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Key Points The currently accepted model of a dorsal–ventral (posterior–anterior) dichotomy of hippocampal function requires revision. There is evidence for two types of long-axis organization: gradients and discrete transitions. Anatomical studies in rodents and primates show that hippocampal extrinsic connectivity is organized as smooth topographical gradients. The relative size of spatial representation by place cells in the rat hippocampus gradually increases from dorsal to ventral. By contrast, rodent gene expression studies demonstrate multiple long-axis functional domains with sharply demarcated borders: at least nine in CA3 and three in CA1 and the dentate gyrus. Hippocampal intrinsic anatomical connectivity, as well as electrophysiological measures of coherence, show abrupt division from the ventral (anterior) one-third to dorsal (posterior) two-thirds of the hippocampus. Hippocampal involvement in unconditioned fear responses is limited to the ventral one-third. When these levels of organization are superimposed, a new model of hippocampal long-axis organization emerges: gradients with discrete domains, the latter dividing the hippocampus into (at least) three portions along the long axis. This model supersedes a simple dissociation of the dorsal from the ventral hippocampus and provides a potential framework for accommodating the multiple functions ascribed to the hippocampus in rodents and primates. It is commonly thought that the dorsal hippocampus is implicated in memory and spatial navigation and the ventral hippocampus in anxiety-related behaviours. On the basis of gene expression, anatomical and electrophysiology studies, Strange and colleagues propose a new model of hippocampal functional anatomy, in which functional long-axis gradients are superimposed on discrete functional domains. The precise functional role of the hippocampus remains a topic of much debate. The dominant view is that the dorsal (or posterior) hippocampus is implicated in memory and spatial navigation and the ventral (or anterior) hippocampus mediates anxiety-related behaviours. However, this 'dichotomy view' may need revision. Gene expression studies demonstrate multiple functional domains along the hippocampal long axis, which often exhibit sharply demarcated borders. By contrast, anatomical studies and electrophysiological recordings in rodents suggest that the long axis is organized along a gradient. Together, these observations suggest a model in which functional long-axis gradients are superimposed on discrete functional domains. This model provides a potential framework to explain and test the multiple functions ascribed to the hippocampus.

This protocol describes how to sequence the transcriptome from a single nucleus. It is particularly suited to cell types that are difficult to isolate as intact whole cells, such as neurons. A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.

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.

With few exceptions, the marked advances in knowledge about the genetic basis of schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. By applying knowledge of the cellular taxonomy of the brain from single-cell RNA sequencing, we evaluated whether the genomic loci implicated in schizophrenia map onto specific brain cell types. We found that the common-variant genomic results consistently mapped to pyramidal cells, medium spiny neurons (MSNs) and certain interneurons, but far less consistently to embryonic, progenitor or glial cells. These enrichments were due to sets of genes that were specifically expressed in each of these cell types. We also found that many of the diverse gene sets previously associated with schizophrenia (genes involved in synaptic function, those encoding mRNAs that interact with FMRP, antipsychotic targets, etc.) generally implicated the same brain cell types. Our results suggest a parsimonious explanation: the common-variant genetic results for schizophrenia point at a limited set of neurons, and the gene sets point to the same cells. The genetic risk associated with MSNs did not overlap with that of glutamatergic pyramidal cells and interneurons, suggesting that different cell types have biologically distinct roles in schizophrenia. Integration of single-cell RNA sequencing with genome-wide association data implicates specific brain cell types in schizophrenia. Gene sets previously associated with schizophrenia implicate the same cell types, which include pyramidal cells and medium spiny neurons.

This Resource paper describes a set of five new reporter mice, derived from Rosa26, driving Cre-dependent strong and ubiquitous expression of fluorescent proteins. In particular, the new mice show clear and specific expression patterns in the adult brain. The mice are available through Jackson Laboratories, and growing expression datasets can be accessed at http://transgenicmouse.alleninstitute.org/ . The Cre/lox system is widely used in mice to achieve cell-type-specific gene expression. However, a strong and universally responding system to express genes under Cre control is still lacking. We have generated a set of Cre reporter mice with strong, ubiquitous expression of fluorescent proteins of different spectra. The robust native fluorescence of these reporters enables direct visualization of fine dendritic structures and axonal projections of the labeled neurons, which is useful in mapping neuronal circuitry, imaging and tracking specific cell populations in vivo . Using these reporters and a high-throughput in situ hybridization platform, we are systematically profiling Cre-directed gene expression throughout the mouse brain in several Cre-driver lines, including new Cre lines targeting different cell types in the cortex. Our expression data are displayed in a public online database to help researchers assess the utility of various Cre-driver lines for cell-type-specific genetic manipulation.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits. Delivering genes to and across the brain vasculature efficiently and specifically across species remains challenging. Here, the authors show that endothelial-specific AAVs with serotype flexibility enable redosing and transform the brain vasculature into an in vivo biofactory in genetically diverse rodents. In primates, these vectors cross the blood-brain-barrier and show broad tropism.

Genes associated with risk for brain disease exhibit characteristic expression patterns that reflect both anatomical and cell type relationships. Brain-wide transcriptomic patterns of disease risk genes provide a molecular-based signature, based on differential co-expression, that is often unique to that disease. Brain diseases can be compared and aggregated based on the similarity of their signatures which often associates diseases from diverse phenotypic classes. Analysis of 40 common human brain diseases identifies 5 major transcriptional patterns, representing tumor-related, neurodegenerative, psychiatric and substance abuse, and 2 mixed groups of diseases affecting basal ganglia and hypothalamus. Further, for diseases with enriched expression in cortex, single-nucleus data in the middle temporal gyrus (MTG) exhibits a cell type expression gradient separating neurodegenerative, psychiatric, and substance abuse diseases, with unique excitatory cell type expression differentiating psychiatric diseases. Through mapping of homologous cell types between mouse and human, most disease risk genes are found to act in common cell types, while having species-specific expression in those types and preserving similar phenotypic classification within species. These results describe structural and cellular transcriptomic relationships of disease risk genes in the adult brain and provide a molecular-based strategy for classifying and comparing diseases, potentially identifying novel disease relationships.

The advancement of single-cell RNA-sequencing technologies has led to an explosion of cell type definitions across multiple organs and organisms. While standards for data and metadata intake are arising, organization of cell types has largely been left to individual investigators, resulting in widely varying nomenclature and limited alignment between taxonomies. To facilitate cross-dataset comparison, the Allen Institute created the common cell type nomenclature (CCN) for matching and tracking cell types across studies that is qualitatively similar to gene transcript management across different genome builds. The CCN can be readily applied to new or established taxonomies and was applied herein to diverse cell type datasets derived from multiple quantifiable modalities. The CCN facilitates assigning accurate yet flexible cell type names in the mammalian cortex as a step toward community-wide efforts to organize multi-source, data-driven information related to cell type taxonomies from any organism.

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets. This cluster also shows strong homology to a putative ET cluster in human temporal cortex, but with a strikingly specific regional signature. Together these results suggest that VENs are a regionally distinctive type of ET neuron. Additionally, we describe the first patch clamp recordings of VENs from neurosurgically-resected tissue that show distinctive intrinsic membrane properties relative to neighboring pyramidal neurons. Little is known about von Economo neurons, which have been described in a subset of mammals and appear to be selectively lost in several human neurological diseases. Here, authors reveal the gene expression profile of these cells and show that they are likely long-distance projection neurons.

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation (“birthdate”) in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an “inside-out” layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.