Functional imaging and anatomical connections in squirrel monkeys reveal parietal-frontal circuits underlying eye movements.

The posterior parietal cortex (PPC) of squirrel monkeys contains subregions where long trains of intracortical microstimulation evoke complex, behaviorally meaningful movements. Recently, we showed that such stimulation of a part of the PPC in the caudal lateral sulcus (LS) elicits eye movements in these monkeys. Here, we studied the functional and anatomical connections of this oculomotor region we call parietal eye field (PEF) with frontal eye field (FEF) and other cortical regions in 2 squirrel monkeys. We demonstrated these connections with intrinsic optical imaging and injections of anatomical tracers. Optical imaging of frontal cortex during stimulation of the PEF evoked focal functional activation within FEF. Tracing studies confirmed the functional PEF-FEF connections. Moreover, tracer injections revealed PEF connections with other PPC regions on the dorsolateral and medial brain surface, cortex in the caudal LS, and visual and auditory cortical association areas. Subcortical projections of PEF were primarily with superior colliculus, and pontine nuclei as well as nuclei of the dorsal posterior thalamus and caudate. These findings suggest that PEF in squirrel monkey is homologous to lateral intraparietal (LIP) area of macaque, supporting the notion that these brain circuits are organized similarly to mediate ethologically relevant oculomotor behaviors.

Modular Organization of Signal Transmission in Primate Somatosensory Cortex.

Axonal patches are known as the major sites of synaptic connections in the cerebral cortex of higher order mammals. However, the functional role of these patches is highly debated. Patches are formed by populations of nearby neurons in a topographic manner and are recognized as the termination fields of long-distance lateral connections within and between cortical areas. In addition, axons form numerous boutons that lie outside the patches, whose function is also unknown. To better understand the functional roles of these two distinct populations of boutons, we compared individual and collective morphological features of axons within and outside the patches of intra-areal, feedforward, and feedback pathways by way of tract tracing in the somatosensory cortex of New World monkeys. We found that, with the exception of tortuosity, which is an invariant property, bouton spacing and axonal convergence properties differ significantly between axons within patch and no-patch domains. Principal component analyses corroborated the clustering of axons according to patch formation without any additional effect by the type of pathway or laminar distribution. Stepwise logistic regression identified convergence and bouton density as the best predictors of patch formation. These findings support that patches are specific sites of axonal convergence that promote the synchronous activity of neuronal populations. On the other hand, no-patch domains could form a neuroanatomical substrate to diversify the responses of cortical neurons.

Head-mounted optical imaging and optogenetic stimulation system for use in behaving primates.

Advances in optical technology have revolutionized studies of brain function in freely behaving mice. Here, we describe an optical imaging and stimulation device for use in primates that easily attaches to an intracranial chamber. It consists of affordable commercially available or 3D-printed components: a monochromatic camera, a small standard lens, a wireless µLED stimulator powered by an induction coil, and an LED array for illumination. We show that the intrinsic imaging performance of this device is comparable to a standard benchtop system in revealing the functional organization of the visual cortex for awake macaques in a primate chair or under anesthesia. Imaging revealed neural modulatory effects of wireless focal optogenetic stimulation aimed at identified functional domains. With a 1 to 2 cm field of view, 100× larger than previously used in primates without head restraint, our device permits widefield optical imaging and optogenetic stimulation for ethological studies in primates.

A thermal nociceptive patch in the S2 cortex of nonhuman primates: a combined functional magnetic resonance imaging and electrophysiology study.

ABSTRACT: Human functional magnetic resonance imaging (fMRI) and behavioral studies have established the roles of cortical areas along the Sylvian fissure in sensing subjective pain. Yet, little is known about how sensory aspects of painful information are represented and processed by neurons in these regions and how their electrophysiological activities are related to fMRI signals. The current study aims to partially address this critical knowledge gap by performing fMRI-guided microelectrode mapping and recording studies in the homologous region of the parietal operculum in squirrel monkeys under light anesthesia. In each animal studied (n = 8), we detected mesoscale mini-networks for heat nociception in cortical regions around the lateral sulcus. Within the network, we discovered a ∼1.5 × 1.5-mm2-sized cortical patch that solely contained heat nociceptive neurons that aligned with the heat fMRI activation locus. These neurons responded slowly to thermal (heat and cold) nociceptive stimuli exclusively, continued firing for several seconds after the succession of stimulation, and exhibited multidigit receptive fields and high spontaneous firing rates. Similar to the fMRI responses, increasing temperatures in the nociceptive range led to a nonlinear increase in firing rates. The finding of a clustering of heat nociceptive neurons provides novel insights into the unique functional organization of thermal nociception in the S2 subregion of the primate brain. With fMRI, it supports the existence of a modality-preferred heat nociceptive patch that is spatially separated and intermingled with touch patches containing neurons with comparable receptive fields and the presence of functionally distinct mini-networks in primate opercular cortex.

Fiberoptic array for multiple channel infrared neural stimulation of the brain.

Significance: We present a new optical method for modulating cortical activity in multiple locations and across multiple time points with high spatial and temporal precision. Our method uses infrared light and does not require dyes or transgenic modifications. It is compatible with a number of other stimulation and recording techniques. Aim: Infrared neural stimulation (INS) has been largely confined to single point stimuli. In this study, we expand upon this approach and develop a rapidly switched fiber array capable of generation of stimulus patterns. Our prototype is capable of stimulating at nine separate locations but is easily scalable. Approach: Our device is made of commercially available components: a solid-state infrared laser, a piezoelectric fiber coupled optical switch, and 200 - µ m diameter optical fibers. We validate it using intrinsic optical signal imaging of INS responses in macaque and squirrel monkey sensory cortical areas. Results: We demonstrate that our switched array can consistently generate responses in primate cortex, consistent with earlier single channel INS investigations. Conclusions: Our device can successfully target the cortical surface, either at one specific region or multiple points spread out across different areas. It is compatible with a host of other imaging and stimulation modalities.

An Open Resource for Non-human Primate Optogenetics.

Optogenetics has revolutionized neuroscience in small laboratory animals, but its effect on animal models more closely related to humans, such as non-human primates (NHPs), has been mixed. To make evidence-based decisions in primate optogenetics, the scientific community would benefit from a centralized database listing all attempts, successful and unsuccessful, of using optogenetics in the primate brain. We contacted members of the community to ask for their contributions to an open science initiative. As of this writing, 45 laboratories around the world contributed more than 1,000 injection experiments, including precise details regarding their methods and outcomes. Of those entries, more than half had not been published. The resource is free for everyone to consult and contribute to on the Open Science Framework website. Here we review some of the insights from this initial release of the database and discuss methodological considerations to improve the success of optogenetic experiments in NHPs.

Optical imaging reveals functional domains in primate sensorimotor cortex.

Motor cortex (M1) and somatosensory cortex (S1) are central to arm and hand control. Efforts to understand encoding in M1 and S1 have focused on temporal relationships between neural activity and movement features. However, it remains unclear how the neural activity is spatially organized within M1 and S1. Optical imaging methods are well-suited for revealing the spatio-temporal organization of cortical activity, but their application is sparse in monkey sensorimotor cortex. Here, we investigate the effectiveness of intrinsic signal optical imaging (ISOI) for measuring cortical activity that supports arm and hand control in a macaque monkey. ISOI revealed spatial domains that were active in M1 and S1 in response to instructed reaching and grasping. The lateral M1 domains overlapped the hand representation and contained a population of neurons with peak firing during grasping. In contrast, the medial M1 domain overlapped the arm representation and a population of neurons with peak firing during reaching. The S1 domain overlapped the hand representations of areas 1 and 2 and a population of neurons with peak firing upon hand contact with the target. Our single unit recordings indicate that ISOI domains report the locations of spatial clusters of functionally related neurons. ISOI is therefore an effective tool for surveilling the neocortex for "hot zones" of activity that supports movement. Combining the strengths of ISOI with other imaging modalities (e.g., fMRI, 2-photon) and with electrophysiological methods can open new frontiers in understanding the spatio-temporal organization of cortical signals involved in movement control.

Mapping mesoscale cortical connectivity in monkey sensorimotor cortex with optical imaging and microstimulation.

To map in vivo cortical circuitry at the mesoscale, we applied a novel approach to map interareal functional connectivity. Electrical intracortical microstimulation (ICMS) in conjunction with optical imaging of intrinsic signals (OIS) was used map functional connections in somatosensory cortical areas in anesthetized squirrel monkeys. ICMS produced activations that were focal and that displayed responses which were stimulation intensity dependent. ICMS in supragranular layers of Brodmann Areas 3b, 1, 2, 3a, and M1 evoked interareal activation patterns that were topographically appropriate and appeared consistent with known anatomical connectivity. Specifically, ICMS revealed Area 3b connections with Area 1; Area 1 connections with Areas 2 and 3a; Area 2 connections with Areas 1, 3a, and M1; Area 3a connections with Areas M1, 1, and 2; and M1 connections with Areas 3a, 1, and 2. These somatosensory connectivity patterns were reminiscent of feedforward patterns observed anatomically, although feedback contributions are also likely present. Further consistent with anatomical connectivity, intra-areal and intra-areal patterns of activation were patchy with patch sizes of 200-300 µm. In summary, ICMS with OIS is a novel approach for mapping interareal and intra-areal connections in vivo. Comparisons with feedforward and feedback anatomical connectivity are discussed.

A 16-channel loop array for in vivo macaque whole-brain imaging at 3 T.

Non-human primates (NHPs) are vital models for neuroscience research. These animals have been widely used in behavioral, electrophysiological, molecular, and more recently, multimodal neuroimaging and neuro-engineering studies. Several RF coil arrays have been designed for functional, high-resolution brain magnetic resonance imaging (MRI), but few have been designed to accommodate multimodal devices. In the present study, a 16-channel array coil was constructed for brain imaging of macaques at 3 Tesla (3 T). To construct this coil, a close-fitting helmet-shaped form was designed to host 16 coil loops for whole-brain coverage. This assembly is mountable onto stereotaxic head frame bars, and the coil functions while the monkey is in the sphinx position with a clear line of vision of stimuli presented from outside of the MRI system. In addition, 4 openings were allocated in the coil housing, allowing multimodal devices to directly access visual cortical regions such as V1-V4 and MT. Coil performance was evaluated in an anesthetized macaque by quantifying and comparing signal-to-noise ratios (SNRs), noise correlations, and g-factor maps to a vendor-supplied human pediatric coil frequently used for NHP MRI. The result from in vivo experiments showed that the NHP coil was well-decoupled, had higher SNRs in cortical regions, and improved data acquisition acceleration capability compared with a vendor-supplied human pediatric coil that has been frequently used in macaque MRI studies. Furthermore, whole-brain anatomic imaging, diffusion tensor imaging and functional brain imaging have also been conducted: the details of brain anatomical structure, such as cerebellum and brainstem, can be clearly visualized in T2-SPACE images; b0 SNR calculated from b0 maps was higher than the human pediatric coil in all regions of interest (ROIs); the time-course SNR (tSNR) map calculated for GRE-EPI images demonstrates that the presented coil can be used for high-resolution functional imaging at 3 T.

A neonatal nonhuman primate model of gestational Zika virus infection with evidence of microencephaly, seizures and cardiomyopathy.

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

Interactions within and between parallel parietal-frontal networks involved in complex motor behaviors in prosimian galagos and a squirrel monkey.

Long-train intracortical microstimulation (ICMS) of motor (M1) and posterior parietal cortices (PPC) in primates reveals cortical domains for different ethologically relevant behaviors. How functional domains interact with each other in producing motor behaviors is not known. In this study, we tested our hypothesis that matching domains interact to produce a specific complex movement, whereas connections between nonmatching domains are involved in suppression of conflicting motor outputs to prevent competing movements. In anesthetized galagos, we used 500-ms trains of ICMS to evoke complex movements from a functional domain in M1 or PPC while simultaneously stimulating another mismatched or matched domain. We considered movements of different and similar directions evoked from chosen cortical sites distant or close to each other. Their trajectories and speeds were analyzed and compared with those evoked by simultaneous stimulation. Stimulation of two sites evoking same or complementary movements produced a similar but more pronounced movement or a combined movement, respectively. Stimulation of two sites representing movements of different directions resulted in partial or total suppression of one of these movements. Thus interactions between domains in M1 and PPC were additive when they were functionally matched across fields or antagonistic between functionally conflicting domains, especially in PPC, suggesting that mismatched domains are involved in mutual suppression. Simultaneous stimulation of unrelated domains (forelimb and face) produced both movements independently. Movements produced by the simultaneous stimulation of sites in domains of two cerebral hemispheres were largely independent, but some interactions were observed.NEW & NOTEWORTHY Long trains of electrical pulses applied simultaneously to two sites in motor cortical areas (M1, PPC) have shown that interactions of functionally matched domains (evoking similar movements) within these areas were additive to produce a specific complex movement. Interactions between functionally mismatched domains (evoking different movements) were mostly antagonistic, suggesting their involvement in mutual suppression of conflicting motor outputs to prevent competing movements. Simultaneous stimulation of unrelated domains (forelimb and face) produced both movements independently.

Focal infrared neural stimulation with high-field functional MRI: A rapid way to map mesoscale brain connectomes.

We have developed a way to map brain-wide networks using focal pulsed infrared neural stimulation in ultrahigh-field magnetic resonance imaging (MRI). The patterns of connections revealed are similar to those of connections previously mapped with anatomical tract tracing methods. These include connections between cortex and subcortical locations and long-range cortico-cortical connections. Studies of local cortical connections reveal columnar-sized laminar activation, consistent with feed-forward and feedback projection signatures. This method is broadly applicable and can be applied to multiple areas of the brain in different species and across different MRI platforms. Systematic point-by-point application of this method may lead to fundamental advances in our understanding of brain connectomes.

Functionally specific optogenetic modulation in primate visual cortex.

In primates, visual perception is mediated by brain circuits composed of submillimeter nodes linked together in specific networks that process different types of information, such as eye specificity and contour orientation. We hypothesized that optogenetic stimulation targeted to cortical nodes could selectively activate such cortical networks. We used viral transfection methods to confer light sensitivity to neurons in monkey primary visual cortex. Using intrinsic signal optical imaging and single-unit electrophysiology to assess effects of targeted optogenetic stimulation, we found that (i) optogenetic stimulation of single ocular dominance columns (eye-specific nodes) revealed preferential activation of nearby same-eye columns but not opposite-eye columns, and (ii) optogenetic stimulation of single orientation domains increased visual response of matching orientation domains and relatively suppressed nonmatching orientation selectivity. These findings demonstrate that optical stimulation of single nodes leads to modulation of functionally specific cortical networks related to underlying neural architecture.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Review of functional and clinical relevance of intrinsic signal optical imaging in human brain mapping.

Intrinsic signal optical imaging (ISOI) within the first decade of its use in humans showed its capacity as a precise functional mapping tool. It is a powerful tool that can be used intraoperatively to help a surgeon to directly identify functional areas of the cerebral cortex. Its use is limited to the intraoperative setting as it requires a craniotomy and durotomy for direct visualization of the brain. It has been applied in humans to study language, somatosensory and visual cortices, cortical hemodynamics, epileptiform activity, and lesion delineation. Despite studies showing clear evidence of its usefulness in clinical care, its clinical use in humans has not grown. Impediments imposed by imaging in a human operating room setting have hindered such work. However, recent studies have been aimed at overcoming obstacles in clinical studies establishing the benefits of its use to patients. This review provides a description of ISOI and its use in human studies with an emphasis on the challenges that have hindered its widespread use and the recent studies that aim to overcome these hurdles. Clinical studies establishing the benefits of its use to patients would serve as the impetus for continued development and use in humans.

Study of single and multidigit activation in monkey somatosensory cortex using voltage-sensitive dye imaging.

Toward the goal of understanding cutaneous sensory integration during manual behavior, we used voltage-sensitive dye (VSD) imaging to study the organization and dynamics of anesthetized monkey primary somatosensory cortex (SI) in response to single and multidigit tactile stimulation. We find that in both macaque and squirrel monkey SI, VSD reveals clear focal digit topography consistent with previous electrophysiological and intrinsic signal imaging studies. VSD also reveals interactions in SI in response to multidigit stimulation. With a tactile funneling paradigm in areas 3b and 1 in squirrel monkeys, VSD reveals two-digit induction of subthreshhold influences, consistent with lateral intracortical inhibition. In response to tactile apparent motion stimuli, VSD reveals preferential response to motion stimuli over static tactile stimuli in both areas 1 and 3b. Comparison of the response at different digit locations to "toward digit" stimuli suggests the presence of direction-selective response in area 1; however, further study is needed. These exciting results indicate that VSD constitutes a powerful tool for studying somatosensory cortical processing in nonhuman primates and should be further developed for future somatosensory studies in awake behaving monkeys.

Is It Time for Synthetic Biodiversity Conservation?

Evidence indicates that, despite some critical successes, current conservation approaches are not slowing the overall rate of biodiversity loss. The field of synthetic biology, which is capable of altering natural genomes with extremely precise editing, might offer the potential to resolve some intractable conservation problems (e.g., invasive species or pathogens). However, it is our opinion that there has been insufficient engagement by the conservation community with practitioners of synthetic biology. We contend that rapid, large-scale engagement of these two communities is urgently needed to avoid unintended and deleterious ecological consequences. To this point we describe case studies where synthetic biology is currently being applied to conservation, and we highlight the benefits to conservation biologists from engaging with this emerging technology.