenetic activation of primate V 1 , 2012 .

[6]  W. Dobelle,et al.  Phosphenes produced by electrical stimulation of human occipital cortex, and their application to the development of a prosthesis for the blind , 1974, The Journal of physiology.

[7]  Xoana G. Troncoso,et al.  Microsaccades Counteract Visual Fading during Fixation , 2005, Neuron.

[8]  Leandro L. Di Stasi,et al.  Abnormal Capillary Vasodynamics Contribute to Ictal Neurodegeneration in Epilepsy , 2017, Scientific Reports.

[9]  Yasmine El-Shamayleh,et al.  Nonhuman Primate Optogenetics: Recent Advances and Future Directions , 2017, The Journal of Neuroscience.

[10]  Fang Liu,et al.  Long-Term Two-Photon Imaging in Awake Macaque Monkey , 2017, Neuron.

[11]  Bruce R. Rosen,et al.  Optogenetically Induced Behavioral and Functional Network Changes in Primates , 2012, Current Biology.

[12]  T. Oertner,et al.  Optical induction of synaptic plasticity using a light-sensitive channel , 2007, Nature Methods.

[13]  Michael C. Avery,et al.  Optogenetic Activation of Normalization in Alert Macaque Visual Cortex , 2015, Neuron.

[14]  P. Schiller,et al.  The role of the monkey superior colliculus in eye movement and vision. , 1972, Investigative ophthalmology.

[15]  Ilana B. Witten,et al.  Recombinase-Driver Rat Lines: Tools, Techniques, and Optogenetic Application to Dopamine-Mediated Reinforcement , 2011, Neuron.

[16]  Samouil L. Farhi,et al.  All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins , 2014, Nature Methods.

[17]  S. Martinez-Conde,et al.  Fixational eye movements across vertebrates: comparative dynamics, physiology, and perception. , 2008, Journal of vision.

[18]  Matthew T. Kaufman,et al.  An optogenetic toolbox designed for primates , 2011, Nature Neuroscience.

[19]  G. Brindley,et al.  The sensations produced by electrical stimulation of the visual cortex , 1968, The Journal of physiology.

[20]  Stefan R. Pulver,et al.  Ultra-sensitive fluorescent proteins for imaging neuronal activity , 2013, Nature.

[21]  Nathan R. Wilson,et al.  Division and subtraction by distinct cortical inhibitory networks in vivo , 2012, Nature.

[22]  Jacob G. Bernstein,et al.  Optogenetic tools for analyzing the neural circuits of behavior , 2011, Trends in Cognitive Sciences.

[23]  David L. Sheinberg,et al.  Optogenetic and Electrical Microstimulation Systematically Bias Visuospatial Choice in Primates , 2014, Current Biology.

[24]  Hiroshi Kawasaki,et al.  Long-Term Two-Photon Calcium Imaging of Neuronal Populations with Subcellular Resolution in Adult Non-human Primates. , 2015, Cell reports.

[25]  Stefan R. Pulver,et al.  Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics , 2013, Front. Mol. Neurosci..

[26]  Mehrdad Jazayeri,et al.  Saccadic eye movements evoked by optogenetic activation of primate V1 , 2012, Nature Neuroscience.

[27]  Lief E. Fenno,et al.  Neocortical excitation/inhibition balance in information processing and social dysfunction , 2011, Nature.

[28]  Rainer W Friedrich,et al.  High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device , 2012, Nature Protocols.

[29]  Ji Dai,et al.  Behavioral Manipulation by Optogenetics in the Nonhuman Primate , 2018, The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry.

[30]  D. Tank,et al.  Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields , 2014, Nature Neuroscience.

[31]  E. J. Tehovnik,et al.  Saccadic eye movements evoked by microstimulation of striate cortex , 2003, The European journal of neuroscience.

[32]  Sharad Ramanathan,et al.  Optical interrogation of neural circuits in Caenorhabditis elegans , 2009, Nature Methods.

[33]  K. Deisseroth,et al.  Millisecond-timescale, genetically targeted optical control of neural activity , 2005, Nature Neuroscience.

[34]  Anna W Roe,et al.  Optogenetics through windows on the brain in the nonhuman primate. , 2013, Journal of neurophysiology.

[35]  Jasper Akerboom,et al.  Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging , 2012, The Journal of Neuroscience.

[36]  William R. Stauffer,et al.  Dopamine Neuron-Specific Optogenetic Stimulation in Rhesus Macaques , 2016, Cell.

[37]  E. Boyden,et al.  Temporally precise single-cell resolution optogenetics , 2017, Nature Neuroscience.

[38]  Michael Häusser,et al.  Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo , 2014, Nature Methods.

[39]  Andreas S Tolias,et al.  In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain , 2017, Nature Methods.

[40]  Karl Deisseroth,et al.  Integration of light-controlled neuronal firing and fast circuit imaging , 2007, Current Opinion in Neurobiology.

[41]  Edward S. Boyden,et al.  Optogenetic Inactivation Modifies Monkey Visuomotor Behavior , 2012, Neuron.

[42]  Benjamin F. Grewe,et al.  Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation , 2012, Nature Methods.

[43]  Winfried Denk,et al.  On the fundamental imaging-depth limit in two-photon microscopy , 2006 .

[44]  C. Kufta,et al.  Feasibility of a visual prosthesis for the blind based on intracortical microstimulation of the visual cortex. , 1996, Brain : a journal of neurology.

[45]  M. Häusser,et al.  All-Optical Interrogation of Neural Circuits , 2015, The Journal of Neuroscience.

[46]  Johannes D. Seelig,et al.  Feature detection and orientation tuning in the Drosophila central complex , 2013, Nature.

[47]  Feng Zhang,et al.  Multimodal fast optical interrogation of neural circuitry , 2007, Nature.

[48]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[49]  Yasmine El-Shamayleh,et al.  Selective Optogenetic Control of Purkinje Cells in Monkey Cerebellum , 2017, Neuron.

[50]  Shay Ohayon,et al.  Deep brain fluorescence imaging with minimally invasive ultra-thin optical fibers , 2017, bioRxiv.

[51]  Azadeh Yazdan-Shahmorad,et al.  A Large-Scale Interface for Optogenetic Stimulation and Recording in Nonhuman Primates , 2016, Neuron.

[52]  J. Bullier,et al.  Visual latencies in areas V1 and V2 of the macaque monkey , 1995, Visual Neuroscience.