Noninvasive focal transgene delivery with viral neuronal tracers in the marmoset monkey.

We establish a reliable method for selectively delivering adeno-associated viral vectors (AAVs) across the blood-brain barrier (BBB) in the marmoset without the need for neurosurgical injection. We focally perturbed the BBB (∼1 × 2 mm) in area 8aD of the frontal cortex in four adult marmoset monkeys using low-intensity transcranial focused ultrasound aided by microbubbles. Within an hour of opening the BBB, either AAV2 or AAV9 was delivered systemically via tail-vein injection. In all four marmosets, fluorescence-encoded neurons were observed at the site of BBB perturbation, with AAV2 showing a sparse distribution of transduced neurons when compared to AAV9. The results are compared to direct intracortical injections of anterograde tracers into area 8aD and similar (albeit sparser) long-range connectivity was observed. With evidence of transduced neurons specific to the region of BBB opening as well as long-distance tracing, we establish a framework for focal noninvasive transgene delivery to the marmoset brain.

Direct interhemispheric cortical communication via thalamic commissures: a new white matter pathway in the primate brain.

Cortical neurons of eutherian mammals project to the contralateral hemisphere, crossing the midline primarily via the corpus callosum and the anterior, posterior, and hippocampal commissures. We recently reported and named the thalamic commissures (TCs) as an additional interhemispheric axonal fiber pathway connecting the cortex to the contralateral thalamus in the rodent brain. Here, we demonstrate that TCs also exist in primates and characterize the connectivity of these pathways with high-resolution diffusion-weighted MRI, viral axonal tracing, and fMRI. We present evidence of TCs in both New World (Callithrix jacchus and Cebus apella) and Old World primates (Macaca mulatta). Further, like rodents, we show that the TCs in primates develop during the embryonic period, forming anatomical and functionally active connections of the cortex with the contralateral thalamus. We also searched for TCs in the human brain, showing their presence in humans with brain malformations, although we could not identify TCs in healthy subjects. These results pose the TCs as a vital fiber pathway in the primate brain, allowing for more robust interhemispheric connectivity and synchrony and serving as an alternative commissural route in developmental brain malformations.

Direct interhemispheric cortical communication via thalamic commissures: a new white-matter pathway in the primate brain.

Cortical neurons of eutherian mammals project to the contralateral hemisphere, crossing the midline primarily via the corpus callosum and the anterior, posterior, and hippocampal commissures. We recently reported an additional commissural pathway in rodents, termed the thalamic commissures (TCs), as another interhemispheric axonal fiber pathway that connects cortex to the contralateral thalamus. Here, we demonstrate that TCs also exist in primates and characterize the connectivity of these pathways with high-resolution diffusion-weighted magnetic resonance imaging, viral axonal tracing, and functional MRI. We present evidence of TCs in both New World (Callithrix jacchus and Cebus apella) and Old World primates (Macaca mulatta). Further, like rodents, we show that the TCs in primates develop during the embryonic period, forming anatomical and functionally active connections of the cortex with the contralateral thalamus. We also searched for TCs in the human brain, showing their presence in humans with brain malformations, although we could not identify TCs in healthy subjects. These results pose the TCs as an important fiber pathway in the primate brain, allowing for more robust interhemispheric connectivity and synchrony and serving as an alternative commissural route in developmental brain malformations.

Heterotopic connectivity of callosal dysgenesis in mice and humans.

The corpus callosum (CC), the largest brain commissure and the primary white matter pathway for interhemispheric cortical connectivity, was traditionally viewed as a predominantly homotopic structure, connecting mirror areas of the cortex. However, new studies verified that most callosal commissural fibers are heterotopic. Recently, we reported that ~75% of the callosal connections in the brains of mice, marmosets, and humans are heterotopic, having an essential role in determining the global properties of brain networks. In the present study, we leveraged high-resolution diffusion-weighted imaging and graph network modeling to investigate the relationship between heterotopic and homotopic callosal fibers in human subjects and in a spontaneous mouse model of Corpus Callosum Dysgenesis (CCD), a congenital developmental CC malformation that leads to widespread whole-brain reorganization. Our results show that the CCD brain is more heterotopic than the normotypical brain, with both mouse and human CCD subjects displaying highly variable heterotopicity maps. CCD mice have a clear heterotopicity cluster in the anterior CC, while hypoplasic humans have strongly variable patterns. Graph network-based connectivity profile showed a direct impact of heterotopic connections on CCD brains altering several network-based statistics. Our collective results show that CCD directly alters heterotopic connections and brain connectivity.

The relevance of heterotopic callosal fibers to interhemispheric connectivity of the mammalian brain.

The corpus callosum (CC) is the largest white matter structure and the primary pathway for interhemispheric brain communication. Investigating callosal connectivity is crucial to unraveling the brain's anatomical and functional organization in health and disease. Classical anatomical studies have characterized the bulk of callosal axonal fibers as connecting primarily homotopic cortical areas. Whenever detected, heterotopic callosal fibers were ascribed to altered sprouting and pruning mechanisms in neurodevelopmental diseases such as CC dysgenesis (CCD). We hypothesized that these heterotopic connections had been grossly underestimated due to their complex nature and methodological limitations. We used the Allen Mouse Brain Connectivity Atlas and high-resolution diffusion-weighted imaging to identify and quantify homotopic and heterotopic callosal connections in mice, marmosets, and humans. In all 3 species, we show that ~75% of interhemispheric callosal connections are heterotopic and comprise the central core of the CC, whereas the homotopic fibers lay along its periphery. We also demonstrate that heterotopic connections have an essential role in determining the global properties of brain networks. These findings reshape our view of the corpus callosum's role as the primary hub for interhemispheric brain communication, directly impacting multiple neuroscience fields investigating cortical connectivity, neurodevelopment, and neurodevelopmental disorders.

An integrated resource for functional and structural connectivity of the marmoset brain.

Comprehensive integration of structural and functional connectivity data is required to model brain functions accurately. While resources for studying the structural connectivity of non-human primate brains already exist, their integration with functional connectivity data has remained unavailable. Here we present a comprehensive resource that integrates the most extensive awake marmoset resting-state fMRI data available to date (39 marmoset monkeys, 710 runs, 12117 mins) with previously published cellular-level neuronal tracing data (52 marmoset monkeys, 143 injections) and multi-resolution diffusion MRI datasets. The combination of these data allowed us to (1) map the fine-detailed functional brain networks and cortical parcellations, (2) develop a deep-learning-based parcellation generator that preserves the topographical organization of functional connectivity and reflects individual variabilities, and (3) investigate the structural basis underlying functional connectivity by computational modeling. This resource will enable modeling structure-function relationships and facilitate future comparative and translational studies of primate brains.

An open access resource for functional brain connectivity from fully awake marmosets.

The common marmoset (Callithrix jacchus) is quickly gaining traction as a premier neuroscientific model. However, considerable progress is still needed in understanding the functional and structural organization of the marmoset brain to rival that documented in longstanding preclinical model species, like mice, rats, and Old World primates. To accelerate such progress, we present the Marmoset Functional Brain Connectivity Resource (marmosetbrainconnectome.org), currently consisting of over 70 h of resting-state fMRI (RS-fMRI) data acquired at 500 µm isotropic resolution from 31 fully awake marmosets in a common stereotactic space. Three-dimensional functional connectivity (FC) maps for every cortical and subcortical gray matter voxel are stored online. Users can instantaneously view, manipulate, and download any whole-brain functional connectivity (FC) topology (at the subject- or group-level) along with the raw datasets and preprocessing code. Importantly, researchers can use this resource to test hypotheses about FC directly - with no additional analyses required - yielding whole-brain correlations for any gray matter voxel on demand. We demonstrate the resource's utility for presurgical planning and comparison with tracer-based neuronal connectivity as proof of concept. Complementing existing structural connectivity resources for the marmoset brain, the Marmoset Functional Brain Connectivity Resource affords users the distinct advantage of exploring the connectivity of any voxel in the marmoset brain, not limited to injection sites nor constrained by regional atlases. With the entire raw database (RS-fMRI and structural images) and preprocessing code openly available for download and use, we expect this resource to be broadly valuable to test novel hypotheses about the functional organization of the marmoset brain.

Corpus callosum dysgenesis causes novel patterns of structural and functional brain connectivity.

Developmental malformations (dysgenesis) of the corpus callosum lead to neurological conditions with a broad range of clinical presentations. Investigating the altered brain connectivity patterns is crucial to understanding both adaptive and maladaptive neuroplasticity in corpus callosum dysgenesis patients. Here, we acquired structural diffusion-weighted and resting-state functional MRI data from a cohort of 11 corpus callosum dysgenesis patients (five with agenesis and six with hypoplasia) and compared their structural and functional connectivity patterns to healthy subjects selected from the Human Connectome Project. We found that these patients have fewer structural inter- and intra-hemispheric brain connections relative to healthy controls. Interestingly, the patients with callosal agenesis have a scant number of inter-hemispheric connections but manage to maintain the full integrity of functional connectivity between the same cortical regions as the healthy subjects. On the other hand, the hypoplasic group presented abnormal structural and functional connectivity patterns relative to healthy controls while maintaining the same total amount of functional connections. These results demonstrate that acallosal patients can compensate for having fewer structural brain connections and present functional adaptation. However, hypoplasics present atypical structural connections to different brain regions, leading to entirely new and abnormal functional brain connectivity patterns.

Direct Interhemispheric Cortical Communication via Thalamic Commissures: A New White-Matter Pathway in the Rodent Brain.

The corpus callosum (CC), the anterior (AC), and the posterior (PC) commissures are the principal axonal fiber bundle pathways that allow bidirectional communication between the brain hemispheres. Here, we used the Allen mouse brain connectivity atlas and high-resolution diffusion-weighted MRI (DWI) to investigate interhemispheric fiber bundles in C57bl6/J mice, the most commonly used wild-type mouse model in biomedical research. We identified 1) commissural projections from the primary motor area through the AC to the contralateral hemisphere; and 2) intrathalamic interhemispheric fiber bundles from multiple regions in the frontal cortex to the contralateral thalamus. This is the first description of direct interhemispheric corticothalamic connectivity from the orbital cortex. We named these newly identified crossing points thalamic commissures. We also analyzed interhemispheric connectivity in the Balb/c mouse model of dysgenesis of the corpus callosum (CCD). Relative to C57bl6/J, Balb/c presented an atypical and smaller AC and weaker interhemispheric corticothalamic communication. These results redefine our understanding of interhemispheric brain communication. Specifically, they establish the thalamus as a regular hub for interhemispheric connectivity and encourage us to reinterpret brain plasticity in CCD as an altered balance between axonal reinforcement and pruning.

Ultrahigh-resolution MRI Reveals Extensive Cortical Demyelination in a Nonhuman Primate Model of Multiple Sclerosis.

Cortical lesions are a primary driver of disability in multiple sclerosis (MS). However, noninvasive detection of cortical lesions with in vivo magnetic resonance imaging (MRI) remains challenging. Experimental autoimmune encephalomyelitis (EAE) in the common marmoset is a relevant animal model of MS for investigating the pathophysiological mechanisms leading to brain damage. This study aimed to characterize cortical lesions in marmosets with EAE using ultrahigh-field (7 T) MRI and histological analysis. Tissue preparation was optimized to enable the acquisition of high-spatial resolution (50-µm isotropic) T2*-weighted images. A total of 14 animals were scanned in this study, and 70% of the diseased animals presented at least one cortical lesion on postmortem imaging. Cortical lesions identified on MRI were verified with myelin proteolipid protein immunostaining. An optimized T2*-weighted sequence was developed for in vivo imaging and shown to capture 65% of cortical lesions detected postmortem. Immunostaining confirmed extensive demyelination with preserved neuronal somata in several cortical areas of EAE animals. Overall, this study demonstrates the relevance and feasibility of the marmoset EAE model to study cortical lesions, among the most important yet least understood features of MS.

Marmoset Brain Mapping V3: Population multi-modal standard volumetric and surface-based templates.

The standard anatomical brain template provides a common space and coordinate system for visualizing and analyzing neuroimaging data from large cohorts of subjects. Previous templates and atlases for the common marmoset brain were either based on data from a single individual or lacked essential functionalities for neuroimaging analysis. Here, we present new population-based in-vivo standard templates and tools derived from multi-modal data of 27 marmosets, including multiple types of T1w and T2w contrast images, DTI contrasts, and large field-of-view MRI and CT images. We performed multi-atlas labeling of anatomical structures on the new templates and constructed highly accurate tissue-type segmentation maps to facilitate volumetric studies. We built fully featured brain surfaces and cortical flat maps to facilitate 3D visualization and surface-based analyses, which are compatible with most surface analyzing tools, including FreeSurfer, AFNI/SUMA, and the Connectome Workbench. Analysis of the MRI and CT datasets revealed significant variations in brain shapes, sizes, and regional volumes of brain structures, highlighting substantial individual variabilities in the marmoset population. Thus, our population-based template and associated tools provide a versatile analysis platform and standard coordinate system for a wide range of MRI and connectome studies of common marmosets. These new template tools comprise version 3 of our Marmoset Brain Mapping Project and are publicly available via marmosetbrainmapping.org/v3.html.

Long-distance aberrant heterotopic connectivity in a mouse strain with a high incidence of callosal anomalies.

Corpus callosum dysgenesis (CCD) is a developmental brain condition in which some white matter fibers fail to find their natural course across the midplane, reorganizing instead to form new aberrant pathways. This type of white matter reorganization is known as long-distance plasticity (LDP). The present work aimed to characterize the Balb/c mouse strain as a model of CCD. We employed high-resolution anatomical MRI in 81 Balb/c and 27 C57bl6 mice to show that the Balb/c mouse strain presents a variance in the size of the CC that is 3.9 times higher than the variance of normotypical C57bl6. We also performed high-resolution diffusion-weighted imaging (DWI) in 8 Balb/c and found that the Balb/c strain shows aberrant white matter bundles, such as the Probst (5/8 animals) and the Sigmoid bundles (7/8 animals), which are similar to those found in humans with CCD. Using a histological tracer technique, we confirmed the existence of these aberrant bundles in the Balb/c strain. Interestingly, we also identified sigmoid-like fibers in the C57bl6 strain, thought to a lesser degree. Next, we used a connectome approach and found widespread brain connectivity differences between Balb/c and C57bl6 strains. The Balb/c strain also exhibited increased variability of global connectivity. These findings suggest that the Balb/c strain presents local and global changes in brain structural connectivity. This strain often presents with callosal abnormalities, along with the Probst and the Sigmoid bundles, making it is an attractive animal model for CCD and LDP in general. Our results also show that even the C57bl6 strain, which typically serves as a normotypical control animal in a myriad of studies, presents sigmoid-fashion pattern fibers laid out in the brain. These results suggest that these aberrant fiber pathways may not necessarily be a pathological hallmark, but instead an alternative roadmap for misguided axons. Such findings offer new insights for interpreting the significance of CCD-associated LDP in humans.

A resource for the detailed 3D mapping of white matter pathways in the marmoset brain.

While the fundamental importance of the white matter in supporting neuronal communication is well known, existing publications of primate brains do not feature a detailed description of its complex anatomy. The main barrier to achieving this is that existing primate neuroimaging data have insufficient spatial resolution to resolve white matter pathways fully. Here we present a resource that allows detailed descriptions of white matter structures and trajectories of fiber pathways in the marmoset brain. The resource includes: (1) the highest-resolution diffusion-weighted MRI data available to date, which reveal white matter features not previously described; (2) a comprehensive three-dimensional white matter atlas depicting fiber pathways that were either omitted or misidentified in previous atlases; and (3) comprehensive fiber pathway maps of cortical connections combining diffusion-weighted MRI tractography and neuronal tracing data. The resource, which can be downloaded from marmosetbrainmapping.org, will facilitate studies of brain connectivity and the development of tractography algorithms in the primate brain.

Dynamic Interhemispheric Desynchronization in Marmosets and Humans With Disorders of the Corpus Callosum.

The corpus callosum, the principal structural avenue for interhemispheric neuronal communication, controls the brain's lateralization. Developmental malformations of the corpus callosum (CCD) can lead to learning and intellectual disabilities. Currently, there is no clear explanation for these symptoms. Here, we used resting-state functional MRI (rsfMRI) to evaluate the dynamic resting-state functional connectivity (rsFC) in both the cingulate cortex (CG) and the sensory areas (S1, S2, A1) in three marmosets (Callithrix jacchus) with spontaneous CCD. We also performed rsfMRI in 10 CCD human subjects (six hypoplasic and four agenesic). We observed no differences in the strength of rsFC between homotopic CG and sensory areas in both species when comparing them to healthy controls. However, in CCD marmosets, we found lower strength of quasi-periodic patterns (QPP) correlation in the posterior interhemispheric sensory areas. We also found a significant lag of interhemispheric communication in the medial CG, suggesting asynchrony between the two hemispheres. Correspondingly, in human subjects, we found that the CG of acallosal subjects had a higher QPP correlation than controls. In comparison, hypoplasic subjects had a lower QPP correlation and a delay of 1.6 s in the sensory regions. These results show that CCD affects the interhemispheric synchrony of both CG and sensory areas and that, in both species, its impact on cortical communication varies along the CC development gradient. Our study shines a light on how CCD misconnects homotopic regions and opens a line of research to explain the causes of the symptoms exhibited by CCD patients and how to mitigate them.

Anatomical and functional investigation of the marmoset default mode network.

The default mode network (DMN) is associated with a wide range of brain functions. In humans, the DMN is marked by strong functional connectivity among three core regions: medial prefrontal cortex (mPFC), posterior parietal cortex (PPC), and the medial parietal and posterior cingulate cortex (PCC). Neuroimaging studies have shown that the DMN also exists in non-human primates, suggesting that it may be a conserved feature of the primate brain. Here, we found that, in common marmosets, the dorsolateral prefrontal cortex (dlPFC; peak at A8aD) has robust fMRI functional connectivity and reciprocal anatomical connections with the posterior DMN core regions (PPC and PCC), while the mPFC has weak connections with the posterior DMN core regions. This strong dlPFC but weak mPFC connectivity in marmoset differs markedly from the stereotypical DMN in humans. The mPFC may be involved in brain functions that are further developed in humans than in other primates.

The Absolute Number of Oligodendrocytes in the Adult Mouse Brain.

The central nervous system is a highly complex network composed of various cell types, each one with different subpopulations. Each cell type has distinct roles for the functional operation of circuits, and ultimately, for brain physiology in general. Since the absolute number of each cell type is considered a proxy of its functional complexity, one approach to better understand how the brain works is to unravel its absolute cellularity and the quantitative relations between cell populations; in other words, how one population of cells is quantitatively structured, in relation to another. Oligodendrocytes are one of these cell types - mainly, they provide electric insulation to axons, optimizing action potential conduction. Their function has recently been revisited and their role extended, one example being their capability of providing trophic support to long axons. To determine the absolute cellularity of oligodendroglia, we have developed a protocol of oligodendrocyte quantification using the isotropic fractionator with a pan-marker for this cell type. We report a detailed assessment of specificity and universality of the oligodendrocyte transcription factor 2 (Olig2), through systematic confocal analyses of the C57BL/6 mouse brain. In addition, we have determined the absolute number (17.4 million) and proportion (about 20%) of this cell type in the brain (and in different brain regions), and tested if this population, at the intraspecific level, scales with the number of neurons in an allometric-based approach. Considering these numbers, oligodendrocytes proved to be the most numerous of glial cells in the mouse brain.

Perinatal Asphyxia and Brain Development: Mitochondrial Damage Without Anatomical or Cellular Losses.

Perinatal asphyxia remains a significant cause of neonatal mortality and is associated with long-term neurodegenerative disorders. In the present study, we evaluated cellular and subcellular damages to brain development in a model of mild perinatal asphyxia. Survival rate in the experimental group was 67%. One hour after the insult, intraperitoneally injected Evans blue could be detected in the fetuses' brains, indicating disruption of the blood-brain barrier. Although brain mass and absolute cell numbers (neurons and non-neurons) were not reduced after perinatal asphyxia immediately and in late brain development, subcellular alterations were detected. Cortical oxygen consumption increased immediately after asphyxia, and remained high up to 7 days, returning to normal levels after 14 days. We observed an increased resistance to mitochondrial membrane permeability transition, and calcium buffering capacity in asphyxiated animals from birth to 14 days after the insult. In contrast to ex vivo data, mitochondrial oxygen consumption in primary cell cultures of neurons and astrocytes was not altered after 1% hypoxia. Taken together, our results demonstrate that although newborns were viable and apparently healthy, brain development is subcellularly altered by perinatal asphyxia. Our findings place the neonate brain mitochondria as a potential target for therapeutic protective interventions.

Cell number changes in Alzheimer's disease relate to dementia, not to plaques and tangles.

Alzheimer's disease is the commonest cause of dementia in the elderly, but its pathological determinants are still debated. Amyloid-ß plaques and neurofibrillary tangles have been implicated either directly as disruptors of neural function, or indirectly by precipitating neuronal death and thus causing a reduction in neuronal number. Alternatively, the initial cognitive decline has been attributed to subtle intracellular events caused by amyloid-ß oligomers, resulting in dementia after massive synaptic dysfunction followed by neuronal degeneration and death. To investigate whether Alzheimer's disease is associated with changes in the absolute cell numbers of ageing brains, we used the isotropic fractionator, a novel technique designed to determine the absolute cellular composition of brain regions. We investigated whether plaques and tangles are associated with neuronal loss, or whether it is dementia that relates to changes of absolute cell composition, by comparing cell numbers in brains of patients severely demented with those of asymptomatic individuals-both groups histopathologically diagnosed as Alzheimer's-and normal subjects with no pathological signs of the disease. We found a great reduction of neuronal numbers in the hippocampus and cerebral cortex of demented patients with Alzheimer's disease, but not in asymptomatic subjects with Alzheimer's disease. We concluded that neuronal loss is associated with dementia and not the presence of plaques and tangles, which may explain why subjects with histopathological features of Alzheimer's disease can be asymptomatic; and exclude amyloid-ß deposits as causes for the reduction of neuronal numbers in the brain. We found an increase of non-neuronal cell numbers in the cerebral cortex and subcortical white matter of demented patients with Alzheimer's disease when compared with asymptomatic subjects with Alzheimer's disease and control subjects, suggesting a reactive glial cell response in the former that may be related to the symptoms they present.

Automatic isotropic fractionation for large-scale quantitative cell analysis of nervous tissue.

Isotropic fractionation is a quantitative technique that allows reliable estimates of absolute numbers of neuronal and non-neuronal brain cells. However, being fast for single small brains, it requires a long time for processing large brains or many small ones, if done manually. To solve this problem, we developed a machine to automate the method, and tested its efficiency, consistency, and reliability as compared with manual processing. The machine consists of a set of electronically controlled rotation and translation motors coupled to tissue grinders, which automatically transform fixed tissue into homogeneous nuclei suspensions. Speed and torque of the motors can be independently regulated by electronic circuits, according to the volume of tissue being processed and its mechanical resistance to fractionation. To test the machine, twelve paraformaldehyde-fixed rat brains and eight human cerebella were separated into two groups, respectively: one processed automatically and the other, manually. Both pairs of groups (rat and human tissue) followed the same, published protocol of the method. We compared the groups according to nuclei morphology, degree of clustering and number of cells. The machine proved superior for yielding faster results due to simultaneous processing in multiple grinders. Quantitative analysis of machine-processed tissue resulted in similar average numbers of total brain cells, neurons, and non-neuronal cells, statistically similar to the manually processed tissue and equivalent to previously published data. We concluded that the machine is more efficient because it utilizes many homogenizers simultaneously, equally consistent in producing high quality material for counting, and quantitatively reliable as compared to manual processing.