Dynamics of directional motor tuning in the primate premotor and primary motor cortices during sensorimotor learning.

Sensorimotor learning requires reorganization of neuronal activity in the premotor cortex (PM) and primary motor cortex (M1). To reveal PM- and M1-specific reorganization in a primate, we conducted calcium imaging in common marmosets while they learned a two-target reaching (pull/push) task after mastering a one-target reaching (pull) task. Throughout learning of the two-target reaching task, the dorsorostral PM (PMdr) showed peak activity earlier than the dorsocaudal PM (PMdc) and M1. During learning, the reaction time in pull trials increased and correlated strongly with the peak timing of PMdr activity. PMdr showed decreasing representation of newly introduced (push) movement, whereas PMdc and M1 maintained high representation of pull and push movements. Many task-related neurons in PMdc and M1 exhibited a strong preference to either movement direction. PMdc neurons dynamically switched their preferred direction depending on their performance in push trials in the early learning stage, whereas M1 neurons stably retained their preferred direction and high similarity of preferred direction between neighbors. These results suggest that in primate sensorimotor learning, dynamic directional motor tuning in PMdc converts the sensorimotor association formed in PMdr to the stable and specific motor representation of M1.

Altered projection-specific synaptic remodeling and its modification by oxytocin in an idiopathic autism marmoset model.

Alterations in the experience-dependent and autonomous elaboration of neural circuits are assumed to underlie autism spectrum disorder (ASD), though it is unclear what synaptic traits are responsible. Here, utilizing a valproic acid-induced ASD marmoset model, which shares common molecular features with idiopathic ASD, we investigate changes in the structural dynamics of tuft dendrites of upper-layer pyramidal neurons and adjacent axons in the dorsomedial prefrontal cortex through two-photon microscopy. In model marmosets, dendritic spine turnover is upregulated, and spines are generated in clusters and survived more often than in control marmosets. Presynaptic boutons in local axons, but not in commissural long-range axons, demonstrate hyperdynamic turnover in model marmosets, suggesting alterations in projection-specific plasticity. Intriguingly, nasal oxytocin administration attenuates clustered spine emergence in model marmosets. Enhanced clustered spine generation, possibly unique to certain presynaptic partners, may be associated with ASD and be a potential therapeutic target.

Change detection in the primate auditory cortex through feedback of prediction error signals.

Although cortical feedback signals are essential for modulating feedforward processing, no feedback error signal across hierarchical cortical areas has been reported. Here, we observed such a signal in the auditory cortex of awake common marmoset during an oddball paradigm to induce auditory duration mismatch negativity. Prediction errors to a deviant tone presentation were generated as offset calcium responses of layer 2/3 neurons in the rostral parabelt (RPB) of higher-order auditory cortex, while responses to non-deviant tones were strongly suppressed. Within several hundred milliseconds, the error signals propagated broadly into layer 1 of the primary auditory cortex (A1) and accumulated locally on top of incoming auditory signals. Blockade of RPB activity prevented deviance detection in A1. Optogenetic activation of RPB following tone presentation nonlinearly enhanced A1 tone response. Thus, the feedback error signal is critical for automatic detection of unpredicted stimuli in physiological auditory processing and may serve as backpropagation-like learning.

The Brain/MINDS Marmoset Connectivity Resource: An open-access platform for cellular-level tracing and tractography in the primate brain.

The primate brain has unique anatomical characteristics, which translate into advanced cognitive, sensory, and motor abilities. Thus, it is important that we gain insight on its structure to provide a solid basis for models that will clarify function. Here, we report on the implementation and features of the Brain/MINDS Marmoset Connectivity Resource (BMCR), a new open-access platform that provides access to high-resolution anterograde neuronal tracer data in the marmoset brain, integrated to retrograde tracer and tractography data. Unlike other existing image explorers, the BMCR allows visualization of data from different individuals and modalities in a common reference space. This feature, allied to an unprecedented high resolution, enables analyses of features such as reciprocity, directionality, and spatial segregation of connections. The present release of the BMCR focuses on the prefrontal cortex (PFC), a uniquely developed region of the primate brain that is linked to advanced cognition, including the results of 52 anterograde and 164 retrograde tracer injections in the cortex of the marmoset. Moreover, the inclusion of tractography data from diffusion MRI allows systematic analyses of this noninvasive modality against gold-standard cellular connectivity data, enabling detection of false positives and negatives, which provide a basis for future development of tractography. This paper introduces the BMCR image preprocessing pipeline and resources, which include new tools for exploring and reviewing the data.

Local and long-distance organization of prefrontal cortex circuits in the marmoset brain.

The prefrontal cortex (PFC) has dramatically expanded in primates, but its organization and interactions with other brain regions are only partially understood. We performed high-resolution connectomic mapping of the marmoset PFC and found two contrasting corticocortical and corticostriatal projection patterns: "patchy" projections that formed many columns of submillimeter scale in nearby and distant regions and "diffuse" projections that spread widely across the cortex and striatum. Parcellation-free analyses revealed representations of PFC gradients in these projections' local and global distribution patterns. We also demonstrated column-scale precision of reciprocal corticocortical connectivity, suggesting that PFC contains a mosaic of discrete columns. Diffuse projections showed considerable diversity in the laminar patterns of axonal spread. Altogether, these fine-grained analyses reveal important principles of local and long-distance PFC circuits in marmosets and provide insights into the functional organization of the primate brain.

Multi-modal brain magnetic resonance imaging database covering marmosets with a wide age range.

Magnetic resonance imaging (MRI) is a non-invasive neuroimaging technique that is useful for identifying normal developmental and aging processes and for data sharing. Marmosets have a relatively shorter life expectancy than other primates, including humans, because they grow and age faster. Therefore, the common marmoset model is effective in aging research. The current study investigated the aging process of the marmoset brain and provided an open MRI database of marmosets across a wide age range. The Brain/MINDS Marmoset Brain MRI Dataset contains brain MRI information from 216 marmosets ranging in age from 1 and 10 years. At the time of its release, it is the largest public dataset in the world. It also includes multi-contrast MRI images. In addition, 91 of 216 animals have corresponding high-resolution ex vivo MRI datasets. Our MRI database, available at the Brain/MINDS Data Portal, might help to understand the effects of various factors, such as age, sex, body size, and fixation, on the brain. It can also contribute to and accelerate brain science studies worldwide.

Tomographic tract tracing and data driven approaches to unravel complex 3D fiber anatomy of DBS relevant prefrontal projections to the diencephalic-mesencephalic junction in the marmoset.

BACKGROUND: Understanding prefrontal cortex projections to diencephalic-mesencephalic junction (DMJ), especially to subthalamic nucleus (STN) and ventral mesencephalic tegmentum (VMT) helps our comprehension of Deep Brain Stimulation (DBS) in major depression (MD) and obsessive-compulsive disorder (OCD). Fiber routes are complex and tract tracing studies in non-human primate species (NHP) have yielded conflicting results. The superolateral medial forebrain bundle (slMFB) is a promising target for DBS in MD and OCD. It has become a focus of criticism owing to its name and its diffusion weighted-imaging based primary description. OBJECTIVE: To investigate DMJ connectivity in NHP with a special focus on slMFB and the limbic hyperdirect pathway utilizing three-dimensional and data driven techniques. METHODS: We performed left prefrontal adeno-associated virus - tracer based injections in the common marmoset monkey (n = 52). Histology and two-photon microscopy were integrated into a common space. Manual and data driven cluster analyses of DMJ, subthalamic nucleus and VMT together, followed by anterior tract tracing streamline (ATTS) tractography were deployed. RESULTS: Typical pre- and supplementary motor hyperdirect connectivity was confirmed. The advanced tract tracing unraveled the complex connectivity to the DMJ. Limbic prefrontal territories directly projected to the VMT but not STN. DISCUSSION: Intricate results of tract tracing studies warrant the application of advanced three-dimensional analyses to understand complex fiber-anatomical routes. The applied three-dimensional techniques can enhance anatomical understanding also in other regions with complex fiber anatomy. CONCLUSION: Our work confirms slMFB anatomy and enfeebles previous misconceptions. The rigorous NHP approach strengthens the role of the slMFB as a target structure for DBS predominantly in psychiatric indications like MD and OCD.

Optimization and validation of diffusion MRI-based fiber tracking with neural tracer data as a reference.

Diffusion-weighted magnetic resonance imaging (dMRI) allows non-invasive investigation of whole-brain connectivity, which can reveal the brain's global network architecture and also abnormalities involved in neurological and mental disorders. However, the reliability of connection inferences from dMRI-based fiber tracking is still debated, due to low sensitivity, dominance of false positives, and inaccurate and incomplete reconstruction of long-range connections. Furthermore, parameters of tracking algorithms are typically tuned in a heuristic way, which leaves room for manipulation of an intended result. Here we propose a general data-driven framework to optimize and validate parameters of dMRI-based fiber tracking algorithms using neural tracer data as a reference. Japan's Brain/MINDS Project provides invaluable datasets containing both dMRI and neural tracer data from the same primates. A fundamental difference when comparing dMRI-based tractography and neural tracer data is that the former cannot specify the direction of connectivity; therefore, evaluating the fitting of dMRI-based tractography becomes challenging. The framework implements multi-objective optimization based on the non-dominated sorting genetic algorithm II. Its performance is examined in two experiments using data from ten subjects for optimization and six for testing generalization. The first uses a seed-based tracking algorithm, iFOD2, and objectives for sensitivity and specificity of region-level connectivity. The second uses a global tracking algorithm and a more refined set of objectives: distance-weighted coverage, true/false positive ratio, projection coincidence, and commissural passage. In both experiments, with optimized parameters compared to default parameters, fiber tracking performance was significantly improved in coverage and fiber length. Improvements were more prominent using global tracking with refined objectives, achieving an average fiber length from 10 to 17 mm, voxel-wise coverage of axonal tracts from 0.9 to 15%, and the correlation of target areas from 40 to 68%, while minimizing false positives and impossible cross-hemisphere connections. Optimized parameters showed good generalization capability for test brain samples in both experiments, demonstrating the flexible applicability of our framework to different tracking algorithms and objectives. These results indicate the importance of data-driven adjustment of fiber tracking algorithms and support the validity of dMRI-based tractography, if appropriate adjustments are employed.

Axonal Projections from Middle Temporal Area to the Pulvinar in the Common Marmoset.

The pulvinar, the largest thalamic nucleus in the primate brain, has connections with a variety of cortical areas and is involved in many aspects of higher brain functions. Among cortico-pulvino-cortical systems, the connection between the middle temporal area (MT) and the pulvinar has been thought to contribute significantly to complex motion recognition. Recently, the common marmoset (Callithrix jacchus), has become a valuable model for a variety of neuroscience studies, including visual neuroscience and translational research of neurological and psychiatric disorders. However, information on projections from MT to the pulvinar in the marmoset brain is scant. We addressed this deficiency by injecting sensitive anterograde viral tracers into MT to examine the distribution of labeled terminations in the pulvinar. The injection sites were placed retinotopically according to visual field coordinates mapped by optical intrinsic imaging. All injections produced anterograde terminal labeling, which was densest in the medial nucleus of the inferior pulvinar (PIm), sparser in the central nucleus of the inferior pulvinar, and weakest in the lateral pulvinar. Within each subnucleus, terminations formed separate retinotopic fields. Most labeled terminals were small but these comingled with a few large terminals, distributed mainly in the dorsomedial part of the PIm. Our results further delineate the organization of projections from MT to the pulvinar in the marmoset as forming parallel complex networks, which may differentially contribute to motion processing. It is interesting that the densest projections from MT target the PIm, the subnucleus recently reported to preferentially receive direct retinal projections.

Rho Guanine Nucleotide Exchange Factors Regulate Horizontal Axon Branching of Cortical Upper Layer Neurons.

Axon branching is a crucial process for cortical circuit formation. However, how the cytoskeletal changes in axon branching are regulated is not fully understood. In the present study, we investigated the role of RhoA guanine nucleotide exchange factors (RhoA-GEFs) in branch formation of horizontally elongating axons (horizontal axons) in the mammalian cortex. In situ hybridization showed that more than half of all known RhoA-GEFs were expressed in the developing rat cortex. These RhoA-GEFs were mostly expressed in the macaque cortex as well. An overexpression study using organotypic cortical slice cultures demonstrated that several RhoA-GEFs strongly promoted horizontal axon branching. Moreover, branching patterns were different between overexpressed RhoA-GEFs. In particular, ARHGEF18 markedly increased terminal arbors, whereas active breakpoint cluster region-related protein (ABR) increased short branches in both distal and proximal regions of horizontal axons. Rho kinase inhibitor treatment completely suppressed the branch-promoting effect of ARHGEF18 overexpression, but only partially affected that of ABR, suggesting that these RhoA-GEFs employ distinct downstream pathways. Furthermore, knockdown of either ARHGEF18 or ABR considerably suppressed axon branching. Taken together, the present study revealed that subsets of RhoA-GEFs differentially promote axon branching of mammalian cortical neurons.

Arm movements induced by noninvasive optogenetic stimulation of the motor cortex in the common marmoset.

Optogenetics is now a fundamental tool for investigating the relationship between neuronal activity and behavior. However, its application to the investigation of motor control systems in nonhuman primates is rather limited, because optogenetic stimulation of cortical neurons in nonhuman primates has failed to induce or modulate any hand/arm movements. Here, we used a tetracycline-inducible gene expression system carrying CaMKII promoter and the gene encoding a Channelrhodopsin-2 variant with fast kinetics in the common marmoset, a small New World monkey. In an awake state, forelimb movements could be induced when Channelrhodopsin-2-expressing neurons in the motor cortex were illuminated by blue laser light with a spot diameter of 1 mm or 2 mm through a cranial window without cortical invasion. Forelimb muscles responded 10 ms to 50 ms after photostimulation onset. Long-duration (500 ms) photostimulation induced discrete forelimb movements that could be markerlessly tracked with charge-coupled device cameras and a deep learning algorithm. Long-duration photostimulation mapping revealed that the primary motor cortex is divided into multiple domains that can induce hand and elbow movements in different directions. During performance of a forelimb movement task, movement trajectories were modulated by weak photostimulation, which did not induce visible forelimb movements at rest, around the onset of task-relevant movement. The modulation was biased toward the movement direction induced by the strong photostimulation. Combined with calcium imaging, all-optical interrogation of motor circuits should be possible in behaving marmosets.

PAT-Probabilistic Axon Tracking for Densely Labeled Neurons in Large 3-D Micrographs.

A major goal of contemporary neuroscience research is to map the structural connectivity of mammalian brain using microscopy imaging data. In this context, the reconstruction of densely labeled axons from two-photon microscopy images is a challenging and important task. The visually overlapping, crossing, and often strongly distorted images of the axons allow many ambiguous interpretations to be made. We address the problem of tracking axons in densely labeled samples of neurons in large image data sets acquired from marmoset brains. Our high-resolution images were acquired using two-photon microscopy and they provided whole brain coverage, occupying terabytes of memory. Both the image distortions and the large data set size frequently make it impractical to apply present-day neuron tracing algorithms to such data due to the optimization of such algorithms to the precise tracing of either single or sparse sets of neurons. Thus, new tracking techniques are needed. We propose a probabilistic axon tracking algorithm (PAT). PAT tackles the tracking of axons in two steps: locally (L-PAT) and globally (G-PAT). L-PAT is a probabilistic tracking algorithm that can tackle distorted, cluttered images of densely labeled axons. L-PAT divides a large micrograph into smaller image stacks. It then processes each image stack independently before mapping the axons in each image to a sparse model of axon trajectories. G-PAT merges the sparse L-PAT models into a single global model of axon trajectories by minimizing a global objective function using a probabilistic optimization method. We demonstrate the superior performance of PAT over standard approaches on synthetic data. Furthermore, we successfully apply PAT to densely labeled axons in large images acquired from marmoset brains.

The relationship between the claustrum and endopiriform nucleus: A perspective towards consensus on cross-species homology.

With the emergence of interest in studying the claustrum, a recent special issue of the Journal of Comparative Neurology dedicated to the claustrum (Volume 525, Issue 6, pp. 1313-1513) brought to light questions concerning the relationship between the claustrum (CLA) and a region immediately ventral known as the endopiriform nucleus (En). These structures have been identified as separate entities in rodents but appear as a single continuous structure in primates. During the recent Society for Claustrum Research meeting, a panel of experts presented data pertaining to the relationship of these regions and held a discussion on whether the CLA and En should be considered (a) separate unrelated structures, (b) separate nuclei within the same formation, or (c) subregions of a continuous structure. This review article summarizes that discussion, presenting comparisons of the cytoarchitecture, neurochemical profiles, genetic markers, and anatomical connectivity of the CLA and En across several mammalian species. In rodents, we conclude that the CLA and the dorsal endopiriform nucleus (DEn) are subregions of a larger complex, which likely performs analogous computations and exert similar effects on their respective cortical targets (e.g., sensorimotor versus limbic). Moving forward, we recommend that the field retain the nomenclature currently employed for this region but should continue to examine the delineation of these structures across different species. Using thorough descriptions of a variety of anatomical features, this review offers a clear definition of the CLA and En in rodents, which provides a framework for identifying homologous structures in primates.

Axonal Projections From the Middle Temporal Area in the Common Marmoset.

Neural activity in the middle temporal (MT) area is modulated by the direction and speed of motion of visual stimuli. The area is buried in a sulcus in the macaque, but exposed to the cortical surface in the marmoset, making the marmoset an ideal animal model for studying MT function. To better understand the details of the roles of this area in cognition, underlying anatomical connections need to be clarified. Because most anatomical tracing studies in marmosets have used retrograde tracers, the axonal projections remain uncharacterized. In order to examine axonal projections from MT, we utilized adeno-associated viral (AAV) tracers, which work as anterograde tracers by expressing either green or red fluorescent protein in infected neurons. AAV tracers were injected into three sites in MT based on retinotopy maps obtained via in vivo optical intrinsic signal imaging. Brains were sectioned and divided into three series, one for fluorescent image scanning and two for myelin and Nissl substance staining to identify specific brain areas. Overall projection patterns were similar across the injections. MT projected to occipital visual areas V1, V2, V3 (VLP) and V4 (VLA) and surrounding areas in the temporal cortex including MTC (V4T), MST, FST, FSTv (PGa/IPa) and TE3. There were also projections to the dorsal visual pathway, V3A (DA), V6 (DM) and V6A, the intraparietal areas AIP, LIP, MIP, frontal A4ab and the prefrontal cortex, A8aV and A8C. There was a visuotopic relationship with occipital visual areas. In a marmoset in which two tracer injections were made, the projection targets did not overlap in A8aV and AIP, suggesting topographic projections from different parts of MT. Most of these areas are known to send projections back to MT, suggesting that they are reciprocally connected with it.

Calcium Transient Dynamics of Neural Ensembles in the Primary Motor Cortex of Naturally Behaving Monkeys.

To understand brain circuits of cognitive behaviors under natural conditions, we developed techniques for imaging neuronal activities from large neuronal populations in the deep layer cortex of the naturally behaving common marmoset. Animals retrieved food pellets or climbed ladders as a miniature fluorescence microscope monitored hundreds of calcium indicator-expressing cortical neurons in the right primary motor cortex. This technique, which can be adapted to other brain regions, can deepen our understanding of brain circuits by facilitating longitudinal population analyses of neuronal representation associated with cognitive naturalistic behaviors and their pathophysiological processes.

Cortical networks of the mouse brain elaborate within the gray matter.

In primates, proximal cortical areas are interconnected via within-cortex "intrinsic" pathway, whereas distant areas are connected via "extrinsic" white matter pathway. To date, such distinction has not been clearly done for small-brained mammals like rodents. In this study, we systematically analyzed the data of Allen Mouse Brain Connectivity Atlas to answer this question and found that the ipsilateral cortical connections in mice are almost exclusively contained within the gray matter, although we observed exceptions for projections from the retrosplenial area and the medial/orbital frontal areas. By analyzing axonal projections within the gray matter using Cortical Box method, which enabled us to investigate the layer patterns across different cortical areas, we obtained the following results. First, widespread axonal projections were observed in both upper and lower layers in the vicinity of injections, whereas highly specific "point-to-point" projections were observed toward remote areas. Second, such long-range projections were predominantly aligned in the anteromedial-posterolateral direction. Third, in the majority of these projections, the connecting axons traveled through layer 6. Finally, the projections from the primary and higher order areas to distant targets preferentially terminated in the middle and superficial layers, respectively, suggesting hierarchical connections similar to those of primates. Overall, our study demonstrated conspicuous differences in gray/white matter segregation of axonal projections between rodents and primates, despite certain similarities in the hierarchical cortical organization.

Two-photon imaging of neuronal activity in motor cortex of marmosets during upper-limb movement tasks.

Two-photon imaging in behaving animals has revealed neuronal activities related to behavioral and cognitive function at single-cell resolution. However, marmosets have posed a challenge due to limited success in training on motor tasks. Here we report the development of protocols to train head-fixed common marmosets to perform upper-limb movement tasks and simultaneously perform two-photon imaging. After 2-5 months of training sessions, head-fixed marmosets can control a manipulandum to move a cursor to a target on a screen. We conduct two-photon calcium imaging of layer 2/3 neurons in the motor cortex during this motor task performance, and detect task-relevant activity from multiple neurons at cellular and subcellular resolutions. In a two-target reaching task, some neurons show direction-selective activity over the training days. In a short-term force-field adaptation task, some neurons change their activity when the force field is on. Two-photon calcium imaging in behaving marmosets may become a fundamental technique for determining the spatial organization of the cortical dynamics underlying action and cognition.

Single-cell bioluminescence imaging of deep tissue in freely moving animals.

Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

Silencing of FUS in the common marmoset (Callithrix jacchus) brain via stereotaxic injection of an adeno-associated virus encoding shRNA.

Fused in sarcoma (FUS) is an RNA binding protein that is involved in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). To establish the common marmoset (Callithrix jacchus) as a model for FTLD, we generated a stereotaxic injection-based marmoset model of FUS-silencing. We designed shRNAs against the marmoset FUS gene and generated an AAV9 virus encoding the most effective shRNA against FUS (shFUS). The AAV encoding shFUS (AAV-shFUS) was introduced into the frontal cortex of young adult marmosets, whereas AAV encoding a control shRNA was injected into the contralateral side. We obtained approximately 70-80% silencing of FUS following AAV-shFUS injection. Interestingly, FUS-silencing provoked a proliferation of astrocytes and microglias. Since FTLD is characterized by various emotional deficits, it would be helpful to establish a marmoset model of FUS-silencing in various brain tissues for investigating the pathomechanism of higher cognitive and behavioral dysfunction.

3D reconstruction of brain section images for creating axonal projection maps in marmosets.

BACKGROUND: The brain of the common marmoset (Callithrix jacchus) is becoming a popular non-human primate model in neuroscience research. Because its brain fiber connectivity is still poorly understood, it is necessary to collect and present connection and trajectory data using tracers to establish a marmoset brain connectivity database. NEW METHOD: To visualize projections and trajectories of axons, brain section images were reconstructed in 3D by registering them to the corresponding block-face brain images taken during brain sectioning. During preprocessing, autofluorescence of the tissue was reduced by applying independent component analysis to a set of fluorescent images taken using different filters. RESULTS: The method was applied to a marmoset dataset after a tracer had been injected into an auditory belt area to fluorescently label axonal projections. Cortical and subcortical connections were clearly reconstructed in 3D. The registration error was estimated to be smaller than 200 µm. Evaluation tests on ICA-based autofluorescence reduction showed a significant improvement in signal and background separation. COMPARISON WITH EXISTING METHODS: Regarding the 3D reconstruction error, the present study shows an accuracy comparable to previous studies using MRI and block-face images. Compared to serial section two-photon tomography, an advantage of the proposed method is that it can be combined with standard histological techniques. The images of differently processed brain sections can be integrated into the original ex vivo brain shape. CONCLUSIONS: The proposed method allows creating 3D axonal projection maps overlaid with brain area annotations based on the histological staining results of the same animal.