Pasko Rakic

In our companion paper, in this issue, we describe our efforts to provide embryonic rhesus macaque ( Macaca mulatta ) brain histology for the study of brain development, with emphasis on cortical development. Here we continue our description of efforts to provide postnatal rhesus macaque brain histology relevant for the study of cellular circuits which continue to mature and change well into postnatal life, in males and females, and which naturally deteriorate in the elderly. The mission of the MacBrain Resource Center (MBRC) in the Department of Neuroscience at Yale University School of Medicine is to provide a cost‐effective means for researchers to conduct de novo studies on this non‐human primate (NHP) brain animal model using materials already in existence and therefore without exorbitant costs and without having to sacrifice additional animals ( https://medicine.yale.edu/neuroscience/macbrain/mission/ ). Here we report on how this mission is being accomplished. Because MBRC materials have been and continue to be gathered from unrelated studies over many years, most methods have already been published. The MBRC divides different types of materials into separate Collections. The present description of histo‐ and immunohistochemical processes is limited to current work that provides materials to populate Collection 6 and is accurate as of May 2026. Collections in the MBRC are dynamic. Of the 8 current MBRC datasets, here we emphasize Collections 5, 6, and 7 and illustrate through examples how different materials are currently being used to conduct research both in cortical and subcortical structures. Many of the electron microscopy (EM) blocks in Collection 5 sampling the brain at numerous regions come from the >100 cases of titrated thymidine ( ³ H‐TdR) injections in Collection 1 in addition to cases in Collections 2 and 3. Altogether, at present there are ~1000 inventoried EM blocks collected from postnatal cases. Collection 6 currently contains >30,000 digital images illustrating 35 different cellular and fiber markers in 32 brains of both sexes ranging from P0 to 32 years of age. Materials in Collections 6 and 7 keep growing as we constantly process and add NHP brains to them. Based on the molecular, genetic, and anatomical similarities between this animal model and human, we underline the importance of archiving and (re‐)using rhesus macaque brains to foster neuroscience research. As far as we know the NHP brain materials in the MBRC Collections constitute the largest datasets of their kind in the world.

The hippocampus participates in crucial functions such as memory consolidation, spatial processing and emotional regulation that require diverse input from multiple cortical areas that is funneled through the upper layers of the entorhinal cortex (EC), mostly from layer II to the dentate gyrus (DG). Traditional models described 200,000 EC layer II neurons projecting to 1 million granule cells (GCs) in the rat, rendering low divergence (1:5), with each EC neuron establishing about 18,000 synapses with GCs and each GC receiving about 4,000 synapses from EC neurons. In this manuscript, we update this model of connectivity incorporating new features described in the last three decades that include updated populations of EC layer II neurons obtained with design-based stereology, a revised definition of EC layer II based on molecular criteria and selecting reelin expressing neurons as the only layer II neurons projecting to the hippocampus. The updated model shows ~80,000 neurons from EC layer II projecting to the DG, ~45,000 from the medial entorhinal cortex (MEC) and ~35,000 from the lateral entorhinal cortex (LEC) with high divergence of 1:20 and 1:30. We also show that EC layer II neurons may establish ~90,000-115,000 synapses on GCs, while GCs receive about 8,000 synapses from EC layer II neurons. We estimate a ~25% redundancy in the connectivity, so each EC neuron may contact ~68,000-86,000 GCs and each GC would be contacted by ~3000 neurons from MEC and 3,000 from LEC. In addition, we quantitatively assess a potential projection of mossy cells to the medial molecular layer described in mice, which could have a potential impact on GC inhibition. Overall, we produced a detailed, complete, and updated quantitative model of EC projections to the DG that reveals a much more divergent and richer projection than previously described, with implications for functional models (e.g.: pattern separation) and more widely for building realistic hippocampal models or establishing comparisons across species.

Datasets for the study of developing Non‐Human Primate (NHP) brain are scarce. Some of the reasons are related to the financial and technical obstacles that make the production of these datasets logistically challenging. NHP embryos of different and verified developmental ages require timed pregnancies and the maintenance of expensive and highly regulated breeding colonies. Here, we document our efforts to provide digital datasets of rhesus macaque ( Macaca mulatta ) embryonic brain histological series of sections that can be used and reused for research. These efforts fall well within the primary mission of the MacBrain Resource Center (MBRC, https://medicine.yale.edu/neuroscience/macbrain/mission/ ), which targets a reduction in the need to sacrifice animals, improved efficiency in the use of resources, the use of male (M) and female (F) specimens to conduct developmental research, and increase archival and sharing of materials for the advancement of neuroscience. First, we describe the already existent tritiated thymidine ( ³ H‐TdR) cases in MBRC Collection 1 and the electron microscopy (EM) blocks of Collection 5, which together contain >100 specimens of different developmental ages. We also report methodological details on, as of July 2025, n = 24 histo‐ and immunohistochemically processed embryonic brains that enrich Collection 6 with thousands of coronal sections stained for n = 38 different cellular and fiber markers. These datasets provide NHP brain histo‐ and immunohistology for comparison with other species and hence help close a gap in the availability of suitable materials for comparative evolution and neurodevelopmental studies. Through examples, we illustrate how different materials are currently being used in de novo research. Finally, we advocate the absolute need to continue using NHPs in the study of neurodevelopment because of the unparalleled molecular, genetic, and anatomical similarities between these animal models and humans.

Human and non‐human primates (NHPs) have a distinct lamination pattern of their primary visual cortex (V1) where layer 4 can be separated into four well‐defined sub‐laminae. Brodmann (1909) classified these into layers 4A, 4B, and 4C. Layer 4C can be further divided into 4Cα and 4Cβ based on inputs from the lateral geniculate magnocellular and parvocellular layers, respectively (Hubel & Wiesel, 1972; Lund, 1973). We have examined the lamination pattern of layer 5 and have found that this layer has a trilaminar structure and have denoted the sublayers as layers 5Aα, 5Aβ, and 5B. This trilaminar division is prominent within the opercular region of the occipital cortex but is usually absent from V1 surrounding the calcarine fissure. In addition, with age we find the trilaminar arrangement of layer 5 within the opercular region interdigitates with a bilaminar (denoted as layers 5Aβ, 5B) pattern. We also show that in a bilaterally enucleated macaque, at embryonic age of 63 days, postnatally, layer 5 is also trilaminar around the occipital cortex and bilaminar around the calcarine fissure. These facts imply that the interaction of the geniculo‐cortical input and local cortical development takes place in the absence of any visual experience, and we propose, functionally that this trilaminar arrangement of layer 5 may play a part in processing of central vision.

Here, to enable researchers to more fully harness the collective discovery potential of multiomic data in the public domain, we have assembled gene-level transcriptomic data from ~200 studies of neocortical development and in vitro models. Applying joint matrix decomposition to mouse, macaque and human data, we define transcriptome dynamics that are conserved across neocortical neurogenesis and identify a program that emerges in ventricular progenitors, is later expressed in neurogenic outer, or basal, radial glia of primates, but is limited to gliogenic precursors in the rodent. Decomposition of adult human neocortical data identified layer-specific signatures in excitatory neurons, enabling the charting of their developmental emergence and protracted maturation, which is in stark contrast to the early peaking expression of layer-defining transcription factors. Interrogation of data from cerebral organoids demonstrated that, although broad elements of in vivo development are recapitulated in vitro, many layer-specific transcriptomic programs in neuronal maturation are absent. We invite cell biologists without coding expertise to use NeMO Analytics in their research and to fuel it with their own emerging data at nemoanalytics.org/landing/neocortex.

The goal of this review is to bring to the attention of the scientific community the opportunities of transmission electron microscopy for analyses of biological subjects and resolving complicated cases of data interpretation. Although procedures for electron microscopy are in general more elaborate (particularly for simultaneous immunolabeling of multiple antigens) compared to fluorescent microscopy, they can help view cellular morpho-functional features undetectable using other methods. In this review, we consider several unexpected and serendipitous discoveries made in our laboratory and fulfilled using unique opportunities provided by electron microscopy of ultrathin sections. We are deliberating the following topics: interpretation of unusual results of immunolabeling; a novel method for in situ identification of cells undergoing mitochondrial disorder and necrosis-like death; the sequence of organelles’ reorganization in dying cells; simultaneous rupture of nuclear and plasma membranes in migrating neurons; and the role of cytoskeleton in lateral expansion of the cerebral cortex.

The NeuroEXPLORER (NX), a next generation brain-dedicated PET scanner, has demonstrated improved spatial resolution and sensitivity compared with previous PET scanners. In this study, we performed synaptic vesicle glycoprotein 2A (SV2A) PET, an in vivo marker of synaptic density, to evaluate the performance of the NX in both fetal and maternal brains of four pregnant rhesus macaques. Results were compared with the previously acquired PET images using miniEXPLORER and Biograph mCT for fetal and maternal brains (four pregnant monkeys per scanner). The NX demonstrated superior performance compared with the other scanners, providing improved image clarity and higher distribution volume ratios (DVRs), which were significant in the occipital region and putamen of the fetal brain ( p < 0.05, FDR uncorrected), as well as across all regions in the maternal brain ( p < 0.05, FDR corrected). The percentage difference in mean regional DVR between maternal and fetal brains ranged from 36.1% in the amygdala to 78.3% in the occipital lobe in the NX, which may reflect regional differences in the phase of synaptic development. This study highlights the NX as a promising non-invasive tool for investigating in utero synaptic development. The presence of in utero motion emphasizes the necessity of motion correction for reliable PET quantification.

The hippocampal formation is a functional entity that includes the hippocampus, subicular complex, and the entorhinal cortex, and has an essential role in learning and memory, emotional processing, and spatial coding. The well‐defined structure of hippocampal fields and the segregation of the connections have made this structure a favorite candidate for functional models that rely on fundamental information such as the number of neurons populating the hippocampal fields. Existing models on the rat rely on neuronal populations obtained from single studies, so we aimed to obtain more representative estimates by analyzing all available data. We identified 89 studies using reliable methodology that provided 264 stereological estimates of principal neuron populations. The resulting averages for males showed 1,000,000 neurons for the granule cell layer (GCL); 50,000 for the hilus; 210,000 for CA3; ~30,000 for CA2; 350,000 for CA1; and 300,000 for the Subiculum. Entorhinal cortex (EC) averages for both sexes showed 108,000 neurons in layer II; 270,000 in layer III; and 340,000 in layer V/VI. Most of those estimates are significantly different from those traditionally used in hippocampal models (e.g., ~2‐fold difference in EC layer II), revealing an updated architecture of the rat hippocampal formation that might help build more realistic models of hippocampal connectivity and function. Comparisons by age or sex were not reliable given the scarce data available from adolescents or females, while comparisons by strain showed inconsistent results, with similar populations in most fields but significant differences in CA3/CA2. The reliability of this finding is discussed.

Understanding the unique features of the human brain compared to non-human primates has long intrigued humankind. The cerebellum refines motor coordination and cognitive functions, contributing to the evolutionary development of human adaptability and dexterity. To identify shared and divergent features across primates, we conducted single-nucleus transcriptomic and chromatin accessibility profiling of the adult cerebellar cortex in humans, chimpanzees, macaques, and marmosets. We revealed human-specific transcriptomic and regulatory features, particularly those involved in synaptogenesis. Notably, we identified an enrichment of the sperm receptor zona pellucida glycoprotein 2 (ZP2) and its potential interactors, known for their roles in gamete interaction, in human granule cells. Experimental data show that ZP2 expression in human granule cells is induced by pontine mossy fibers, reducing synaptic proteins at pontocerebellar glomerular synapses, and decreasing cerebellar neuron electrophysiological activity. This unexpected co-option of ZP2 in human-specific synapse regulation provides insights into the evolutionary specialization of the human cerebellum.

The implications of the early phases of human telencephalic development, involving neural stem cells (NSCs), in the etiology of cortical disorders remain elusive. Here, we explore the expression dynamics of cortical and neuropsychiatric disorder-associated genes in datasets generated from human NSCs across telencephalic fate transitions in vitro and in vivo. We identify risk genes expressed in brain organizers and sequential gene regulatory networks throughout corticogenesis, revealing disease-specific critical phases when NSCs may be more vulnerable to gene dysfunction and converging signaling across multiple diseases. Further, we simulate the impact of risk transcription factor (TF) depletions on neural cell trajectories traversing human corticogenesis and observe a spatiotemporal-dependent effect for each perturbation. Finally, single-cell transcriptomics of autism-affected patient-derived NSCs in vitro reveals recurrent expression alteration of TFs orchestrating brain patterning and NSC lineage commitment. This work opens perspectives to explore human brain dysfunction at early phases of development.