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The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. 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 based on the combination of two single-cell-level, whole-brain-scale datasets: a single- cell RNA-sequencing (scRNA-seq) dataset of ∼7 million cells profiled, and a spatially resolved transcriptomic dataset of ∼4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature 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. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain 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 deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

Variation in cortical cytoarchitecture is the basis for histology-based definition of cortical areas, such as Brodmann areas. Single cell transcriptomics enables higher-resolution characterization of cell types in human cortex, which we used to revisit the idea of the canonical cortical microcircuit and to understand functional areal specialization. Deeply sampled single nucleus RNA-sequencing of eight cortical areas spanning cortical structural variation showed highly consistent cellular makeup for 24 coarse cell subclasses. However, proportions of excitatory neuron subclasses varied strikingly, reflecting differences in intra- and extracortical connectivity across primary sensorimotor and association cortices. Astrocytes and oligodendrocytes also showed differences in laminar organization across areas. Primary visual cortex showed dramatically different organization, including major differences in the ratios of excitatory to inhibitory neurons, expansion of layer 4 excitatory neuron types and specialized inhibitory neurons. Finally, gene expression variation in conserved neuron subclasses predicts differences in synaptic function across areas. Together these results provide a refined cellular and molecular characterization of human cortical cytoarchitecture that reflects functional connectivity and predicts areal specialization.

The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer’s disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease., Combined patch clamp recording, biocytin staining and single-cell RNA-sequencing of human neurocortical neurons shows an expansion of glutamatergic neuron types relative to mouse that characterizes the greater complexity of the human neocortex.

Humans have unique cognitive abilities among primates, including language, but their molecular, cellular, and circuit substrates are poorly understood. We used comparative single nucleus transcriptomics in adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets from the middle temporal gyrus (MTG) to understand human-specific features of cellular and molecular organization. Human, chimpanzee, and gorilla MTG showed highly similar cell type composition and laminar organization, and a large shift in proportions of deep layer intratelencephalic-projecting neurons compared to macaque and marmoset. Species differences in gene expression generally mirrored evolutionary distance and were seen in all cell types, although chimpanzees were more similar to gorillas than humans, consistent with faster divergence along the human lineage. Microglia, astrocytes, and oligodendrocytes showed accelerated gene expression changes compared to neurons or oligodendrocyte precursor cells, indicating either relaxed evolutionary constraints or positive selection in these cell types. Only a few hundred genes showed human-specific patterning in all or specific cell types, and were significantly enriched near human accelerated regions (HARs) and conserved deletions (hCONDELS) and in cell adhesion and intercellular signaling pathways. These results suggest that relatively few cellular and molecular changes uniquely define adult human cortical structure, particularly by affecting circuit connectivity and glial cell function.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. 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.

The neocortex is disproportionately expanded in human compared to mouse, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers that selectively make connections within the cortex and other telencephalic structures. Single-cell transcriptomic analyses of human and mouse cortex show an increased diversity of glutamatergic neuron types in supragranular cortex in human and pronounced gradients as a function of cortical depth. To probe the functional and anatomical correlates of this transcriptomic diversity, we describe a robust Patch-seq platform using neurosurgically-resected human tissues. We characterize the morphological and physiological properties of five transcriptomically defined human glutamatergic supragranular neuron types. Three of these types have properties that are specialized compared to the more homogeneous properties of transcriptomically defined homologous mouse neuron types. The two remaining supragranular neuron types, located exclusively in deep layer 3, do not have clear mouse homologues in supragranular cortex but are transcriptionally most similar to deep layer mouse intratelencephalic-projecting neuron types. Furthermore, we reveal the transcriptomic types in deep layer 3 that express high levels of non-phosphorylated heavy chain neurofilament protein that label long-range neurons known to be selectively depleted in Alzheimer’s disease. Together, these results demonstrate the power of transcriptomic cell type classification, provide a mechanistic underpinning for increased complexity of cortical function in human cortical evolution, and implicate discrete transcriptomic cell types as selectively vulnerable in disease.

The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup of this region, whose similarity mirrors evolutionary distance and is consistent between the transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal types allowed the generation of a cross-species consensus cell type classification and inference of conserved cell type properties across species. Despite overall conservation, many species specializations were apparent, including differences in cell type proportions, gene expression, DNA methylation, and chromatin state. Few cell type marker genes were conserved across species, providing a short list of candidate genes and regulatory mechanisms responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human primate and human and characterization of 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.

SUMMARYThe isocortex and hippocampal formation are two major structures in the mammalian brain that play critical roles in perception, cognition, emotion and learning. Both structures contain multiple regions, for many of which the cellular composition is still poorly understood. In this study, we used two complementary single-cell RNA-sequencing approaches, SMART-Seq and 10x, to profile ∼1.2 million cells covering all regions in the adult mouse isocortex and hippocampal formation, and derived a cell type taxonomy comprising 379 transcriptomic types. The completeness of coverage enabled us to define gene expression variations across the entire spatial landscape without significant gaps. We found that cell types are organized in a hierarchical manner and exhibit varying degrees of discrete or continuous relatedness with each other. Such molecular relationships correlate strongly with the spatial distribution patterns of the cell types, which can be region-specific, or shared across multiple regions, or part of one or more gradients along with other cell types. Glutamatergic neuron types have much greater diversity than GABAergic neuron types, both molecularly and spatially, and they define regional identities as well as inter-region relationships. For example, we found that glutamatergic cell types between the isocortex and hippocampal formation are highly distinct from each other yet possess shared molecular signatures and corresponding layer specificities, indicating their homologous relationships. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation for the first time, and begins to shed light on its underlying relationship with the development, evolution, connectivity and function of these two brain structures.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1–3. 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 regions4. 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.

Single cell transcriptomics has transformed the characterization of brain cell identity by providing quantitative molecular signatures for large, unbiased samples of brain cell populations. With the proliferation of taxonomies based on individual datasets, a major challenge is to integrate and validate results toward defining biologically meaningful cell types. We used a battery of single-cell transcriptome and epigenome measurements generated by the BRAIN Initiative Cell Census Network (BICCN) to comprehensively assess the molecular signatures of cell types in the mouse primary motor cortex (MOp). We further developed computational and statistical methods to integrate these multimodal data and quantitatively validate the reproducibility of the cell types. The reference atlas, based on more than 600,000 high quality single-cell or -nucleus samples assayed by six molecular modalities, is a comprehensive molecular account of the diverse neuronal and non-neuronal cell types in MOp. Collectively, our study indicates that the mouse primary motor cortex contains over 55 neuronal cell types that are highly replicable across analysis methods, sequencing technologies, and modalities. We find many concordant multimodal markers for each cell type, as well as thousands of genes and gene regulatory elements with discrepant transcriptomic and epigenomic signatures. These data highlight the complex molecular regulation of brain cell types and will directly enable design of reagents to target specific MOp cell types for functional analysis.

Neurons are frequently classified into distinct groups or cell types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 3,700 GABAergic mouse visual cortical neurons and reconstructed the local morphologies of 350 of those neurons. We found that most transcriptomic types (t-types) occupy specific laminar positions within mouse visual cortex, and many of those t-types exhibit consistent electrophysiological and morphological features. We observed that these properties could vary continuously between t-types, which limited the ability to predict specific t-types from other data modalities. Despite that, the data support the presence of at least 20 interneuron met-types that have congruent morphological, electrophysiological, and transcriptomic properties.HighlightsPatch-seq data obtained from >3,700 GABAergic cortical interneuronsComprehensive characterization of morpho-electric features of transcriptomic types20 interneuron met-types that have congruent properties across data modalitiesDifferent Sst met-types preferentially innervate different cortical layers

Neurons are frequently classified into distinct groups or cell types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 3,700 GABAergic mouse visual cortical neurons and reconstructed the local morphologies of 350 of those neurons. We found that most transcriptomic types (t-types) occupy specific laminar positions within mouse visual cortex, and many of those t-types exhibit consistent electrophysiological and morphological features. We observed that these properties could vary continuously between t- types, which limited the ability to predict specific t-types from other data modalities. Despite that, the data support the presence of at least 20 interneuron met-types that have congruent morphological, electrophysiological, and transcriptomic properties.

The isocortex and hippocampal formation are two major structures in the mammalian brain that play critical roles in perception, cognition, emotion and learning. Using single-cell RNA-sequencing approaches, we profiled ~1.2 million cells covering all regions in the adult mouse isocortex and hippocampal formation. The cell types are organized hierarchically and exhibit varying degrees of discrete or continuous variations. Such molecular relationships correlate strongly with the spatial distribution patterns of the cell types, which can be region-specific, shared across multiple regions, or part of one or more gradients. Glutamatergic neuron types display much greater diversity than GABAergic neuron types, both molecularly and spatially, and define regional identities as well as inter-region relationships. Our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation for the first time, and begins to shed light on its underlying relationship with the development, evolution, connectivity and function of these two brain structures.

Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain.

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled ∼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.

Neurons are frequently classified into distinct types on the basis of structural, physiological, or genetic attributes. To better constrain the definition of neuronal cell types, we characterized the transcriptomes and intrinsic physiological properties of over 4,200 mouse visual cortical GABAergic interneurons and reconstructed the local morphologies of 517 of those neurons. We find that most transcriptomic types (t-types) occupy specific laminar positions within visual cortex, and, for most types, the cells mapping to a t-type exhibit consistent electrophysiological and morphological properties. These properties display both discrete and continuous variation among t-types. Through multimodal integrated analysis, we define 28 met-types that have congruent morphological, electrophysiological, and transcriptomic properties and robust mutual predictability. We identify layer-specific axon innervation pattern as a defining feature distinguishing different met-types. These met-types represent a unified definition of cortical GABAergic interneuron types, providing a systematic framework to capture existing knowledge and bridge future analyses across different modalities.

Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.

Elucidating the cellular architecture of the human neocortex is central to understanding our cognitive abilities and susceptibility to disease. Here we applied single nucleus RNA-sequencing to perform a comprehensive analysis of cell types in the middle temporal gyrus of human cerebral cortex. We identify a highly diverse set of excitatory and inhibitory neuronal types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to a similar mouse cortex single cell RNA-sequencing dataset revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of human cell type properties. Despite this general conservation, we also find extensive differences between homologous human and mouse cell types, including dramatic alterations in proportions, laminar distributions, gene expression, and morphology. These species-specific features emphasize the importance of directly studying human brain.

Transcriptional profiling of complex tissues by RNA-sequencing of single nuclei presents some advantages over whole cell analysis. It enables unbiased cellular coverage, lack of cell isolation-based transcriptional effects, and application to archived frozen specimens. Using a well-matched pair of single-nucleus RNA-seq (snRNA-seq) and single-cell RNA-seq (scRNA-seq) SMART-Seq v4 datasets from mouse visual cortex, we demonstrate that similarly high-resolution clustering of closely related neuronal types can be achieved with both methods if intronic sequences are included in nuclear RNA-seq analysis. More transcripts are detected in individual whole cells (∼11,000 genes) than nuclei (∼7,000 genes), but the majority of genes have similar detection across cells and nuclei. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.

Neocortex contains a multitude of cell types segregated into layers and functionally distinct regions. To investigate the diversity of cell types across the mouse neocortex, we analyzed 12,714 cells from the primary visual cortex (VISp), and 9,035 cells from the anterior lateral motor cortex (ALM) by deep single-cell RNA-sequencing (scRNA-seq), identifying 116 transcriptomic cell types. These two regions represent distant poles of the neocortex and perform distinct functions. We define 50 inhibitory transcriptomic cell types, all of which are shared across both cortical regions. In contrast, 49 of 52 excitatory transcriptomic types were found in either VISp or ALM, with only three present in both. By combining single cell RNA-seq and retrograde labeling, we demonstrate correspondence between excitatory transcriptomic types and their region-specific long-range target specificity. This study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct regions of the mouse cortex.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.