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.

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.

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.

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.

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.

Spatiotemporal trajectories of reactivation of somatosensory cortex by direct and secondary pathways after dorsal column lesions in squirrel monkeys.

After lesions of the somatosensory dorsal column (DC) pathway, the cortical hand representation can become unresponsive to tactile stimuli, but considerable responsiveness returns over weeks of post-lesion recovery. The reactivation suggests that preserved subthreshold sensory inputs become potentiated and axon sprouting occurs over time to mediate recovery. Here, we studied the recovery process in 3 squirrel monkeys, using high-resolution cerebral blood volume-based functional magnetic resonance imaging (CBV-fMRI) mapping of contralateral somatosensory cortex responsiveness to stimulation of distal finger pads with low and high level electrocutaneous stimulation (ES) before and 2, 4, and 6weeks after a mid-cervical level contralateral DC lesion. Both low and high intensity ES of digits revealed the expected somatotopy of the area 3b hand representation in pre-lesion monkeys, while in areas 1 and 3a, high intensity stimulation was more effective in activating somatotopic patterns. Six weeks post-lesion, and irrespective of the severity of loss of direct DC inputs (98%, 79%, 40%), somatosensory cortical area 3b of all three animals showed near complete recovery in terms of somatotopy and responsiveness to low and high intensity ES. However there was significant variability in the patterns and amplitudes of reactivation of individual digit territories within and between animals, reflecting differences in the degree of permanent and/or transient silencing of primary DC and secondary inputs 2weeks post-lesion, and their spatio-temporal trajectories of recovery between 2 and 6weeks. Similar variations in the silencing and recovery of somatotopy and responsiveness to high intensity ES in areas 3a and 1 are consistent with individual differences in damage to and recovery of DC and spinocuneate pathways, and possibly the potentiation of spinothalamic pathways. Thus, cortical deactivation and subsequent reactivation depends not only on the degree of DC lesion, but also on the severity and duration of loss of secondary as well as primary inputs revealed by low and high intensity ES.

Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum.

The "dark matter of life" describes microbes and even entire divisions of bacterial phyla that have evaded cultivation and have yet to be sequenced. We present a genome from the globally distributed but elusive candidate phylum TM6 and uncover its metabolic potential. TM6 was detected in a biofilm from a sink drain within a hospital restroom by analyzing cells using a highly automated single-cell genomics platform. We developed an approach for increasing throughput and effectively improving the likelihood of sampling rare events based on forming small random pools of single-flow-sorted cells, amplifying their DNA by multiple displacement amplification and sequencing all cells in the pool, creating a "mini-metagenome." A recently developed single-cell assembler, SPAdes, in combination with contig binning methods, allowed the reconstruction of genomes from these mini-metagenomes. A total of 1.07 Mb was recovered in seven contigs for this member of TM6 (JCVI TM6SC1), estimated to represent 90% of its genome. High nucleotide identity between a total of three TM6 genome drafts generated from pools that were independently captured, amplified, and assembled provided strong confirmation of a correct genomic sequence. TM6 is likely a Gram-negative organism and possibly a symbiont of an unknown host (nonfree living) in part based on its small genome, low-GC content, and lack of biosynthesis pathways for most amino acids and vitamins. Phylogenomic analysis of conserved single-copy genes confirms that TM6SC1 is a deeply branching phylum.

[Neuronal connections within the hand representation in areas 3b and 1 of the somatosensory cortex in primates]. / Neuronalis összeköttetések a szomatoszenzoros kérgi área 3b és área 1 kézreprezentációs területén foemlosökben.

INTRODUCTION: The close functional relationship between areas 3b and 1 of the somatosensory cortex is based on their reciprocal connections indicating that tactile sensation depends on the interaction of these two areas. AIM: The aim of the authors was to explore this neuronal circuit at the level of the distal finger pad representation. METHOD: The study was made by bidirectional tract tracing aided by neurophysiological mapping in squirrel monkeys (Saimiri sciureus). RESULTS: Inter-areal connections between the two areas preferred the homologues representations. However, intra-areal connections were formed between the neighboring finger pad representations supporting the physiological observations. Interestingly, the size of the local input area of the injected cortical micro-region, which differed in the two areas, represented the same skin area. CONCLUSIONS: The authors propose that intra-areal connections are important in integrating information across fingers, while inter-areal connections are important in maintaining input localization during hand movement. Orv. Hetil., 2016, 157(33), 1320-1325.

Infrared neural stimulation of primary visual cortex in non-human primates.

Infrared neural stimulation (INS) is an alternative neurostimulation modality that uses pulsed infrared light to evoke spatially precise neural activity that does not require direct contact with neural tissue. With these advantages INS has the potential to increase our understanding of specific neural pathways and impact current diagnostic and therapeutic clinical applications. In order to develop this technique, we investigate the feasibility of INS (λ=1.875µm, fiber diameter=100-400µm) to activate and modulate neural activity in primary visual cortex (V1) of Macaque monkeys. Infrared neural stimulation was found to evoke localized neural responses as evidenced by both electrophysiology and intrinsic signal optical imaging (OIS). Single unit recordings acquired during INS indicated statistically significant increases in neuron firing rates that demonstrate INS evoked excitatory neural activity. Consistent with this, INS stimulation led to focal intensity-dependent reflectance changes recorded with OIS. We also asked whether INS is capable of stimulating functionally specific domains in visual cortex and of modulating visually evoked activity in visual cortex. We found that application of INS via 100µm or 200µm fiber optics produced enhancement of visually evoked OIS response confined to the eye column where INS was applied and relative suppression of the other eye column. Stimulating the cortex with a 400µm fiber, exceeding the ocular dominance width, led to relative suppression, consistent with involvement of inhibitory surrounds. This study is the first to demonstrate that INS can be used to either enhance or diminish visual cortical response and that this can be done in a functional domain specific manner. INS thus holds great potential for use as a safe, non-contact, focally specific brain stimulation technology in primate brains.

Distinct fine-scale fMRI activation patterns of contra- and ipsilateral somatosensory areas 3b and 1 in humans.

Inter-areal and ipsilateral cortical responses to tactile stimulation have not been well described in human S1 cortex. By taking advantage of the high signal-to-noise ratio at 7 T, we quantified blood oxygenation level dependent (BOLD) response patterns and time courses to tactile stimuli on individual distal finger pads at a fine spatial scale, and examined whether there are inter-areal (area 3b versus area 1) and interhemispheric response differences to unilateral tactile stimulation in healthy human subjects. We found that 2-Hz tactile stimulation of individual fingertips evoked detectable BOLD signal changes in both contralateral and ipsilateral area 3b and area 1. Contralateral digit activations were organized in an orderly somatotopic manner, and BOLD responses in area 3b were more digit selective than those in area 1. However, the area of cortex that was responsive to stimulation of a single digit (stimulus-response field) was similar across areas. In the ipsilateral hemisphere, response magnitudes in both areas 3b and 1 were significantly weaker than those of the contralateral hemisphere. Digit activations exhibited no clear somatotopic organizational pattern in either area 3b or area 1, yet digit selectivity was retained in area 1 but not in area 3b. The observation of distinct digit-selective responses of contralateral area 3b versus area 1 supports a higher order function of contralateral area 1 in spatial integration. In contrast, ipsilateral cortices may play a less discriminative role in the perception of unilateral tactile sensation in humans.

Optical imaging in galagos reveals parietal-frontal circuits underlying motor behavior.

The posterior parietal cortex (PPC) of monkeys and prosimian galagos contains a number of subregions where complex, behaviorally meaningful movements, such as reaching, grasping, and body defense, can be evoked by electrical stimulation with long trains of electrical pulses through microelectrodes. Shorter trains of pulses evoke no or simple movements. One possibility for the difference in effectiveness of intracortical microstimulation is that long trains activate much larger regions of the brain. Here, we show that long-train stimulation of PPC does not activate widespread regions of frontal motor and premotor cortex but instead, produces focal, somatotopically appropriate activations of frontal motor and premotor cortex. Shorter stimulation trains activate the same frontal foci but less strongly, showing that longer stimulus trains do not produce less specification. Because the activated sites in frontal cortex correspond to the locations of direct parietal-frontal anatomical connections from the stimulated PPC subregions, the results show the usefulness of optical imaging in conjunction with electrical stimulation in showing functional pathways between nodes in behavior-specific cortical networks. Thus, long-train stimulation is effective in evoking ethologically relevant sequences of movements by activating nodes in a cortical network for a behaviorally relevant period rather than spreading activation in a nonspecific manner.

Optical imaging of cortical networks via intracortical microstimulation.

Understanding cortical organization is key to understanding brain function. Distinct neural networks underlie the functional organization of the cerebral cortex; however, little is known about how different nodes in the cortical network interact during perceptual processing and motor behavior. To study cortical network function we examined whether the optical imaging of intrinsic signals (OIS) reveals the functional patterns of activity evoked by electrical cortical microstimulation. We examined the effects of current amplitude, train duration, and depth of cortical stimulation on the hemodynamic response to electrical microstimulation (250-Hz train, 0.4-ms pulse duration) in anesthetized New World monkey somatosensory cortex. Electrical stimulation elicited a restricted cortical response that varied according to stimulation parameters and electrode depth. Higher currents of stimulation recruited more areas of cortex than smaller currents. The largest cortical responses were seen when stimulation was delivered around cortical layer 4. Distinct local patches of activation, highly suggestive of local projections, around the site of stimulation were observed at different depths of stimulation. Thus we find that specific electrical stimulation parameters can elicit activation of single cortical columns and their associated columnar networks, reminiscent of anatomically labeled networks. This novel functional tract tracing method will open new avenues for investigating relationships of local cortical organization.

Voltage-sensitive dye imaging reveals shifting spatiotemporal spread of whisker-induced activity in rat barrel cortex.

In rats, navigating through an environment requires continuous information about objects near the head. Sensory information such as object location and surface texture are encoded by spike firing patterns of single neurons within rat barrel cortex. Although there are many studies using single-unit electrophysiology, much less is known regarding the spatiotemporal pattern of activity of populations of neurons in barrel cortex in response to whisker stimulation. To examine cortical response at the population level, we used voltage-sensitive dye (VSD) imaging to examine ensemble spatiotemporal dynamics of barrel cortex in response to stimulation of single or two adjacent whiskers in urethane-anesthetized rats. Single whisker stimulation produced a poststimulus fluorescence response peak within 12-16 ms in the barrel corresponding to the stimulated whisker (principal whisker). This fluorescence subsequently propagated throughout the barrel field, spreading anisotropically preferentially along a barrel row. After paired whisker stimulation, the VSD signal showed sublinear summation (less than the sum of 2 single whisker stimulations), consistent with previous electrophysiological and imaging studies. Surprisingly, we observed a spatial shift in the center of activation occurring over a 10- to 20-ms period with shift magnitudes of 1-2 barrels. This shift occurred predominantly in the posteromedial direction within the barrel field. Our data thus reveal previously unreported spatiotemporal patterns of barrel cortex activation. We suggest that this nontopographical shift is consistent with known functional and anatomic asymmetries in barrel cortex and that it may provide an important insight for understanding barrel field activation during whisking behavior.

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.

Differentiation of somatosensory cortices by high-resolution fMRI at 7 T.

This study aimed to evaluate the ability of BOLD signals at high MRI field (7 T) to map fine-scale single-digit activations in subdivisions (areas 3b and 1) of the human primary somatosensory cortex (SI) in individual subjects. We acquired BOLD fMRI data from cortical areas around the central suclus in six healthy human subjects while stimulating individual finger pads with 2-Hz air puffs. Discrete, single-digit responses were identified in an area along the posterior bank of the central sulcus corresponding to area 3b and in an area along the crest of the postcentral gyrus corresponding to area 1. In single subjects, activations of digits 1 to 4 in both areas 3b and 1 were organized in a somatotopic manner. The separation of digit representations was measured for adjacent digits and was approximately 1.6 times greater in area 3b than in area 1. Within individual subjects, the cortical responses to single-digit stimulations and the magnitude of the BOLD signals were reproducible across imaging runs and were comparable across subjects. Our findings demonstrate that BOLD fMRI at 7 T is capable of revealing the somatotopic organization of single-digit activations with good within-subject reliability and reproducibility, and activation maps can be acquired within a reasonably short time window, which are essential characteristics for several neurological applications within patient populations.

Pulsed infrared light alters neural activity in rat somatosensory cortex in vivo.

Pulsed infrared light has shown promise as an alternative to electrical stimulation in applications where contact free or high spatial precision stimulation is desired. Infrared neural stimulation (INS) is well characterized in the peripheral nervous system; however, to date, research has been limited in the central nervous system. In this study, pulsed infrared light (λ=1.875 µm, pulse width=250 µs, radiant exposure=0.01-0.55 J/cm(2), fiber size=400 µm, repetition rate=50-200 Hz) was used to stimulate the somatosensory cortex of anesthetized rats, and its efficacy was assessed using intrinsic optical imaging and electrophysiology techniques. INS was found to evoke an intrinsic response of similar magnitude to that evoked by tactile stimulation (0.3-0.4% change in intrinsic signal magnitude). A maximum deflection in the intrinsic signal was measured to range from 0.05% to 0.4% in response to INS, and the activated region of cortex measured approximately 2mm in diameter. The intrinsic signal magnitude increased with faster laser repetition rates and increasing radiant exposures. Single unit recordings indicated a statistically significant decrease in neuronal firing that was observed at the onset of INS stimulation (0.5s stimulus) and continued up to 1s after stimulation onset. The pattern of neuronal firing differed from that observed during tactile stimulation, potentially due to a different spatial integration field of the pulsed infrared light compared to tactile stimulation. The results demonstrate that INS can be used safely and effectively to manipulate neuronal firing.

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.

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.