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.

Color and luminance processing in V1 complex cells and artificial neural networks.

Object recognition by natural and artificial visual systems benefits from the identification of object boundaries. A useful cue for the detection of object boundaries is the superposition of luminance and color edges. To gain insight into the suitability of this cue for object recognition, we examined convolutional neural network models that had been trained to recognize objects in natural images. We focused specifically on units in the second convolutional layer whose activations are invariant to the spatial phase of a sinusoidal grating. Some of these units were tuned for a nonlinear combination of color and luminance, which is broadly consistent with a role in object boundary detection. Others were tuned for luminance alone, but very few were tuned for color alone. A literature review reveals that V1 complex cells have a similar distribution of tuning. We speculate that this pattern of sensitivity provides an efficient basis for object recognition, perhaps by mitigating the effects of lighting on luminance contrast polarity. The absence of a contrast polarity-invariant representation of chromaticity alone suggests that it is redundant with other representations.

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.

Anatomical and molecular characterization of parvalbumin-cholecystokinin co-expressing inhibitory interneurons: implications for neuropsychiatric conditions.

Inhibitory interneurons are crucial to brain function and their dysfunction is implicated in neuropsychiatric conditions. Emerging evidence indicates that cholecystokinin (CCK)-expressing interneurons (CCK+) are highly heterogenous. We find that a large subset of parvalbumin-expressing (PV+) interneurons express CCK strongly; between 40 and 56% of PV+ interneurons in mouse hippocampal CA1 express CCK. Primate interneurons also exhibit substantial PV/CCK co-expression. Mouse PV+/CCK+ and PV+/CCK- cells show distinguishable electrophysiological and molecular characteristics. Analysis of single nuclei RNA-seq and ATAC-seq data shows that PV+/CCK+ cells are a subset of PV+ cells, not of synuclein gamma positive (SNCG+) cells, and that they strongly express oxidative phosphorylation (OXPHOS) genes. We find that mitochondrial complex I and IV-associated OXPHOS gene expression is strongly correlated with CCK expression in PV+ interneurons at both the transcriptomic and protein levels. Both PV+ interneurons and dysregulation of OXPHOS processes are implicated in neuropsychiatric conditions, including autism spectrum (ASD) disorder and schizophrenia (SCZ). Analysis of human brain samples from patients with these conditions shows alterations in OXPHOS gene expression. Together these data reveal important molecular characteristics of PV-CCK co-expressing interneurons and support their implication in neuropsychiatric conditions.

Preferential transduction of parvalbumin-expressing cortical neurons by AAV-mDLX5/6 vectors.

A major goal of modern neuroscience is to understand the functions of the varied neuronal types that comprise the mammalian brain. Toward this end, some types of neurons can be targeted and manipulated with enhancer-bearing AAV vectors. These vectors hold great promise to advance basic and translational neuroscience, but to realize this potential, their selectivity must be characterized. In this study, we investigated the selectivity of AAV vectors carrying an enhancer of the murine Dlx5 and Dlx6 genes. Vectors were injected into the visual cortex of two macaque monkeys, the frontal cortex of two others, and the somatosensory/motor cortex of three rats. Post-mortem immunostaining revealed that parvalbumin-expressing neurons were transduced efficiently in all cases but calretinin-expressing neurons were not. We speculate that this specificity is a consequence of differential activity of this DLX5/6 enhancer in adult neurons of different developmental lineages.

Correction: Temporal information loss in the macaque early visual system.

[This corrects the article DOI: 10.1371/journal.pbio.3000570.].

Cognitive neuroscience: Mental replay in monkeys.

Cognition allows sensory experiences to inform later actions in flexible ways. A new study shows that, in a cognitively demanding task, monkeys store visual information in short-term memory and replay it when they need it to make a decision.

Deficits in decision-making induced by parietal cortex inactivation are compensated at two timescales.

Perceptual decisions arise through the transformation of samples of evidence into a commitment to a proposition or plan of action. Such transformation is thought to involve cortical circuits capable of computation over timescales associated with working memory, attention, and planning. Neurons in the lateral intraparietal area (LIP) play a role in these functions, and much of what is known about the neurobiology of decision-making has been influenced by studies of LIP and its network of connections. However, the causal role of LIP remains controversial. In this study, we used pharmacological and chemogenetic methods to inactivate LIP in one brain hemisphere of four rhesus monkeys. This inactivation produced biases in decisions, but the effects dissipated despite persistent neural inactivation, implying compensation by unaffected areas. Compensation occurred rapidly within an experimental session and more gradually across sessions. These findings resolve disparate studies and inform the interpretation of focal perturbations of brain function.

Coding of chromatic spatial contrast by macaque V1 neurons.

Color perception relies on comparisons between adjacent lights, but how the brain performs these comparisons is poorly understood. To elucidate the underlying mechanisms, we recorded spiking responses of individual V1 neurons in macaque monkeys to pairs of stimuli within the classical receptive field (RF). We estimated the spatial-chromatic RF of each neuron and then presented customized colored edges using a closed-loop technique. We found that many double-opponent (DO) cells, which have spatially and chromatically opponent RFs, responded to chromatic contrast as a weighted sum, akin to how other V1 neurons responded to luminance contrast. Yet other neurons integrated chromatic signals nonlinearly, confirming that linear signal integration is not an obligate property of V1 neurons. The functional similarity of cone-opponent DO cells and cone non-opponent simple cells suggests that these two groups may share a common underlying circuitry, promotes the construction of image-computable models for full-color image representation, and sheds new light on V1 complex cells.

Single-cell and single-nucleus RNA-seq uncovers shared and distinct axes of variation in dorsal LGN neurons in mice, non-human primates, and humans.

Abundant evidence supports the presence of at least three distinct types of thalamocortical (TC) neurons in the primate dorsal lateral geniculate nucleus (dLGN) of the thalamus, the brain region that conveys visual information from the retina to the primary visual cortex (V1). Different types of TC neurons in mice, humans, and macaques have distinct morphologies, distinct connectivity patterns, and convey different aspects of visual information to the cortex. To investigate the molecular underpinnings of these cell types, and how these relate to differences in dLGN between human, macaque, and mice, we profiled gene expression in single nuclei and cells using RNA-sequencing. These efforts identified four distinct types of TC neurons in the primate dLGN: magnocellular (M) neurons, parvocellular (P) neurons, and two types of koniocellular (K) neurons. Despite extensively documented morphological and physiological differences between M and P neurons, we identified few genes with significant differential expression between transcriptomic cell types corresponding to these two neuronal populations. Likewise, the dominant feature of TC neurons of the adult mouse dLGN is high transcriptomic similarity, with an axis of heterogeneity that aligns with core vs. shell portions of mouse dLGN. Together, these data show that transcriptomic differences between principal cell types in the mature mammalian dLGN are subtle relative to the observed differences in morphology and cortical projection targets. Finally, alignment of transcriptome profiles across species highlights expanded diversity of GABAergic neurons in primate versus mouse dLGN and homologous types of TC neurons in primates that are distinct from TC neurons in mouse.

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.

Windows and periscopes into primate behavior.

Calcium imaging of neurons in monkeys making reaches is complicated by brain movements and limited by shallow imaging depth. In a pair of recent studies, Trautmann et al., 2021 and Bollimunta et al. (2021) present complementary solutions to these problems.

Temporal filtering of luminance and chromaticity in macaque visual cortex.

Contrast sensitivity peaks near 10 Hz for luminance modulations and at lower frequencies for modulations between equiluminant lights. This difference is rooted in retinal filtering, but additional filtering occurs in the cerebral cortex. To measure the cortical contributions to luminance and chromatic temporal contrast sensitivity, signals in the lateral geniculate nucleus (LGN) were compared to the behavioral contrast sensitivity of macaque monkeys. Long wavelength-sensitive (L) and medium wavelength-sensitive (M) cones were modulated in phase to produce a luminance modulation (L + M) or in counterphase to produce a chromatic modulation (L - M). The sensitivity of LGN neurons was well matched to behavioral sensitivity at low temporal frequencies but was approximately 7 times greater at high temporal frequencies. Similar results were obtained for L + M and L - M modulations. These results show that differences in the shapes of the luminance and chromatic temporal contrast sensitivity functions are due almost entirely to pre-cortical mechanisms.

Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex.

Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.

Spatial receptive field structure of double-opponent cells in macaque V1.

The spatial processing of color is important for visual perception. Double-opponent (DO) cells likely contribute to this processing by virtue of their spatially opponent and cone-opponent receptive fields (RFs). However, the representation of visual features by DO cells in the primary visual cortex of primates is unclear because the spatial structure of their RFs has not been fully characterized. To fill this gap, we mapped the RFs of DO cells in awake macaques with colorful, dynamic white noise patterns. The spatial RF of each neuron was fitted with a Gabor function and three versions of the difference of Gaussians (DoG) function. The Gabor function provided the more accurate description for most DO cells, a result that is incompatible with a center-surround RF organization. A nonconcentric version of the DoG function, in which the RFs have a circular center and a crescent-shaped surround, performed nearly as well as the Gabor model thus reconciling results from previous reports. For comparison, we also measured the RFs of simple cells. We found that the superiority of the Gabor fits over DoG fits was slightly more decisive for simple cells than for DO cells. The implications of these results on biological image processing and visual perception are discussed.NEW & NOTEWORTHY Double-opponent cells in macaque area V1 respond to spatial chromatic contrast in visual scenes. What information they carry is debated because their receptive field organization has not been characterized thoroughly. Using white noise analysis and statistical model comparisons, De and Horwitz show that many double-opponent receptive fields can be captured by either a Gabor model or a center-with-an-asymmetric-surround model but not by a difference of Gaussians model.

Signals Related to Color in the Early Visual Cortex.

Visual images can be described in terms of the illuminants and objects that are causal to the light reaching the eye, the retinal image, its neural representation, or how the image is perceived. Respecting the differences among these distinct levels of description can be challenging but is crucial for a clear understanding of color vision. This article approaches color by reviewing what is known about its neural representation in the early visual cortex, with a brief description of signals in the eye and the thalamus for context. The review focuses on the properties of single neurons and advances the general theme that experimental approaches based on knowledge of feedforward signals have promoted greater understanding of the neural code for color than approaches based on correlating single-unit responses with color perception. New data from area V1 illustrate the strength of the feedforward approach. Future directions for progress in color neurophysiology are discussed: techniques for improved single-neuron characterization, for investigations of neural populations and small circuits, and for the analysis of natural image statistics.

Viral Vectors for Neural Circuit Mapping and Recent Advances in Trans-synaptic Anterograde Tracers.

Viral tracers are important tools for neuroanatomical mapping and genetic payload delivery. Genetically modified viruses allow for cell-type-specific targeting and overcome many limitations of non-viral tracers. Here, we summarize the viruses that have been developed for neural circuit mapping, and we provide a primer on currently applied anterograde and retrograde viral tracers with practical guidance on experimental uses. We also discuss and highlight key technical and conceptual considerations for developing new safer and more effective anterograde trans-synaptic viral vectors for neural circuit analysis in multiple species.

Fast and reversible neural inactivation in macaque cortex by optogenetic stimulation of GABAergic neurons.

Optogenetic techniques for neural inactivation are valuable for linking neural activity to behavior but they have serious limitations in macaques. To achieve powerful and temporally precise neural inactivation, we used an adeno-associated viral (AAV) vector carrying the channelrhodopsin-2 gene under the control of a Dlx5/6 enhancer, which restricts expression to GABAergic neurons. We tested this approach in the primary visual cortex, an area where neural inactivation leads to interpretable behavioral deficits. Optical stimulation modulated spiking activity and reduced visual sensitivity profoundly in the region of space represented by the stimulated neurons. Rebound firing, which can have unwanted effects on neural circuits following inactivation, was not observed, and the efficacy of the optogenetic manipulation on behavior was maintained across >1000 trials. We conclude that this inhibitory cell-type-specific optogenetic approach is a powerful and spatiotemporally precise neural inactivation tool with broad utility for probing the functional contributions of cortical activity in macaques.

Temporal information loss in the macaque early visual system.

Stimuli that modulate neuronal activity are not always detectable, indicating a loss of information between the modulated neurons and perception. To identify where in the macaque visual system information about periodic light modulations is lost, signal-to-noise ratios were compared across simulated cone photoreceptors, lateral geniculate nucleus (LGN) neurons, and perceptual judgements. Stimuli were drifting, threshold-contrast Gabor patterns on a photopic background. The sensitivity of LGN neurons, extrapolated to populations, was similar to the monkeys' at low temporal frequencies. At high temporal frequencies, LGN sensitivity exceeded the monkeys' and approached the upper bound set by cone photocurrents. These results confirm a loss of high-frequency information downstream of the LGN. However, this loss accounted for only about 5% of the total. Phototransduction accounted for essentially all of the rest. Together, these results show that low temporal frequency information is lost primarily between the cones and the LGN, whereas high-frequency information is lost primarily within the cones, with a small additional loss downstream of the LGN.

Primate optogenetics: Progress and prognosis.

Monkeys are a premier model organism for neuroscience research. Activity in the central nervous systems of monkeys can be recorded and manipulated while they perform complex perceptual, motor, or cognitive tasks. Conventional techniques for manipulating neural activity in monkeys are too coarse to address many of the outstanding questions in primate neuroscience, but optogenetics holds the promise to overcome this hurdle. In this article, we review the progress that has been made in primate optogenetics over the past 5 years. We emphasize the use of gene regulatory sequences in viral vectors to target specific neuronal types, and we present data on vectors that we engineered to target parvalbumin-expressing neurons. We conclude with a discussion of the utility of optogenetics for treating sensorimotor hearing loss and Parkinson's disease, areas of translational neuroscience in which monkeys provide unique leverage for basic science and medicine.