Advanced Circuit and Cellular Imaging Methods in Nonhuman Primates.

Novel genetically encoded tools and advanced microscopy methods have revolutionized neural circuit analyses in insects and rodents over the last two decades. Whereas numerous technical hurdles originally barred these methodologies from success in nonhuman primates (NHPs), current research has started to overcome those barriers. In some cases, methodological advances developed with NHPs have even surpassed their precursors. One such advance includes new ultra-large imaging windows on NHP cortex, which are larger than the entire rodent brain and allow analysis unprecedented ultra-large-scale circuits. NHP imaging chambers now remain patent for periods longer than a mouse's lifespan, allowing for long-term all-optical interrogation of identified circuits and neurons over timeframes that are relevant to human cognitive development. Here we present some recent imaging advances brought forth by research teams using macaques and marmosets. These include technical developments in optogenetics; voltage-, calcium- and glutamate-sensitive dye imaging; two-photon and wide-field optical imaging; viral delivery; and genetic expression of indicators and light-activated proteins that result in the visualization of tens of thousands of identified cortical neurons in NHPs. We describe a subset of the many recent advances in circuit and cellular imaging tools in NHPs focusing here primarily on the research presented during the corresponding mini-symposium at the 2019 Society for Neuroscience annual meeting.

Flexible modeling of large-scale neural network stimulation: electrical and optical extensions to The Virtual Electrode Recording Tool for EXtracellular Potentials (VERTEX).

Computational models that predict effects of neural stimulation can be used as a preliminary tool to inform in-vivo research, reducing the costs, time, and ethical considerations involved. However, current models do not support the diverse neural stimulation techniques used in-vivo, including the expanding selection of electrodes, stimulation modalities, and stimulation paradigms. To develop a more comprehensive software, we created several extensions to The Virtual Electrode Recording Tool for EXtracellular Potentials (VERTEX), the MATLAB-based neural stimulation tool from Newcastle University. VERTEX simulates input currents in a large population of multi-compartment neurons within a small cortical slice to model electric field stimulation, while recording local field potentials (LFPs) and spiking activity. Our extensions to its existing electric field stimulation framework include multiple pairs of parametrically defined electrodes and biphasic, bipolar stimulation delivered at programmable delays. To support the growing use of optogenetic approaches for targeted neural stimulation, we introduced a feature that models optogenetic stimulation through an additional VERTEX input function that converts irradiance to currents at optogenetically responsive neurons. Finally, we added extensions to allow complex stimulation protocols including paired-pulse, spatiotemporal patterned, and closed-loop stimulation. We demonstrated our novel features using VERTEX's built-in functionalities, illustrating how these extensions can be used to efficiently and systematically test diverse, targeted, and individualized stimulation patterns.

A Neural Implant Design Toolbox for Nonhuman Primates.

This paper describes an in-house method of 3D brain and skull modeling from magnetic resonance imaging (MRI) tailored for nonhuman primate (NHP) neurosurgical planning. This automated, computational software-based technique provides an efficient way of extracting brain and skull features from MRI files as opposed to traditional manual extraction techniques using imaging software. Furthermore, the procedure provides a method for visualizing the brain and craniotomized skull together for intuitive, virtual surgical planning. This generates a drastic reduction in time and resources from those required by past work, which relied on iterative 3D printing. The skull modeling process creates a footprint that is exported into modeling software to design custom-fit cranial chambers and headposts for surgical implantation. Custom-fit surgical implants minimize gaps between the implant and the skull that could introduce complications, including infection or decreased stability. By implementing these pre-surgical steps, surgical and experimental complications are reduced. These techniques can be adapted for other surgical processes, facilitating more efficient and effective experimental planning for researchers and, potentially, neurosurgeons.

Inferring Neural Communication Dynamics from Field Potentials Using Graph Diffusion Autoregression.

Estimating dynamic network communication is attracting increased attention, spurred by rapid advancements in multi-site neural recording technologies and efforts to better understand cognitive processes. Yet, traditional methods, which infer communication from statistical dependencies among distributed neural recordings, face core limitations: they do not model neural interactions in a biologically plausible way, neglect spatial information from the recording setup, and yield predominantly static estimates that cannot capture rapid changes in the brain. To address these issues, we introduce a graph diffusion autoregressive model. Designed for distributed field potential recordings, our model combines vector autoregression with a network communication process to produce a high-resolution communication signal. We successfully validated the model on simulated neural activity and recordings from subdural and intracortical micro-electrode arrays placed in macaque sensorimotor cortex demonstrating its ability to describe rapid communication dynamics induced by optogenetic stimulation, changes in resting state communication, and the trial-by-trial variability during a reach task.

A large-scale optogenetic neurophysiology platform for improving accessibility in NHP behavioral experiments.

Optogenetics has been a powerful scientific tool for two decades, yet its integration with non-human primate (NHP) electrophysiology has been limited due to several technical challenges. These include a lack of electrode arrays capable of supporting large-scale and long-term optical access, inaccessible viral vector delivery methods for transfection of large regions of cortex, a paucity of hardware designed for large-scale patterned cortical illumination, and inflexible designs for multi-modal experimentation. To address these gaps, we introduce a highly accessible platform integrating optogenetics and electrophysiology for behavioral and neural modulation with neurophysiological recording in NHPs. We employed this platform in two rhesus macaques and showcased its capability of optogenetically disrupting reaches, while simultaneously monitoring ongoing electrocorticography activity underlying the stimulation-induced behavioral changes. The platform exhibits long-term stability and functionality, thereby facilitating large-scale electrophysiology, optical imaging, and optogenetics over months, which is crucial for translationally relevant multi-modal studies of neurological and neuropsychiatric disorders.

Early Intervention with Electrical Stimulation Reduces Neural Damage After Stroke in Non-human Primates.

Ischemic stroke is a neurological condition that results in significant mortality and long-term disability for adults, creating huge health burdens worldwide. For stroke patients, acute intervention offers the most critical therapeutic opportunity as it can reduce irreversible tissue injury and improve functional outcomes. However, currently available treatments within the acute window are highly limited. Although emerging neuromodulation therapies have been tested for chronic stroke patients, acute stimulation is rarely studied due to the risk of causing adverse effects related to ischemia-induced electrical instability. To address this gap, we combined electrophysiology and histology tools to investigate the effects of acute electrical stimulation on ischemic neural damage in non-human primates. Specifically, we induced photothrombotic lesions in the monkey sensorimotor cortex while collecting electrocorticography (ECoG) signals through a customized neural interface. Gamma activity in ECoG was used as an electrophysiological marker to track the effects of stimulation on neural activation. Meanwhile, histological analysis including Nissl, cFos, and microglial staining was performed to evaluate the tissue response to ischemic injury. Comparing stimulated monkeys to controls, we found that theta-burst stimulation administered directly adjacent to the ischemic infarct at 1 hour post-stroke briefly inhibits peri-infarct neuronal activation as reflected by decreased ECoG gamma power and cFos expression. Meanwhile, lower microglial activation and smaller lesion volumes were observed in animals receiving post-stroke stimulation. Together, these results suggest that acute electrical stimulation can be used safely and effectively as an early stroke intervention to reduce excitotoxicity and inflammation, thus mitigating neural damage and enhancing stroke outcomes.

Protocol to study ischemic stroke by photothrombotic lesioning in the cortex of non-human primates.

Neurorehabilitation strategies for ischemic stroke have shown promise for functional recovery, yet minimal tools are available to study rehabilitation techniques in non-human primates (NHPs). Here, we present a protocol to study rehabilitation techniques in NHPs using a photothrombotic technique, a form of optical focal lesioning. We also describe steps for simultaneous neurophysiological recording and in vivo validation through vascular flow imaging. This interface can examine emerging neurorehabilitation strategies in the post-stroke environment in NHPs that are evolutionarily close to humans. For complete details on the use and execution of this protocol, please refer to Khateeb et al. (2022).6.

Type-2 Diabetes Alters Hippocampal Neural Oscillations and Disrupts Synchrony between the Hippocampus and Cortex.

Type 2 diabetes mellitus (T2DM) increases the risk of neurological diseases, yet how brain oscillations change as age and T2DM interact is not well characterized. To delineate the age and diabetic effect on neurophysiology, we recorded local field potentials with multichannel electrodes spanning the somatosensory cortex and hippocampus (HPC) under urethane anesthesia in diabetic and normoglycemic control mice, at 200 and 400 days of age. We analyzed the signal power of brain oscillations, brain state, sharp wave associate ripples (SPW-Rs), and functional connectivity between the cortex and HPC. We found that while both age and T2DM were correlated with a breakdown in long-range functional connectivity and reduced neurogenesis in the dentate gyrus and subventricular zone, T2DM further slowed brain oscillations and reduced theta-gamma coupling. Age and T2DM also prolonged the duration of SPW-Rs and increased gamma power during SPW-R phase. Our results have identified potential electrophysiological substrates of hippocampal changes associated with T2DM and age. The perturbed brain oscillation features and diminished neurogenesis may underlie T2DM-accelerated cognitive impairment.

Type-2 diabetes alters hippocampal neural oscillations and disrupts synchrony between hippocampus and cortex.

Type 2 diabetes mellitus (T2DM) increases the risk of neurological diseases, yet how brain oscillations change as age and T2DM interact is not well characterized. To delineate the age and diabetic effect on neurophysiology, we recorded local field potentials with multichannel electrodes spanning the somatosensory cortex and hippocampus (HPC) under urethane anesthesia in diabetic and normoglycemic control mice, at 200 and 400 days of age. We analyzed the signal power of brain oscillations, brain state, sharp wave associate ripples (SPW-Rs), and functional connectivity between the cortex and HPC. We found that while both age and T2DM were correlated with a breakdown in long-range functional connectivity and reduced neurogenesis in the dentate gyrus and subventricular zone, T2DM further slowed brain oscillations and reduced theta-gamma coupling. Age and T2DM also prolonged the duration of SPW-Rs and increased gamma power during SPW-R phase. Our results have identified potential electrophysiological substrates of hippocampal changes associated with T2DM and age. The perturbed brain oscillation features and diminished neurogenesis may underlie T2DM-accelerated cognitive impairment.

Large-scale multimodal surface neural interfaces for primates.

Deciphering the function of neural circuits can help with the understanding of brain function and treating neurological disorders. Progress toward this goal relies on the development of chronically stable neural interfaces capable of recording and modulating neural circuits with high spatial and temporal precision across large areas of the brain. Advanced innovations in designing high-density neural interfaces for small animal models have enabled breakthrough discoveries in neuroscience research. Developing similar neurotechnology for larger animal models such as nonhuman primates (NHPs) is critical to gain significant insights for translation to humans, yet still it remains elusive due to the challenges in design, fabrication, and system-level integration of such devices. This review focuses on implantable surface neural interfaces with electrical and optical functionalities with emphasis on the required technological features to realize scalable multimodal and chronically stable implants to address the unique challenges associated with nonhuman primate studies.

Network structure mediates functional reorganization induced by optogenetic stimulation of non-human primate sensorimotor cortex.

Because aberrant network-level functional connectivity underlies a variety of neural disorders, the ability to induce targeted functional reorganization would be a profound development toward therapies for neural disorders. Brain stimulation has been shown to induce large-scale network-wide functional connectivity changes (FCC), but the mapping from stimulation to the induced changes is unclear. Here, we develop a model which jointly considers the stimulation protocol and the cortical network structure to accurately predict network-wide FCC in response to optogenetic stimulation of non-human primate primary sensorimotor cortex. We observe that the network structure has a much stronger effect than the stimulation protocol on the resulting FCC. We also observe that the mappings from these input features to the FCC diverge over frequency bands and successive stimulations. Our framework represents a paradigm shift for targeted neural stimulation and can be used to interrogate, improve, and develop stimulation-based interventions for neural disorders.

Neuroprotective Effects of Electrical Stimulation Following Ischemic Stroke in Non-Human Primates.

Brain stimulation has emerged as a novel therapy for ischemic stroke, a major cause of brain injury that often results in lifelong disability. Although past works in rodents have demonstrated protective effects of stimulation following stroke, few of these results have been replicated in humans due to the anatomical differences between rodent and human brains and a limited understanding of stimulation-induced network changes. Therefore, we combined electrophysiology and histology to study the neuroprotective mechanisms of electrical stimulation following cortical ischemic stroke in non-human primates. To produce controlled focal lesions, we used the photothrombotic method to induce targeted vasculature damage in the sensorimotor cortices of two macaques while collecting electrocorticography (ECoG) signals bilaterally. In another two monkeys, we followed the same lesioning procedures and applied repeated electrical stimulation via an ECoG electrode adjacent to the lesion. We studied the protective effects of stimulation on neural dynamics using ECoG signal power and coherence. In addition, we performed histological analysis to evaluate the differences in lesion volume. In comparison to controls, the ECoG signals showed decreased gamma power across the sensorimotor cortices in stimulated animals. Meanwhile, Nissl staining revealed smaller lesion volumes for the stimulated group, suggesting that electrical stimulation may exert neuroprotection by suppressing post-ischemic neural activity. With the similarity between NHP and human brains, this study paves the path for developing effective stimulation-based therapy for acute stroke in clinical studies.

Demonstration of an Optimized Large-scale Optogenetic Cortical Interface for Non-human Primates.

Optogenetics is a powerful neuroscientific tool which allows neurons to be modulated by optical stimulation. Despite widespread optogenetic experimentation in small animal models, optogenetics in non-human primates (NHPs) remains a niche field, particularly at the large scales necessary for multi-regional neural research. We previously published a large-scale, chronic optogenetic cortical interface for NHPs which was successful but came with a number of limitations. In this work, we present an optimized interface which improves upon the stability and scale of our previous interface while using more easily replicable methods to increase our system's availability to the scientific community. Specifically, we (1) demonstrate the long-term (~3 months) optical access to the brain achievable using a commercially-available transparent artificial dura with embedded electrodes, (2) showcase large-scale optogenetic expression achievable with simplified (magnetic resonance-free) surgical techniques, and (3) effectively modulated the expressing areas at large scales (~1 cm2) by light emitting diode (LED) arrays assembled in-house.

Pathological changes of brain oscillations following ischemic stroke.

Brain oscillations recorded in the extracellular space are among the most important aspects of neurophysiology data reflecting the activity and function of neurons in a population or a network. The signal strength and patterns of brain oscillations can be powerful biomarkers used for disease detection and prediction of the recovery of function. Electrophysiological signals can also serve as an index for many cutting-edge technologies aiming to interface between the nervous system and neuroprosthetic devices and to monitor the efficacy of boosting neural activity. In this review, we provided an overview of the basic knowledge regarding local field potential, electro- or magneto- encephalography signals, and their biological relevance, followed by a summary of the findings reported in various clinical and experimental stroke studies. We reviewed evidence of stroke-induced changes in hippocampal oscillations and disruption of communication between brain networks as potential mechanisms underlying post-stroke cognitive dysfunction. We also discussed the promise of brain stimulation in promoting post stroke functional recovery via restoring neural activity and enhancing brain plasticity.

A versatile toolbox for studying cortical physiology in primates.

Lesioning and neurophysiological studies have facilitated the elucidation of cortical functions and mechanisms of functional recovery following injury. Clinical translation of such studies is contingent on their employment in non-human primates (NHPs), yet tools for monitoring and modulating cortical physiology are incompatible with conventional lesioning techniques. To address these challenges, we developed a toolbox validated in seven macaques. We introduce the photothrombotic method for inducing focal cortical lesions, a quantitative model for designing experiment-specific lesion profiles and optical coherence tomography angiography (OCTA) for large-scale (~5 cm2) monitoring of vascular dynamics. We integrate these tools with our electrocorticographic array for large-scale monitoring of neural dynamics and testing stimulation-based interventions. Advantageously, this versatile toolbox can be incorporated into established chronic cranial windows. By combining optical and electrophysiological techniques in the NHP cortex, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, integrate physiological and behavioral findings, and develop neurorehabilitative treatments. MOTIVATION The primate neocortex encodes for complex functions and behaviors, the physiologies of which are yet to be fully understood. Such an understanding in both healthy and diseased states can be crucial for the development of effective neurorehabilitative strategies. However, there is a lack of a comprehensive and adaptable set of tools that enables the study of multiple physiological phenomena in healthy and injured brains. Therefore, we developed a toolbox with the capability to induce targeted cortical lesions, monitor dynamics of underlying cortical microvasculature, and record and stimulate neural activity. With this toolbox, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, test stimulation-based interventions, and integrate physiological and behavioral findings.

Improving the Efficacy and Accessibility of Intracranial Viral Vector Delivery in Non-Human Primates.

Non-human primates (NHPs) are precious resources for cutting-edge neuroscientific research, including large-scale viral vector-based experimentation such as optogenetics. We propose to improve surgical outcomes by enhancing the surgical preparation practices of convection-enhanced delivery (CED), which is an efficient viral vector infusion technique for large brains such as NHPs'. Here, we present both real-time and next-day MRI data of CED in the brains of ten NHPs, and we present a quantitative, inexpensive, and practical bench-side model of the in vivo CED data. Our bench-side model is composed of food coloring infused into a transparent agar phantom, and the spread of infusion is optically monitored over time. Our proposed method approximates CED infusions into the cortex, thalamus, medial temporal lobe, and caudate nucleus of NHPs, confirmed by MRI data acquired with either gadolinium-based or manganese-based contrast agents co-infused with optogenetic viral vectors. These methods and data serve to guide researchers and surgical team members in key surgical preparations for intracranial viral delivery using CED in NHPs, and thus improve expression targeting and efficacy and, as a result, reduce surgical risks.

Multi-modal artificial dura for simultaneous large-scale optical access and large-scale electrophysiology in non-human primate cortex.

Objective.Non-human primates (NHPs) are critical for development of translational neural technologies because of their neurological and neuroanatomical similarities to humans. Large-scale neural interfaces in NHPs with multiple modalities for stimulation and data collection poise us to unveil network-scale dynamics of both healthy and unhealthy neural systems. We aim to develop a large-scale multi-modal interface for NHPs for the purpose of studying large-scale neural phenomena including neural disease, damage, and recovery.Approach.We present a multi-modal artificial dura (MMAD) composed of flexible conductive traces printed into transparent medical grade polymer. Our MMAD provides simultaneous neurophysiological recordings and optical access to large areas of the cortex (∼3 cm2) and is designed to mitigate photo-induced electrical artifacts. The MMAD is the centerpiece of the interfaces we have designed to support electrocorticographic recording and stimulation, cortical imaging, and optogenetic experiments, all at the large-scales afforded by the brains of NHPs. We performed electrical and optical experiments bench-side andin vivowith macaques to validate the utility of our MMAD.Main results.Using our MMAD we present large-scale electrocorticography from sensorimotor cortex of three macaques. Furthermore, we validated surface electrical stimulation in one of our animals. Our bench-side testing showed up to 90% reduction of photo-induced artifacts with our MMAD. The transparency of our MMAD was confirmed both via bench-side testing (87% transmittance) and viain vivoimaging of blood flow from the underlying microvasculature using optical coherence tomography angiography.Significance.Our results indicate that our MMAD supports large-scale electrocorticography, large-scale cortical imaging, and, by extension, large-scale optical stimulation. The MMAD prepares the way for both acute and long-term chronic experiments with complimentary data collection and stimulation modalities. When paired with the complex behaviors and cognitive abilities of NHPs, these assets prepare us to study large-scale neural phenomena including neural disease, damage, and recovery.

Local field potentials identify features of cortico-hippocampal communication impacted by stroke and environmental enrichment therapy.

Objective. Cognitive and memory impairments are common sequelae after stroke, yet how middle cerebral artery (MCA) stroke chronically affects the neural activity of the hippocampus, a brain region critical for memory but remote from the stroke epicenter, is poorly understood. Environmental enrichment (EE) improves cognition following stroke; however, the electrophysiology that underlies this behavioral intervention is still elusive.Approach.We recorded extracellular local field potentials simultaneously from sensorimotor cortex and hippocampus in rats during urethane anesthesia following MCA occlusion and subsequent EE treatment.Main results.We found that MCA stroke significantly impacted the electrophysiology in the hippocampus, in particular it disrupted characteristics of sharp-wave associated ripples (SPW-Rs) altered brain state, and disrupted phase amplitude coupling (PAC) within the hippocampus and between the cortex and hippocampus. Importantly, we show that EE mitigates stroke-induced changes to SPW-R characteristics but does not restore hippocampal brain state or PAC.Significance.These results begin to uncover the complex interaction between cognitive deficit following stroke and EE treatment, providing a testbed to assess different strategies for therapeutics following stroke.

Autonomous cage-side system for remote training of non-human primates.

BACKGROUND: Training non-human primates (NHPs) for translational medical experimentation is an essential yet time consuming process. To increase training efficiency, some training systems have been designed for NHPs to use at their home cages. Several autonomous cage-side tablet-based systems have been proposed, but none of these systems allow for remote monitoring and task modification while also being wireless, low-cost, light weight, and portable. NEW METHOD: Here we present ACTS: an Autonomous Cage-side Training System which meets all these criteria. ACTS consists of 1) a touchscreen tablet and a speaker attached to the subject's home cage, 2) an inexpensive reward system made from a slightly modified fish feeder, and 3), a laptop operating the system wirelessly and remotely via a router. RESULTS: We were able to test the system and wirelessly train two macaques in their home cages. Remote access enabled us to control ACTS from up to 90 m, through up to 3 walls, and through a floor of a building. The device is compatible with different reward pellet sizes and could run about two hours with a ∼4 mm pellet size. The animals were able to generalize the task when transferred to a traditional experimental rig. COMPARISON WITH EXISTING METHODS: The low cost and modest skill required to build and implement ACTS lowers the barrier for NHP researchers and caregivers to deploy autonomous, remotely controlled tablet-based cage-side systems. CONCLUSION: ACTS can be used for low-cost, wireless cage-side training of NHPs being prepared for translational medical experimentation.

Experimental cortical stroke induces aberrant increase of sharp-wave-associated ripples in the hippocampus and disrupts cortico-hippocampal communication.

The functional consequences of ischemic stroke in the remote brain regions are not well characterized. The current study sought to determine changes in hippocampal oscillatory activity that may underlie the cognitive impairment observed following distal middle cerebral artery occlusion (dMCAO) without causing hippocampal structural damage. Local field potentials were recorded from the dorsal hippocampus and cortex in urethane-anesthetized rats with multichannel silicon probes during dMCAO and reperfusion, or mild ischemia induced by bilateral common carotid artery occlusion (CCAO). Bilateral change of brain state was evidenced by reduced theta/delta amplitude ratio and shortened high theta duration following acute dMCAO but not CCAO. An aberrant increase in the occurrence of sharp-wave-associated ripples (150-250 Hz), crucial for memory consolidation, was only detected after dMCAO reperfusion, coinciding with an increased occurrence of high-frequency discharges (250-450 Hz). dMCAO also significantly affected the modulation of gamma amplitude in the cortex coupled to hippocampal theta phase, although both hippocampal theta and gamma power were temporarily decreased during dMCAO. Our results suggest that MCAO may disrupt the balance between excitatory and inhibitory circuits in the hippocampus and alter the function of cortico-hippocampal network, providing a novel insight in how cortical stroke affects function in remote brain regions.