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

<div><p>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.</p></div

Latencies of saccades triggered by optogenetic activation versus visual stimulation.

<p>(A) Saccades triggered by visual stimuli (single 22-ms flash) in Monkey M1. The gray area denotes the stimulation period. (B) Saccades triggered by Opto Stim (single laser pulse with durations of 22, 44, and 66 ms, respectively) on the target (C1V1-expressing) cortical area of Monkey M1. Orange and blue lines represent horizontal and vertical eye positions, respectively. (C) Eye traces when the stimulation laser (66-ms pulse) was targeted to an unlabeled region of cortex (Mistargeted Stim). (D) Average saccade distance to the fixation point in response to visual (Left, green curve) versus optogenetic (Right, red curve) stimulation (gray denotes stimulation period; mean ± s.e.m., <i>n</i> = 94 and 96 trials, respectively). Visually induced saccades crossed the 1-degree threshold with an average latency of 119 ms, whereas Opto Stim–induced saccades crossed the 1-degree threshold with an average latency of approximately 90 ms. (E) Success rates of reactions under Opto Stim with varying laser pulse durations. (F) Saccades triggered by visual stimuli (single 22-ms flash) in Monkey M2. (G) Saccades triggered by Opto Stim (laser pulse train, at 15 Hz, 22 ms, 0.8 mW) on the target (C1V1-expressing) cortical region. (H) Saccades during Opto Stim can be sorted into two groups as a function of latency. (Left) Saccades driven by the first laser pulse of 22 ms (latency: approximately 96 ms). (Right) Saccades that failed to initiate on first pulse but instead initiated to the second laser pulse. (I) Eye traces when the stimulation laser pulses were targeted to an unlabeled region of cortex. (J) Average saccade distance to the fixation point in response to Visual Stim (Left, green curve; mean ± s.e.m., <i>n</i> = 50) versus Opto Stim (Right, red curve; mean ± s.e.m., <i>n</i> = 15). (K) Saccadic stimulation success rates as a function of Opto Stim versus cumulative pulse durations. Data can be found at <a href="https://github.com/EastRainju/Opto-TP" target="_blank">https://github.com/EastRainju/Opto-TP</a>. C1V1, ChR1/VChR1.</p

Behavior induced by visual versus optogenetic stimulation in V1.

<p>(A) “GO”/“NO GO” visual object detecting task. Monkeys were trained to report the onset of a visual cue (either a visual stimulus—Visual Stim—or an artificial visual perception induced by optogenetic stimulus—Opto Stim) by producing an eye movement. Each trial began when the NHP fixated the central fixation point. In the GO condition, monkeys were required to make a saccade within 500 ms of the cue onset to obtain a juice reward. In the NO GO condition, no stimulus was presented, and monkeys were tasked with maintaining fixation for 2,000 ms to get a juice reward. (B) An apparatus used for redirecting laser pulses towards a nearby cortical area not transduced with C1V1 (Mistargeted Stim, see insert). (C) Analysis of behavior in two monkeys (M1 & M2) during the Visual Stim block (No Stim [blue] and Visual Stim [green]) versus the Opto Stim block (No Stim [blue], Opto Stim [red], and Mistargeted Stim [teal]). The trial numbers that we recorded for the five conditions (No Stim, Visual Stim, No Stim, Opto Stim, Mis Stim) for Monkey M1 were {70, 94, 244, 96, 62}, respectively, and {32, 50, 139, 51, 14} for Monkey M2, respectively. (D) Saccadic trajectories in the Visual Stim block. The green dashed circles indicate the location of the Visual Stim, and the red dashed circles denote the receptive fields of C1V1-expressing sites. (E) Saccadic trajectories in the Opto Stim block. With Opto Stim, both monkeys uniformly targeted their eye movements to the receptive fields of the C1V1-expressing site. Data can be found at <a href="https://github.com/EastRainju/Opto-TP" target="_blank">https://github.com/EastRainju/Opto-TP</a>. C1V1, ChR1/VChR1; NHP, nonhuman primate; PMT, photomultiplier; V1, primary visual cortex.</p

Long-term stability of AOI in macaque V1.

<p>(A) 2P images of a V1 neuronal population on Day 204 and Day 292 after virus injection. (B) The same neuronal population was activated with visual stimuli on Day 158 and Day 292. (C) Repeated optical stimulation evoked stable neuronal activity in 5 example cells (colored circles in A-B) on Day 158 and Day 292 (mean ± s.e.m., <i>n</i> = 9 trials). Laser stimulation was identical to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005839#pbio.2005839.g001" target="_blank">Fig 1</a>. (D) Visual stimulation also evoked repeatable neuronal activity in the same 5 cells (from A-C) on Day 158 and Day 292 (mean ± s.e.m., <i>n</i> = 10 and 5 trials, respectively). (E) Multilayer 2P volume from the cortical surface to 500-μm depth. (F) Optogenetic calcium responses in different layers (five layers from 150-μm to 350-μm depth, three neurons from each layer) under 1P stimulation. (G) 2P images averaged across photostimulation experiments on Days 158 and 292, respectively. The orange ROIs indicate neurons that were activated by both visual and laser stimulation, on both recording days, targeted for further analysis. (H) Neuronal response correlations to optical stimulation on Days 158 and 292. Each point represents a single neuron, and the orange solid line is their corresponding regression line. The dashed line is the unity line. R² = 0.83 when fitted to the unity line, indicating that there was no significant difference between responses with long-term repeated stimulation. (I) Neuronal orientation tuning correlations on Days 158 and 292. The orientation tuned neurons were picked by ANOVA with <i>P</i> < 0.05. Data can be found at <a href="https://github.com/EastRainju/Opto-TP" target="_blank">https://github.com/EastRainju/Opto-TP</a>. 1P, single-photon; 2P, two-photon; AOI, all-optical interrogation; ROI, region of interest; V1, primary visual cortex.</p

AOI with single-cell resolution in V1.

<p>(A) 2P image of a targeted neuron co-expressing GCaMP5G and C1V1 (orange box, on Day 400 at 161 μm). (B) Calcium responses of the targeted neuron following spiral 2P optogenetic stimulation at each of the 5 × 5 grid locations from (A). The target neuron responded only to directly focused 2P stimulation, indicating that 2P stimulation is spatially precise. (C) Average fluorescence traces from the neuron when targeting 2P stimulation directly at the soma (orange) versus the surrounding parenchyma (blue; mean ± s.e.m., <i>n</i> = 15 trials). (D) Fluorescence traces of three other neurons under 2P stimulation (first two on Day 570 and third on Day 400). (E) 2P image containing four targeted neurons co-expressing GCaMP5G and C1V1 (on Day 570 at 189 μm). (F) Calcium responses of the targeted neurons (from E) when they were sequentially activated with spiral 2P stimulation. Each row demonstrates one of four neurons’ activity (numbered as in E) during stimulation on either the same or alternate numbered neuron (columns). (G) 2P images of the targeted neurons (E) and their individual average fluorescence traces when each of them was targeted (orange) versus not targeted (blue; mean ± s.e.m., <i>n</i> = 10 trials). Data can be found at <a href="https://github.com/EastRainju/Opto-TP" target="_blank">https://github.com/EastRainju/Opto-TP</a>. 2P, two-photon; AOI, all-optical interrogation; C1V1, ChR1/VChR1; GCaMP, genetically encoded calmodulin protein; V1, primary visual cortex.</p

AOI of a V1 neuronal population in awake macaque.

<p>(A) 2P image of V1 neurons expressing C1V1–ts–EYFP and GCaMP6s. The colored regions of interest (ROIs) indicate neurons that responded to both visual and optical stimuli, targeted for further analysis. (B) Top, a differential image of GCaMP6s fluorescence (stimulated-baseline [F-F0], averaged across all stimulations), driven by visual stimuli consisting of gratings or colored patches. Bottom, calcium signals from 10 neurons (colors from panel A) in response to 9 varied visual stimuli (presentation times in gray). (C) Top, widefield optogenetic stimulation (0.8 mW/mm², 30 Hz and 25% duty ratio) evoked robust responses in the same neurons. Bottom, 8 sequential identical optogenetic stimulations evoked equivalent responses in each cycle. (D) Responses of two neurons to their preferred visual stimuli (Left pink cell, color patch response; Left green cell, drifting grating response; mean ± s.e.m., <i>n</i> = 10 trials) versus photostimulation in the same cells (Right, mean ± s.e.m., <i>n</i> = 9 trials). (E) Neuronal population responses (<i>N</i> = 26) to their preferred visual stimuli versus laser stimulation. Green dots stand for cells that have similar responses to visual versus laser stimuli (<i>p</i> > 0.05), red dots for cells that have significantly stronger responses to visual stimuli versus laser stimulation (<i>p</i> < 0.05), and blue dots vice versa (<i>p</i> < 0.05). (F) Neuronal population responses (<i>N</i> = 26) from the first versus last four trials from a single experiment. (G) Laser intensity dose-response curve (mean ± s.e.m., <i>n</i> = 9 trials, <i>N</i> = 30 neurons). Optogenetic stimulation saturates at approximately 0.8 mW/mm². Data can be found at <a href="https://github.com/EastRainju/Opto-TP" target="_blank">https://github.com/EastRainju/Opto-TP</a>. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005839#pbio.2005839.s002" target="_blank">S2 Fig</a>. 1P, single-photon; 2P, two-photon; AOI, all-optical interrogation; C1V1, ChR1/VChR1; GCaMP, genetically encoded calmodulin protein; ROI, region of interest.</p

Saccadic parameters in PD patients, PSP patients and healthy controls.

<p>Saccade rates, magnitudes, peak velocity-magnitude relationship slopes and vertical components (of saccade direction) are indicated. Bars represent the average value across subjects of each group and the error bars indicate the standard error of the mean. Asterisks show significance (p<0.05, t-test).</p

Characteristics of fixational saccades across subject groups.

<p>First row, saccadic peak velocity/magnitude relationships. Second row, saccadic duration/magnitude relationships. Third row, saccade magnitude distributions. Fourth row, polar histograms of saccade directions. Each graph shows the combined data for all subjects in each group.</p

Clinical features of all patients.

*<p>On clinical examination, most patients and age-matched controlled subjects showed mild limitation of upgaze and mild impairment of convergence and smooth pursuit. HYS: Hoehn-Yahr Scale for Parkinsons’ disease <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058535#pone.0058535-Hoehn1" target="_blank">[18]</a> **All PSP patients showed impaired smooth pursuit and vergence eye movements.</p

Examples of saccadic intrusions in a control subject, a PSP patient, a PD patient, a CBS patient, a MSA patient and a SCASI patient.

<p>SWJs were present in all subject groups, although they were smaller and less frequent in healthy controls. Each trace represents a 5 s recording of horizontal eye positions containing SWJs. Horizontal position and timescales for all traces are as in the bottom trace.</p