Single-cell spatial transcriptome reveals cell-type organization in the macaque cortex.

Elucidating the cellular organization of the cerebral cortex is critical for understanding brain structure and function. Using large-scale single-nucleus RNA sequencing and spatial transcriptomic analysis of 143 macaque cortical regions, we obtained a comprehensive atlas of 264 transcriptome-defined cortical cell types and mapped their spatial distribution across the entire cortex. We characterized the cortical layer and region preferences of glutamatergic, GABAergic, and non-neuronal cell types, as well as regional differences in cell-type composition and neighborhood complexity. Notably, we discovered a relationship between the regional distribution of various cell types and the region's hierarchical level in the visual and somatosensory systems. Cross-species comparison of transcriptomic data from human, macaque, and mouse cortices further revealed primate-specific cell types that are enriched in layer 4, with their marker genes expressed in a region-dependent manner. Our data provide a cellular and molecular basis for understanding the evolution, development, aging, and pathogenesis of the primate brain.

Vaccine protection against the SARS-CoV-2 Omicron variant in macaques.

The rapid spread of the SARS-CoV-2 Omicron (B.1.1.529) variant, including in highly vaccinated populations, has raised important questions about the efficacy of current vaccines. In this study, we show that the mRNA-based BNT162b2 vaccine and the adenovirus-vector-based Ad26.COV2.S vaccine provide robust protection against high-dose challenge with the SARS-CoV-2 Omicron variant in cynomolgus macaques. We vaccinated 30 macaques with homologous and heterologous prime-boost regimens with BNT162b2 and Ad26.COV2.S. Following Omicron challenge, vaccinated macaques demonstrated rapid control of virus in bronchoalveolar lavage, and most vaccinated animals also controlled virus in nasal swabs. However, 4 vaccinated animals that had moderate Omicron-neutralizing antibody titers and undetectable Omicron CD8+ T cell responses failed to control virus in the upper respiratory tract. Moreover, virologic control correlated with both antibody and T cell responses. These data suggest that both humoral and cellular immune responses contribute to vaccine protection against a highly mutated SARS-CoV-2 variant.

3D Genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis.

Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.

Representational geometry of perceptual decisions in the monkey parietal cortex.

Lateral intraparietal (LIP) neurons represent formation of perceptual decisions involving eye movements. In circuit models for these decisions, neural ensembles that encode actions compete to form decisions. Consequently, representation and readout of the decision variables (DVs) are implemented similarly for decisions with identical competing actions, irrespective of input and task context differences. Further, DVs are encoded as partially potentiated action plans through balance of activity of action-selective ensembles. Here, we test those core principles. We show that in a novel face-discrimination task, LIP firing rates decrease with supporting evidence, contrary to conventional motion-discrimination tasks. These opposite response patterns arise from similar mechanisms in which decisions form along curved population-response manifolds misaligned with action representations. These manifolds rotate in state space based on context, indicating distinct optimal readouts for different tasks. We show similar manifolds in lateral and medial prefrontal cortices, suggesting similar representational geometry across decision-making circuits.

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.

Cloning of Macaque Monkeys by Somatic Cell Nuclear Transfer.

Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2 healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42 surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts.

A Common Space Approach to Comparative Neuroscience.

Comparative neuroscience is entering the era of big data. New high-throughput methods and data-sharing initiatives have resulted in the availability of large, digital data sets containing many types of data from ever more species. Here, we present a framework for exploiting the new possibilities offered. The multimodality of the data allows vertical translations, which are comparisons of different aspects of brain organization within a single species and across scales. Horizontal translations compare particular aspects of brain organization across species, often by building abstract feature spaces. Combining vertical and horizontal translations allows for more sophisticated comparisons, including relating principles of brain organization across species by contrasting horizontal translations, and for making formal predictions of unobtainable data based on observed results in a model species.

Inactivation of face-selective neurons alters eye movements when free viewing faces.

During free viewing, faces attract gaze and induce specific fixation patterns corresponding to the facial features. This suggests that neurons encoding the facial features are in the causal chain that steers the eyes. However, there is no physiological evidence to support a mechanistic link between face-encoding neurons in high-level visual areas and the oculomotor system. In this study, we targeted the middle face patches of the inferior temporal (IT) cortex in two macaque monkeys using an functional magnetic resonance imaging (fMRI) localizer. We then utilized muscimol microinjection to unilaterally suppress IT neural activity inside and outside the face patches and recorded eye movements while the animals free viewing natural scenes. Inactivation of the face-selective neurons altered the pattern of eye movements on faces: The monkeys found faces in the scene but neglected the eye contralateral to the inactivation hemisphere. These findings reveal the causal contribution of the high-level visual cortex in eye movements.

Longitudinal mapping of the development of cortical thickness and surface area in rhesus macaques during the first three years.

Studying dynamic spatiotemporal patterns of early brain development in macaque monkeys is critical for understanding the cortical organization and evolution in humans, given the phylogenetic closeness between humans and macaques. However, due to huge challenges in the analysis of early brain Magnetic Resonance Imaging (MRI) data typically with extremely low contrast and dynamic imaging appearances, our knowledge of the early macaque cortical development remains scarce. To fill this critical gap, this paper characterizes the early developmental patterns of cortical thickness and surface area in rhesus macaques by leveraging advanced computing tools tailored for early developing brains based on a densely sampled longitudinal dataset with 140 rhesus macaque MRI scans seamlessly covering from birth to 36 mo of age. The average cortical thickness exhibits an inverted U-shaped trajectory with peak thickness at around 4.3 mo of age, which is remarkably in line with the age of peak thickness at 14 mo in humans, considering the around 3:1 age ratio of human to macaque. The total cortical surface area in macaques increases monotonically but with relatively lower expansions than in humans. The spatial distributions of thicker and thinner regions are quite consistent during development, with gyri having a thicker cortex than sulci. By 4 mo of age, over 81% of cortical vertices have reached their peaks in thickness, except for the insula and medial temporal cortices, while most cortical vertices keep expanding in surface area, except for the occipital cortex. These findings provide important insights into early brain development and evolution in primates.

Encoding of 3D physical dimensions by face-selective cortical neurons.

Neurons throughout the primate inferior temporal (IT) cortex respond selectively to visual images of faces and other complex objects. The response magnitude of neurons to a given image often depends on the size at which the image is presented, usually on a flat display at a fixed distance. While such size sensitivity might simply reflect the angular subtense of retinal image stimulation in degrees, one unexplored possibility is that it tracks the real-world geometry of physical objects, such as their size and distance to the observer in centimeters. This distinction bears fundamentally on the nature of object representation in IT and on the scope of visual operations supported by the ventral visual pathway. To address this question, we assessed the response dependency of neurons in the macaque anterior fundus (AF) face patch to the angular versus physical size of faces. We employed a macaque avatar to stereoscopically render three-dimensional (3D) photorealistic faces at multiple sizes and distances, including a subset of size/distance combinations designed to cast the same size retinal image projection. We found that most AF neurons were modulated principally by the 3D physical size of the face rather than its two-dimensional (2D) angular size on the retina. Further, most neurons responded strongest to extremely large and small faces, rather than to those of normal size. Together, these findings reveal a graded encoding of physical size among face patch neurons, providing evidence that category-selective regions of the primate ventral visual pathway participate in a geometric analysis of real-world objects.

Comparative connectomics reveals noncanonical wiring for color vision in human foveal retina.

The Old World macaque monkey and New World common marmoset provide fundamental models for human visual processing, yet the human ancestral lineage diverged from these monkey lineages over 25 Mya. We therefore asked whether fine-scale synaptic wiring in the nervous system is preserved across these three primate families, despite long periods of independent evolution. We applied connectomic electron microscopy to the specialized foveal retina where circuits for highest acuity and color vision reside. Synaptic motifs arising from the cone photoreceptor type sensitive to short (S) wavelengths and associated with "blue-yellow" (S-ON and S-OFF) color-coding circuitry were reconstructed. We found that distinctive circuitry arises from S cones for each of the three species. The S cones contacted neighboring L and M (long- and middle-wavelength sensitive) cones in humans, but such contacts were rare or absent in macaques and marmosets. We discovered a major S-OFF pathway in the human retina and established its absence in marmosets. Further, the S-ON and S-OFF chromatic pathways make excitatory-type synaptic contacts with L and M cone types in humans, but not in macaques or marmosets. Our results predict that early-stage chromatic signals are distinct in the human retina and imply that solving the human connectome at the nanoscale level of synaptic wiring will be critical for fully understanding the neural basis of human color vision.

Neuronal Population Encoding of Identity in Primate Prefrontal Cortex.

The ventrolateral prefrontal cortex (VLPFC) shows robust activation during the perception of faces and voices. However, little is known about what categorical features of social stimuli drive neural activity in this region. Since perception of identity and expression are critical social functions, we examined whether neural responses to naturalistic stimuli were driven by these two categorical features in the prefrontal cortex. We recorded single neurons in the VLPFC, while two male rhesus macaques (Macaca mulatta) viewed short audiovisual videos of unfamiliar conspecifics making expressions of aggressive, affiliative, and neutral valence. Of the 285 neurons responsive to the audiovisual stimuli, 111 neurons had a main effect (two-way ANOVA) of identity, expression, or their interaction in their stimulus-related firing rates; however, decoding of expression and identity using single-unit firing rates rendered poor accuracy. Interestingly, when decoding from pseudo-populations of recorded neurons, the accuracy for both expression and identity increased with population size, suggesting that the population transmitted information relevant to both variables. Principal components analysis of mean population activity across time revealed that population responses to the same identity followed similar trajectories in the response space, facilitating segregation from other identities. Our results suggest that identity is a critical feature of social stimuli that dictates the structure of population activity in the VLPFC, during the perception of vocalizations and their corresponding facial expressions. These findings enhance our understanding of the role of the VLPFC in social behavior.

Pupillary Responses Reflect Dynamic Changes in Multiple Cognitive Factors During Associative Learning in Primates.

Associative learning involves complex interactions of multiple cognitive factors. While adult subjects can articulate these factors verbally, for model animals such as macaques, we rely on behavioral outputs. In our study, we used pupillary responses as an alternative measure to capture these underlying cognitive changes. We recorded the dynamic changes in the pupils of three male macaques when they learned the associations between visual stimuli and reward sizes under the classical Pavlovian experimental paradigm. We found that during the long-term learning process, the gradual changes in the pupillary response reflect the changes in the cognitive state of the animals. The pupillary response can be explained by a linear combination of components corresponding to multiple cognitive factors. These components reflect the impact of visual stimuli on the pupils, the prediction of reward values associated with the visual stimuli, and the macaques' understanding of the current experimental reward rules. The changing patterns of these factors during interday and intraday learning clearly demonstrate the enhancement of current reward-stimulus association and the weakening of previous reward-stimulus association. Our study shows that the dynamic response of pupils can serve as an objective indicator to characterize the psychological changes of animals, understand their learning process, and provide important tools for exploring animal behavior during the learning process.

Value-Based Search Efficiency Is Encoded in the Substantia Nigra Reticulata Firing Rate, Spiking Irregularity and Local Field Potential.

Recent results show that valuable objects can pop out in visual search, yet its neural mechanisms remain unexplored. Given the role of substantia nigra reticulata (SNr) in object value memory and control of gaze, we recorded its single-unit activity while male macaque monkeys engaged in efficient or inefficient search for a valuable target object among low-value objects. The results showed that efficient search was concurrent with stronger inhibition and higher spiking irregularity in the target-present (TP) compared with the target-absent (TA) trials in SNr. Importantly, the firing rate differentiation of TP and TA trials happened within ∼100 ms of display onset, and its magnitude was significantly correlated with the search times and slopes (search efficiency). Time-frequency analyses of local field potential (LFP) after display onset revealed significant modulations of the gamma band power with search efficiency. The greater reduction of SNr firing in TP trials in efficient search can create a stronger disinhibition of downstream superior colliculus, which in turn can facilitate saccade to obtain valuable targets in competitive environments.

Macaque antibodies targeting Marburg virus glycoprotein induced by multivalent immunization.

Marburg virus infection in humans is associated with case fatality rates that can reach up to 90%, but to date, there are no approved vaccines or monoclonal antibody (mAb) countermeasures. Here, we immunized Rhesus macaques with multivalent combinations of filovirus glycoprotein (GP) antigens belonging to Marburg, Sudan, and Ebola viruses to generate monospecific and cross-reactive antibody responses against them. From the animal that developed the highest titers of Marburg virus GP-specific neutralizing antibodies, we sorted single memory B cells using a heterologous Ravn virus GP probe and cloned and characterized a panel of 34 mAbs belonging to 28 unique lineages. Antibody specificities were assessed by overlapping pepscan and binding competition analyses, revealing that roughly a third of the lineages mapped to the conserved receptor binding region, including potent neutralizing lineages that were confirmed by negative stain electron microscopy to target this region. Additional lineages targeted a protective region on GP2, while others were found to possess cross-filovirus reactivity. Our study advances the understanding of orthomarburgvirus glycoprotein antigenicity and furthers efforts to develop candidate antibody countermeasures against these lethal viruses. IMPORTANCE: Marburg viruses were the first filoviruses characterized to emerge in humans in 1967 and cause severe hemorrhagic fever with average case fatality rates of ~50%. Although mAb countermeasures have been approved for clinical use against the related Ebola viruses, there are currently no approved countermeasures against Marburg viruses. We successfully isolated a panel of orthomarburgvirus GP-specific mAbs from a macaque immunized with a multivalent combination of filovirus antigens. Our analyses revealed that roughly half of the antibodies in the panel mapped to regions on the glycoprotein shown to protect from infection, including the host cell receptor binding domain and a protective region on the membrane-anchoring subunit. Other antibodies in the panel exhibited broad filovirus GP recognition. Our study describes the discovery of a diverse panel of cross-reactive macaque antibodies targeting orthomarburgvirus and other filovirus GPs and provides candidate immunotherapeutics for further study and development.

Neuronal composition of processing modules in human V1: laminar density for neuronal and non-neuronal populations and a comparison with macaque.

The neuronal composition of homologous brain regions in different primates is important for understanding their processing capacities. Primary visual cortex (V1) has been widely studied in different members of the catarrhines. Neuronal density is considered to be central in defining the structure-function relationship. In human, there are large variations in the reported neuronal density from prior studies. We found the neuronal density in human V1 was 79,000 neurons/mm3, which is 35% of the neuronal density previously determined in macaque V1. Laminar density was proportionally similar between human and macaque. In V1, the ocular dominance column (ODC) contains the circuits for the emergence of orientation preference and spatial processing of a point image in many mammalian species. Analysis of the total neurons in an ODC and of the full number of neurons in macular vision (the central 15°) indicates that humans have 1.3× more neurons than macaques even though the density of neurons in macaque is 3× the density in human V1. We propose that the number of neurons in a functional processing unit rather than the number of neurons under a mm2 of cortex is more appropriate for cortical comparisons across species.

Effects of multiple anesthetic exposures on rhesus macaque brain development: a longitudinal structural MRI analysis.

Concerns about the potential neurotoxic effects of anesthetics on developing brain exist. When making clinical decisions, the timing and dosage of anesthetic exposure are critical factors to consider due to their associated risks. In our study, we investigated the impact of repeated anesthetic exposures on the brain development trajectory of a cohort of rhesus monkeys (n = 26) over their first 2 yr of life, utilizing longitudinal magnetic resonance imaging data. We hypothesized that early or high-dose anesthesia exposure could negatively influence structural brain development. By employing the generalized additive mixed model, we traced the longitudinal trajectories of brain volume, cortical thickness, and white matter integrity. The interaction analysis revealed that age and cumulative anesthetic dose were variably linked to white matter integrity but not to morphometric measures. Early high-dose exposure was associated with increased mean, axial, and radial diffusivities across all white matter regions, compared to late-low-dose exposure. Our findings indicate that early or high-dose anesthesia exposure during infancy disrupts structural brain development in rhesus monkeys. Consequently, the timing of elective surgeries and procedures that require anesthesia for children and pregnant women should be strategically planned to account for the cumulative dose of volatile anesthetics, aiming to minimize the potential risks to brain development.

Lateral expansion of the mammalian cerebral cortex is related to anchorage of centrosomes in apical neural progenitors.

The centrosome is the main microtubule organizing center in stem cells, and its mother centriole, anchored to the cell membrane, serves as the basal body of the primary cilium. Prolonged anchorage of centrosomes and primary cilia to the apical segment of the membrane of apical neural progenitor cells is considered vital for interkinetic nuclear translocation and repetitive cycling in the ventricular zone. In contrast, the basolateral anchorage of primary cilia has been regarded as the first step in delamination and conversion of apical to basal neural progenitor cells or neurons. Using electron microscopy analysis of serial sections, we show that centrosomes, in a fraction of cells, anchor to the basolateral cell membrane immediately after cell division and before development of cilia. In other cells, centrosomes situate freely in the cytoplasm, increasing their probability of subsequent apical anchorage. In mice, anchored centrosomes in the cells shortly after mitosis predominate during the entire cerebral neurogenesis, whereas in macaque monkeys, cytoplasmic centrosomes are more numerous. Species-specific differences in the ratio of anchored and free cytoplasmic centrosomes appear to be related to prolonged neurogenesis in the ventricular zone that is essential for lateral expansion of the cerebral cortex in primates.

Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques.

Human T cell leukemia/T-lymphotropic virus type 1 (HTLV-1) infection occurs by cell-to-cell transmission and can induce fatal adult T cell leukemia. Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of 10 vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the 2 vaccinated macaques without NAb induction and 10 unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.