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The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties 1 – 3 . Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions—in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.  A transcriptomic cell-type atlas of the whole adult mouse brain with ~5,300 clusters built from single-cell and spatial transcriptomic datasets with more than eight million cells reveals remarkable cell type diversity across the brain and unique cell type characteristics of different brain regions. 

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals 1 . Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations. An examination of motor cortex in humans, marmosets and mice reveals a generally conserved cellular makeup that is likely to extend to many mammalian species, but also differences in gene expression, DNA methylation and chromatin state that lead to species-dependent specializations.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain 1 – 3 . With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas—containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities—is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions 4 . We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis. The authors describe an integrated atlas of the diverse cell types in the mouse primary motor cortex.

Alzheimer’s disease (AD) is the leading cause of dementia in older adults. Although AD progression is characterized by stereotyped accumulation of proteinopathies, the affected cellular populations remain understudied. Here we use multiomics, spatial genomics and reference atlases from the BRAIN Initiative to study middle temporal gyrus cell types in 84 donors with varying AD pathologies. This cohort includes 33 male donors and 51 female donors, with an average age at time of death of 88 years. We used quantitative neuropathology to place donors along a disease pseudoprogression score. Pseudoprogression analysis revealed two disease phases: an early phase with a slow increase in pathology, presence of inflammatory microglia, reactive astrocytes, loss of somatostatin + inhibitory neurons, and a remyelination response by oligodendrocyte precursor cells; and a later phase with exponential increase in pathology, loss of excitatory neurons and Pvalb + and Vip + inhibitory neuron subtypes. These findings were replicated in other major AD studies. The affected cellular populations during Alzheimer’s disease progression remain understudied. Here the authors use a cohort of 84 donors, quantitative neuropathology and multimodal datasets from the BRAIN Initiative. Their pseudoprogression analysis revealed two disease phases.

Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization 1 – 5 . First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties. The BRAIN Initiative Cell Census Network has constructed a multimodal cell census and atlas of the mammalian primary motor cortex in a landmark effort towards understanding brain cell-type diversity, neural circuit organization and brain function.

Biological ageing can be defined as a gradual loss of homeostasis across various aspects of molecular and cellular function 1 , 2 . Mammalian brains consist of thousands of cell types 3 , which may be differentially susceptible or resilient to ageing. Here we present a comprehensive single-cell RNA sequencing dataset containing roughly 1.2 million high-quality single-cell transcriptomes of brain cells from young adult and aged mice of both sexes, from regions spanning the forebrain, midbrain and hindbrain. High-resolution clustering of all cells results in 847 cell clusters and reveals at least 14 age-biased clusters that are mostly glial types. At the broader cell subclass and supertype levels, we find age-associated gene expression signatures and provide a list of 2,449 unique differentially expressed genes (age-DE genes) for many neuronal and non-neuronal cell types. Whereas most age-DE genes are unique to specific cell types, we observe common signatures with ageing across cell types, including a decrease in expression of genes related to neuronal structure and function in many neuron types, major astrocyte types and mature oligodendrocytes, and an increase in expression of genes related to immune function, antigen presentation, inflammation, and cell motility in immune cell types and some vascular cell types. Finally, we observe that some of the cell types that demonstrate the greatest sensitivity to ageing are concentrated around the third ventricle in the hypothalamus, including tanycytes, ependymal cells, and certain neuron types in the arcuate nucleus, dorsomedial nucleus and paraventricular nucleus that express genes canonically related to energy homeostasis. Many of these types demonstrate both a decrease in neuronal function and an increase in immune response. These findings suggest that the third ventricle in the hypothalamus may be a hub for ageing in the mouse brain. Overall, this study systematically delineates a dynamic landscape of cell-type-specific transcriptomic changes in the brain associated with normal ageing that will serve as a foundation for the investigation of functional changes in ageing and the interaction of ageing and disease. A comprehensive single-cell RNA sequencing study delineates cell-type-specific transcriptomic changes in the brain associated with normal ageing that will inform the investigation into functional changes and the interaction of ageing and disease.

Background Dysfunction of the Basal Ganglia is implicated in several neurodegenerative diseases such as Parkinson’s and Huntington’s. A substructure of the Basal Ganglia, the caudate nucleus, is observed to have diffuse amyloid plaques in Alzheimer’s disease (AD), in Thal phase III. Additionally, literature suggests the presence of AD ‐related tangles. Functionally, the caudate is known to be involved in cognitive functions impacted by AD such as memory. The caudate also receives signals and has efferent projections to significantly affected regions in AD such as cortex and hippocampus respectively. Despite these connections, caudate nucleus remains understudied in AD. Method AT8 (pTau) and 6e10 (Aβ) immunohistochemical staining was performed on the caudate from 42 donors with only canonical proteionopathies and no comorbities. Single nucleus RNA and ATAC‐seq (multiome or singleome) was collected for all donors in the cohort. Spatial transcriptomics was performed on a subset of 5 Thal I‐III and 5 Thal IV‐V donors, with post‐hoc immunostaining of AT8 and 6e10. Cells were labeled using deep learning with a reference caudate dataset from healthy BRAIN Initiative donors. Changes in expression and cell type abundance were modeled in terms of levels of AT8 and 6e10 using Bayesian and general linear mixed effects models respectively. Result We identified caudate specific pTau associated abundance increases in astrocyte and microglia types. These microglia types were not the stereotypical disease associated types described in cortex. We also identified pTau associated abundance decreases in oliogodendrocyte subtypes consistent with cortex. Almost all neuronal populations in the caudate show little change in their cellular abundances. Most effects in cellular composition or differential expression were observed specifically with respect to level of pTau and not Aβ. Conclusion AD’s impact in caudate head contrasts with established effects on the cortex. Regionally unique increases in certain non‐neuronal populations suggest a caudate specific response to AD. Additionally, little neuronal loss, even with respect to significant pTau pathology suggests either environmental or cellular factors that confer resilience, or distinct pTau conditions in the caudate. Finally, our data suggests that the predominantly diffuse plaques in caudate are not sufficient for a plaque induced response in microglia.

Dysfunction of the Basal Ganglia is implicated in several neurodegenerative diseases such as Parkinson's and Huntington's. A substructure of the Basal Ganglia, the caudate nucleus, is observed to have diffuse amyloid plaques in Alzheimer's disease (AD), in Thal phase III. Additionally, literature suggests the presence of AD -related tangles. Functionally, the caudate is known to be involved in cognitive functions impacted by AD such as memory. The caudate also receives signals and has efferent projections to significantly affected regions in AD such as cortex and hippocampus respectively. Despite these connections, caudate nucleus remains understudied in AD. AT8 (pTau) and 6e10 (Aβ) immunohistochemical staining was performed on the caudate from 42 donors with only canonical proteionopathies and no comorbities. Single nucleus RNA and ATAC-seq (multiome or singleome) was collected for all donors in the cohort. Spatial transcriptomics was performed on a subset of 5 Thal I-III and 5 Thal IV-V donors, with post-hoc immunostaining of AT8 and 6e10. Cells were labeled using deep learning with a reference caudate dataset from healthy BRAIN Initiative donors. Changes in expression and cell type abundance were modeled in terms of levels of AT8 and 6e10 using Bayesian and general linear mixed effects models respectively. We identified caudate specific pTau associated abundance increases in astrocyte and microglia types. These microglia types were not the stereotypical disease associated types described in cortex. We also identified pTau associated abundance decreases in oliogodendrocyte subtypes consistent with cortex. Almost all neuronal populations in the caudate show little change in their cellular abundances. Most effects in cellular composition or differential expression were observed specifically with respect to level of pTau and not Aβ. AD's impact in caudate head contrasts with established effects on the cortex. Regionally unique increases in certain non-neuronal populations suggest a caudate specific response to AD. Additionally, little neuronal loss, even with respect to significant pTau pathology suggests either environmental or cellular factors that confer resilience, or distinct pTau conditions in the caudate. Finally, our data suggests that the predominantly diffuse plaques in caudate are not sufficient for a plaque induced response in microglia.

Alzheimer's disease (AD) is clinically characterized by a progressive cognitive decline associated with stereotyped accumulation of amyloid-beta (Aβ) plaques and hyperphosphorylated Tau (pTau) tangles across brain regions. While histopathology has revealed patterns of regional involvement, the molecular and cellular events that accompany and potentially drive this progression remain incompletely understood. We applied single nucleus RNA sequencing (RNAseq), ATAC-seq, and Multiome profiling to over 7 million high-quality nuclei from 10 brain regions-spanning medial and lateral entorhinal cortices, hippocampus, multiple temporal and frontal cortical areas, and primary visual cortex-sampled from the same cohort of 84 aged human donors across the AD spectrum (3 regions from all donors, 7 from those without severe co-morbidities). We predicted the cell-type for each nucleus by mapping to an expanded BRAIN initiative cell-type taxonomy, which included AD-associated non-neuronal states and ∼70 brain region-specific neuronal types. These datasets were paired with regional quantitative measurements of Aβ (6e10), pTau (AT8), and other protein pathologies, as well as cellular stains for neurons, microglia, and astrocytes. We inferred two distinct protein pathology accumulation patterns across brain regions: neocortical areas accumulated Aβ prior to pTau, whereas hippocampus and entorhinal cortex had early and, in some cases, substantial pTau burden independent of Aβ. Among neocortical regions, accumulation of AT8 beyond the temporal medial lobe strongly associated with dementia. In analyzing cell-type abundance differences associated with higher levels of pTau pathology, we identified shared motifs of selective neuronal loss consistent with our previous observations from the middle temporal gyrus. These included loss of L2/3 and L5 intratelencephalic excitatory neurons and several types of inhibitory interneurons (e.g., Vip, Sst, Pvalb). These same vulnerable inhibitory types were also reduced in hippocampal and entorhinal regions, when present, and we observed parallel increases in astrocyte, microglial, and oligodendrocyte precursor cells. Additionally, we observed a decrease in specific, regionally specialized neuron subtypes in the hippocampus, entorhinal cortex, and visual cortex. Our multimodal, multi-region single-cell atlas reveals common and region-specific patterns of cellular vulnerability in AD. These cell-types, particularly those commonly affected in distinct neural circuits, could serve as candidate therapeutic targets and biomarkers.