The human brain is subdivided into distinct anatomical structures, including the neocortex, which in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish early brain regions and cortical areas, but how early patterns result in finer and more discrete spatial differences remains poorly understood1. Here we use single-cell RNA sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Within the neocortex, we find that early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization approach, we find that laminar gene-expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene-expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.

Cerebrovascular diseases are a leading cause of death and neurologic disability. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of cerebrovascular cells in humans. We profiled transcriptomes of 181,388 cells to define a cell atlas of the adult human cerebrovasculature, including endothelial cell molecular signatures with arteriovenous segmentation and expanded perivascular cell diversity. By leveraging this reference, we investigated cellular and molecular perturbations in brain arteriovenous malformations, a leading cause of stroke in young people, and identified pathologic endothelial transformations with abnormal vascular patterning and the ontology of vascularly derived inflammation. Here, we illustrate the interplay between vascular and immune cells that contributes to brain hemorrhage and catalog opportunities for targeting angiogenic and inflammatory programs in vascular malformations.

The human brain is subdivided into distinct anatomical structures. The neocortex, one of these structures, enables higher-order sensory, associative, and cognitive functions, and in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish an early blueprint for the specification of brain regions and cortical areas. Furthermore, recent studies have uncovered distinct transcriptomic signatures between opposing poles of the developing neocortex1. However, how early, broad developmental patterns result in finer and more discrete spatial differences across the adult human brain remains poorly understood2. Here, we use single-cell RNA-sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Our data reveal that distinct cell subtypes are predominantly brain-structure specific. Within the neocortex, we find that even early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization (smFISH) approach, we validated the expression pattern of area-specific neuronal genes and also discover that laminar gene expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.

The mechanism by which the inhaled anesthetic isoflurane produces amnesia and immobility is not understood. Isoflurane modulates GABA A receptors (GABA A -Rs) in a manner that makes them plausible targets. We asked whether GABA A -R ␣2 subunits contribute to a site of anesthetic action in vivo. Previous studies demonstrated that Ser270 in the second transmembrane domain is involved in the modulation of GABA A -Rs by volatile anesthetics and alcohol, either as a binding site or a critical allosteric residue. We engineered GABA A -Rs with two mutations in the ␣2 subunit, changing Ser270 to His and Leu277 to Ala. Recombinant receptors with these mutations demonstrated normal affinity for GABA, but substantially reduced responses to isoflurane. We then produced mutant (knockin) mice in which this mutated subunit replaced the wildtype ␣2 subunit. The adult mutant mice were overtly normal, although there was evidence of enhanced neonatal mortality and fear conditioning. Electrophysiological recordings from dentate granule neurons in brain slices confirmed the decreased actions of isoflurane on mutant receptors contributing to inhibitory synaptic currents. The loss of righting reflex EC 50 for isoflurane did not differ between genotypes, but time to regain the righting reflex was increased in N 2 generation knockins. This effect was not observed at the N 4 generation. Isoflurane produced immobility (as measured by tail clamp) and amnesia (as measured by fear conditioning) in both wild-type and mutant mice, and potencies (EC 50 ) did not differ between the strains for these actions of isoflurane. Thus, immobility or amnesia does not require isoflurane potentiation of the ␣2 subunit.

Background General anesthesia produces multiple endpoints including immobility, hypnosis, sedation and amnesia. Tonic inhibition via γ–aminobutyric acid type A receptors (GABAA-Rs) may play a role in mediating behavioral endpoints that are suppressed by low concentrations of anesthetics (e.g., hypnosis and amnesia). GABAA-Rs containing the α4 subunit are highly concentrated in the hippocampus and thalamus, and when combined with δ subunits they mediate tonic inhibition, which is sensitive to low concentrations of isoflurane. Methods The present study used a GABAA α4 receptor knockout mouse line to evaluate the contribution of α4-containing GABAAreceptors to the effects of immobility, hypnosis and amnesia produced by isoflurane. Knockout mice and their wild-type counterparts were assessed on three behavioral tests: conditional fear (to assess amnesia), loss of righting reflex (to assess hypnosis), and MAC, the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation in 50% of subjects (to assess immobility). Results Genetic inactivation of the α4 subunit reduced the amnestic effect of isoflurane, minimally affected loss of righting reflex, and had no effect on immobility. Conclusions These results lend support to the hypothesis that different sites of action mediate different anesthetic endpoints and suggest that α4-containing GABAA-R are important mediators of the amnestic effect of isoflurane on hippocampal-dependent declarative memory.