Closed-Loop Optogenetic Perturbation of Macaque Oculomotor Cerebellum: Evidence for an Internal Saccade Model.

Internal models are essential for the production of accurate movements. The accuracy of saccadic eye movements is thought to be mediated by an internal model of oculomotor mechanics encoded in the cerebellum. The cerebellum may also be part of a feedback loop that predicts the displacement of the eyes and compares it to the desired displacement in real time to ensure that saccades land on target. To investigate the role of the cerebellum in these two aspects of saccade production, we delivered saccade-triggered light pulses to channelrhodopsin-2-expressing Purkinje cells in the oculomotor vermis (OMV) of two male macaque monkeys. Light pulses delivered during the acceleration phase of ipsiversive saccades slowed the deceleration phase. The long latency of these effects and their scaling with light pulse duration are consistent with an integration of neural signals at or downstream of the stimulation site. In contrast, light pulses delivered during contraversive saccades reduced saccade velocity at short latency and were followed by a compensatory reacceleration which caused gaze to land on or near the target. We conclude that the contribution of the OMV to saccade production depends on saccade direction; the ipsilateral OMV is part of a forward model that predicts eye displacement, whereas the contralateral OMV is part of an inverse model that creates the force required to move the eyes with optimal peak velocity for the intended displacement.

Encoding of action by the Purkinje cells of the cerebellum.

Execution of accurate eye movements depends critically on the cerebellum, suggesting that the major output neurons of the cerebellum, Purkinje cells, may predict motion of the eye. However, this encoding of action for rapid eye movements (saccades) has remained unclear: Purkinje cells show little consistent modulation with respect to saccade amplitude or direction, and critically, their discharge lasts longer than the duration of a saccade. Here we analysed Purkinje-cell discharge in the oculomotor vermis of behaving rhesus monkeys (Macaca mulatta) and found neurons that increased or decreased their activity during saccades. We estimated the combined effect of these two populations via their projections to the caudal fastigial nucleus, and uncovered a simple-spike population response that precisely predicted the real-time motion of the eye. When we organized the Purkinje cells according to each cell's complex-spike directional tuning, the simple-spike population response predicted both the real-time speed and direction of saccade multiplicatively via a gain field. This suggests that the cerebellum predicts the real-time motion of the eye during saccades via the combined inputs of Purkinje cells onto individual nucleus neurons. A gain-field encoding of simple spikes emerges if the Purkinje cells that project onto a nucleus neuron are not selected at random but share a common complex-spike property.

Elimination of the error signal in the superior colliculus impairs saccade motor learning.

When movements become dysmetric, the resultant motor error induces a plastic change in the cerebellum to correct the movement, i.e., motor adaptation. Current evidence suggests that the error signal to the cerebellum is delivered by complex spikes originating in the inferior olive (IO). To prove a causal link between the IO error signal and motor adaptation, several studies blocked the IO, which, unfortunately, affected not only the adaptation but also the movement itself. We avoided this confound by inactivating the source of an error signal to the IO. Several studies implicate the superior colliculus (SC) as the source of the error signal to the IO for saccade adaptation. When we inactivated the SC, the metrics of the saccade to be adapted were unchanged, but saccade adaptation was impaired. Thus, an intact rostral SC is necessary for saccade adaptation. Our data provide experimental evidence for the cerebellar learning theory that requires an error signal to drive motor adaptation.

How cerebellar motor learning keeps saccades accurate.

The neuronal substrate underlying the learning of a sophisticated task has been difficult to study. However, the advent of a behavioral paradigm that deceives the saccadic system into thinking it is making an error has allowed the mechanisms of the adaptation that corrects this error to be revealed in a primate. The neural elements that fashion the command signal for the generation of accurate saccades involve subcortical structures in the brain stem and cerebellum. In this review we show that sites in both those structures also are involved with the gradual adaptation of saccade size, a form of motor learning. Pharmacological manipulation of the oculomotor vermis (lobules VIc and VII) impairs mechanisms that either increase or decrease saccade size during adaptation. The net saccade-related simple spike (SS) activity of its Purkinje cells is correlated with the changes in saccade characteristics that occur during adaptation. These changes in SS activity are driven by an error signal delivered over climbing fibers, which create complex spikes whose probability of occurrence reflects the motor error between the actual and desired saccade size. These climbing fibers originate in the part of the inferior olive that receives projections from the superior colliculus (SC). Disabling the SC prevents adaptation and stimulation of the SC just after a normal saccade produces a surrogate error signal that drives adaptation without an actual visual error. Therefore, the SC provides not only the initial command that generates a saccade, as shown by others, but also the error signal that ensures that saccades remain accurate.

Signals driving the adaptation of saccades that require spatial updating.

Saccades adapt to persistent natural or artificially imposed dysmetrias. The characteristics and circuitry of saccade adaptation have been revealed using a visually guided task (VGT) where the vectors of the target step and the intended saccade command are the same. However, in real life, another saccade occasionally intervenes before the saccade to the target occurs. This necessitates an updating of the intended saccade to account for the intervening saccadic displacement, which dissociates the visual target signal and the intended saccade command. We determined whether the adaptation process is similar for VGT and updated saccades by studying the transfer of adaptation between them. The ultimate visual target was dissociated from the intended saccade command with double-step saccade tasks (DSTs) in which two targets are flashed sequentially at different locations while the monkey maintains fixation. The resulting saccades toward the first and second targets occur in the dark. The transfer of visually guided saccade adaptation to the second saccades of a DST and vice versa depended on the eccentricity of the second visual target, and not the second saccade command. If a target with the same eccentricity as the adapted target appears briefly during the intersaccadic interval of a DST, more adaptation transfers. Because a brief appearance of the visual target either before the first saccade or during the intersaccadic interval influences how much adaptation transfer the second saccade will express, the processing of adaptation and DST updating may overlap. NEW & NOTEWORTHY Adaptation and the spatial updating of saccades are thought to be independent processes. When we dissociate the visual target and the intended saccade command, the transfer of visually guided saccade adaptation to the saccades of the double-step saccade tasks (DST) and vice versa is driven by a visual not motor error. The visual target has an effect until the second saccade of a DST occurs. Therefore, the processing of adaptation and the spatial updating of saccades may overlap.

Adaptation and adaptation transfer characteristics of five different saccade types in the monkey.

Shifts in the direction of gaze are accomplished by different kinds of saccades, which are elicited under different circumstances. Saccade types include targeting saccades to simple jumping targets, delayed saccades to visible targets after a waiting period, memory-guided (MG) saccades to remembered target locations, scanning saccades to stationary target arrays, and express saccades after very short latencies. Studies of human cases and neurophysiological experiments in monkeys suggest that separate pathways, which converge on a common locus that provides the motor command, generate these different types of saccade. When behavioral manipulations in humans cause targeting saccades to have persistent dysmetrias as might occur naturally from growth, aging, and injury, they gradually adapt to reduce the dysmetria. Although results differ slightly between laboratories, this adaptation generalizes or transfers to all the other saccade types mentioned above. Also, when one of the other types of saccade undergoes adaptation, it often transfers to another saccade type. Similar adaptation and transfer experiments, which allow inferences to be drawn about the site(s) of adaptation for different saccade types, have yet to be done in monkeys. Here we show that simian targeting and MG saccades adapt more than express, scanning, and delayed saccades. Adaptation of targeting saccades transfers to all the other saccade types. However, the adaptation of MG saccades transfers only to delayed saccades. These data suggest that adaptation of simian targeting saccades occurs on the pathway common to all saccade types. In contrast, only the delayed saccade command passes through the adaptation site of the MG saccade.

Cerebellar fastigial nucleus influence on ipsilateral abducens activity during saccades.

To characterize the cerebellar influence on neurons in the abducens (ABD) nucleus, we recorded ABD neurons before and after we inactivated the caudal part of the ipsilateral cerebellar fastigial nucleus (cFN) with muscimol injection. cFN activity influences the horizontal component of saccades. cFN inactivation increased the activity of most ipsilateral ABD neurons (19/22 in 2 monkeys) during ipsiversive (hypermetric) saccades, primarily by increasing burst duration. During contraversive (hypometric) saccades, the off-direction pause of most (10/15) ABD neurons was shorter than normal because of the early resumption of ABD activity. Early ABD firing caused the early contraction of antagonist muscles that reduced eye rotation and made contraversive saccades hypometric. Thus the cerebellum controls ipsilateral ABD activity by truncating on-direction bursts during ipsiversive saccades and extending off-direction pauses during contraversive saccades. We conclude that cFN output keeps saccades accurate by controlling when ABD on-direction bursts and off-direction pauses end.

Primate Eye Tracking with Carbon-Nanotube-Paper-Composite Based Capacitive Sensors and Machine Learning Algorithms.

BACKGROUND: Accurate real-time eye tracking is crucial in oculomotor system research. While the scleral search coil system is the gold standard, its implantation procedure and bulkiness pose challenges. Camera-based systems are affected by ambient lighting and require high computational and electric power. New Method This study presents a novel eye tracker using proximity capacitive sensors made of carbon-nanotube-paper-composite (CPC). These sensors detect femtofarad-level capacitance changes caused by primate corneal movement during horizontal and vertical eye rotations. Data processing and machine learning algorithms are evaluated to enhance the accuracy of gaze angle prediction. RESULTS: The system performance is benchmarked against the scleral coil during smooth pursuits, saccades tracking, and fixations. The eye tracker demonstrates up to 0.97 correlation with the coil in eye tracking and is capable of estimating gaze angle with a median absolute error as low as 0.30°. Comparison The capacitive eye tracker demonstrates good consistency and accuracy in comparison to the gold-standard scleral search coil method. CONCLUSIONS: This lightweight, non-invasive capacitive eye tracker offers potential as an alternative to traditional coil and camera-based systems in oculomotor research and vision science.

Closed-loop optogenetic perturbation of macaque oculomotor cerebellum: evidence for an internal saccade model.

Internal models are essential for the production of accurate movements. The accuracy of saccadic eye movements is thought to be mediated by an internal model of oculomotor mechanics encoded in the cerebellum. The cerebellum may also be part of a feedback loop that predicts the displacement of the eyes and compares it to the desired displacement in real time to ensure that saccades land on target. To investigate the role of the cerebellum in these two aspects of saccade production, we delivered saccade-triggered light pulses to channelrhodopsin-2-expressing Purkinje cells in the oculomotor vermis (OMV) of two macaque monkeys. Light pulses delivered during the acceleration phase of ipsiversive saccades slowed the deceleration phase. The long latency of these effects and their scaling with light pulse duration are consistent with an integration of neural signals at or downstream of the stimulation site. In contrast, light pulses delivered during contraversive saccades reduced saccade velocity at short latency and were followed by a compensatory reacceleration which caused gaze to land near or on the target. We conclude that the contribution of the OMV to saccade production depends on saccade direction; the ipsilateral OMV is part of a forward model that predicts eye displacement, whereas the contralateral OMV is part of an inverse model that creates the force required to move the eyes with optimal peak velocity for the intended displacement.

Conserved circuits for direction selectivity in the primate retina.

The detection of motion direction is a fundamental visual function and a classic model for neural computation. In the non-primate retina, direction selectivity arises in starburst amacrine cell (SAC) dendrites, which provide selective inhibition to direction-selective retinal ganglion cells (dsRGCs). Although SACs are present in primates, their connectivity and the existence of dsRGCs remain open questions. Here, we present a connectomic reconstruction of the primate ON SAC circuit from a serial electron microscopy volume of the macaque central retina. We show that the structural basis for the SACs' ability to confer directional selectivity on postsynaptic neurons is conserved. SACs selectively target a candidate homolog to the mammalian ON-sustained dsRGCs that project to the accessory optic system (AOS) and contribute to gaze-stabilizing reflexes. These results indicate that the capacity to compute motion direction is present in the retina, which is earlier in the primate visual system than classically thought.

Changes in simple spike activity of some Purkinje cells in the oculomotor vermis during saccade adaptation are appropriate to participate in motor learning.

Adaptation of saccadic eye movements provides an excellent motor learning model to study theories of neuronal plasticity. When primates make saccades to a jumping target, a backward step of the target during the saccade can make it appear to overshoot. If this deception continues for many trials, saccades gradually decrease in amplitude to go directly to the back-stepped target location. We used this adaptation paradigm to evaluate the Marr-Albus hypothesis that such motor learning occurs at the Purkinje (P)-cell of the cerebellum. We recorded the activity of identified P-cells in the oculomotor vermis, lobules VIc and VII. After documenting the on and off error directions of the complex spike activity of a P-cell, we determined whether its saccade-related simple spike (SS) activity changed during saccade adaptation in those two directions. Before adaptation, 57 of 61 P-cells exhibited a clear burst, pause, or a combination of both for saccades in one or both directions. Sixty-two percent of all cells, including two of the four initially unresponsive ones, behaved differently for saccades whose size changed because of adaptation than for saccades of similar sizes gathered before adaptation. In at least 42% of these, the changes were appropriate to decrease saccade amplitude based on our current knowledge of cerebellum and brainstem saccade circuitry. Changes in activity during adaptation were not compensating for the potential fatigue associated with performing many saccades. Therefore, many P-cells in the oculomotor vermis exhibit changes in SS activity specific to adapted saccades and therefore appropriate to induce adaptation.

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain.

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

Subthreshold activation of the superior colliculus drives saccade motor learning.

How the brain learns and maintains accurate precision movements is currently unknown. At times throughout life, rapid gaze shifts (saccades) become inaccurate, but the brain makes gradual adjustments so they again stop on target. Previously, we showed that complex spikes (CSs) in Purkinje cells of the oculomotor cerebellum report the direction and amplitude by which saccades are in error. Anatomical studies indicate that this error signal could originate in the superior colliculus (SC). Here, we deliver subthreshold electrical stimulation of the SC after the saccade lands to signal an apparent error. The size of saccades in the same direction as the simulated error gradually increase; those in the opposite direction decrease. The electrically adapted saccades endure after stimulation is discontinued, exhibit an adaptation field, can undergo changes in direction, and depend on error timing. These electrically induced adaptations were virtually identical with those produced by the visually induced adaptations that we report here for comparable visual errors in the same monkeys. Therefore, our experiments reveal that an additional role for the SC in the generation of saccades is to provide a vector error signal that drives dysmetric saccades to adapt. Moreover, the characteristics of the electrically induced adaptation reflect those of error-related CS activity in the oculomotor cerebellum, suggesting that CS activity serves as the learning signal. We speculate that CS activity may serve as the error signal that drives other kinds of motor learning as well.

Behavior of the oculomotor vermis for five different types of saccade.

Single unit and lesion studies have implicated the oculomotor vermis of the cerebellum in the control of targeting saccades to jumping visual targets. However, saccades can be made in a variety of other target situations where they can occur with different reaction times (express or delayed saccades) in response to a remembered target location (memory-guided saccades) or between several targets that are always visible (scanning saccades). Here we ask whether the oculomotor vermis contributes to generating all these types of saccades by examining the simple spike discharge of its Purkinje cells. Twenty-six of 32 P-cells (81%) exhibited qualitatively similar phasic firing patterns for targeting, express, scanning, delayed, and memory-guided saccades. The remaining six exhibited a different pattern for just scanning saccades. Although a sensitive test of discharge patterns revealed significant differences for some pairs of saccade types in ∼29% of P-cells, there was no cell-to-cell consistency as to which pairs were associated with different patterns. Also, a less sensitive comparison identified substantially fewer cells (∼15%) with different patterns. Thus the lack of any consistent difference in firing for different saccade types leads us to conclude that the oculomotor vermis is not likely to contribute differently to targeting, express, scanning, delayed, or memory-guided saccades.

Encoding of error and learning to correct that error by the Purkinje cells of the cerebellum.

The primary output cells of the cerebellar cortex, Purkinje cells, make kinematic predictions about ongoing movements via high-frequency simple spikes, but receive sensory error information about that movement via low-frequency complex spikes (CS). How is the vector space of sensory errors encoded by this low-frequency signal? Here we measured Purkinje cell activity in the oculomotor vermis of animals during saccades, then followed the chain of events from experience of visual error, generation of CS, modulation of simple spikes, and ultimately change in motor output. We found that while error direction affected the probability of CS, error magnitude altered its temporal distribution. Production of CS changed the simple spikes on the next trial, but regardless of the actual visual error, this change biased the movement only along a vector that was parallel to the Purkinje cell's preferred error. From these results, we inferred the anatomy of a sensory-to-motor adaptive controller that transformed visual error vectors into motor-corrections.

Complex spike activity of purkinje cells in the oculomotor vermis during behavioral adaptation of monkey saccades.

Throughout life, the oculomotor system can correct itself when saccadic eye movements become inaccurate. This adaptation mechanism can be engaged in the laboratory by displacing the target when the saccade toward it is in flight. Forward and backward target displacements cause gradual increases and decreases in saccade amplitude, respectively. Equipped with this paradigm, we asked whether Purkinje cells (P-cells) in the vermis of the oculomotor cerebellum, lobules VIc and VII, changed their complex spike (CS) discharge during the behavioral adaptation of horizontal saccades. We tested the hypothesis that CS activity would change only when a targeting saccade caused an error in eye position relative to the target, i.e., during the error interval between the primary and corrective saccades. We examined only those P-cells whose simple spike activity exhibited either a burst or pause with saccades in several directions. Approximately 80% of such P-cells exhibited an increase in CS activity during the error interval when the adaptation paradigm imposed horizontal eye-position errors in one direction and a decrease in activity for errors in the other. As adaptation progressed and errors were reduced, there was no consistent change in the CS activity. These data suggest that the CS activity of P-cells in the oculomotor vermis signals the direction but not the magnitude of eye-position error during saccade adaptation. Our results are consistent with cerebellar learning models that have been proposed to explain adaptation of the vestibulo-ocular reflex so similar mechanisms may also underlie plasticity of this precision voluntary oculomotor behavior.

Change in sensitivity to visual error in superior colliculus during saccade adaptation.

Saccadic eye movements provide a valuable model to study the brain mechanisms underlying motor learning. If a target is displaced surreptitiously while a saccade is underway, the saccade appears to be in error. If the error persists gradual neuronal adjustments cause the eye movement again to land near the target. This saccade adaptation typically follows an exponential time course, i.e., adaptation speed slows as adaptation progresses, indicating that the sensitivity to error decreases during adaptation. Previous studies suggested that the superior colliculus (SC) sends error signals to drive saccade adaptation. The objective of this study is to test whether the SC error signal is related to the decrease in the error sensitivity during adaptation. We show here that the visual activity of SC neurons, which is induced by a constant visual error that drives adaptation, decreases during saccade adaptation. This decrease of sensitivity to visual error was not correlated with the changes of primary saccade amplitude. Therefore, a possible interpretation of this result is that the reduction of visual sensitivity of SC neurons contributes an error sensitivity signal that could help control the saccade adaptation process.

Selective reward affects the rate of saccade adaptation.

In this study we tested whether a selective reward could affect the adaptation of saccadic eye movements in monkeys. We induced the adaptation of saccades by displacing the target of a horizontal saccade vertically as the eye moved toward it, thereby creating an apparent vertical dysmetria. The repeated upward target displacement caused the originally horizontal saccade to gradually deviate upward over the course of several hundred trials. We induced this directional adaptation in both right- and leftward saccades in every experiment (n=20). In half of the experiments (n=10), we rewarded monkeys only when they made leftward saccades and in the other half (n=10) only for rightward saccades. The reaction time of saccades in the rewarded direction was shorter and we, like others, interpreted this change as a sign of the reward's preferential effect in that direction. Saccades in the rewarded direction showed more rapid adaptation of their directions than did saccades in the non-rewarded direction, indicating that the selective reward increased the speed of saccade adaptation. The differences in adaptation speed were reflected in changes in saccade metrics, which were usually more noticeable in the deceleration phases of saccades than in their acceleration phases. Because previous studies have shown that the oculomotor cerebellum is involved with saccade deceleration and also participates in saccade adaptation, it is possible that selective reward could influence cerebellar plasticity.

Selective Optogenetic Control of Purkinje Cells in Monkey Cerebellum.

Purkinje cells of the primate cerebellum play critical but poorly understood roles in the execution of coordinated, accurate movements. Elucidating these roles has been hampered by a lack of techniques for manipulating spiking activity in these cells selectively-a problem common to most cell types in non-transgenic animals. To overcome this obstacle, we constructed AAV vectors carrying the channelrhodopsin-2 (ChR2) gene under the control of a 1 kb L7/Pcp2 promoter. We injected these vectors into the cerebellar cortex of rhesus macaques and tested vector efficacy in three ways. Immunohistochemical analyses confirmed selective ChR2 expression in Purkinje cells. Neurophysiological recordings confirmed robust optogenetic activation. Optical stimulation of the oculomotor vermis caused saccade dysmetria. Our results demonstrate the utility of AAV-L7-ChR2 for revealing the contributions of Purkinje cells to circuit function and behavior, and they attest to the feasibility of promoter-based, targeted, genetic manipulations in primates.

Effect of inactivation and disinhibition of the oculomotor vermis on saccade adaptation.

The ability to adapt a variety of motor acts to compensate for persistent natural or artificially induced errors in movement accuracy requires the cerebellum. For adaptation of the rapid shifts in the direction of gaze called saccades, the oculomotor vermis (OMV) of the cerebellum must be intact. We disrupted the neural circuitry of the OMV by manipulating gamma aminobutyric acid (GABA), the transmitter used by many neurons in the vermis. We injected either muscimol, an agonist of GABA, to inactivate the OMV or bicuculline, an antagonist, to block GABA inhibition. Our previous study showed that muscimol injections cause ipsiversive saccades to fall short of their targets, whereas bicuculline injections cause most ipsiversive saccades to overshoot. Once these dysmetrias had stabilized, we tested the monkey's ability to adapt saccade size to intra-saccadic target steps that produced a consistent saccade under-shoot (amplitude increase adaptation required) or overshoot (amplitude decrease adaptation required). Injections of muscimol abolished the amplitude increase adaptation of ipsiversive saccades, but had either no effect, or occasionally facilitated, amplitude decrease adaptation. In contrast, injections of bicuculline impaired amplitude decrease adaptation and usually facilitated amplitude increase adaptation. Neither drug produced consistent effects on the adaptation of contraversive saccades. Taken together, these data suggest that OMV activity is necessary for amplitude increase adaptation, whereas amplitude decrease adaptation may involve the inhibitory circuits within the OMV.