Elucidating the cellular architecture of the human cerebral cortex 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 cortex. We identified 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 similar mouse cortex single cell RNA-sequencing datasets 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.

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To profile morpho-electric properties of mammalian neurons systematically, we established a single cell characterization pipeline using standardized patch clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly-accessible online database, the Allen Cell Types Database, to display these data sets. Intrinsic physiological and morphological properties were measured from over 1,800 neurons from the adult laboratory mouse visual cortex. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We establish a taxonomy of morphologically-and electrophysiologically-defined cell types for this region of cortex with 17 e-types and 35 m-types, as well as an initial correspondence with previously-defined transcriptomic cell types using the same transgenic mouse lines. INTRODUCTION Neurons of the mammalian neocortex exhibit diverse physiological and morphological characteristics. Classifying these neurons into cell types, following Plato's dictum to "carve

IntroductionNatural killer (NK) cells comprise 5% to 20% of human peripheral blood lymphoid cells and are a critical component of the immune system, providing protection against viral infections and contributing to tumor immune surveillance. NK-cell activity is regulated by an intricate balance of signals transmitted by inhibitory and activating receptors. 1,2 Functionally distinct NK-cell subsets can be defined based on the level of CD56 and CD16 coexpression. 3 CD56 bright CD16 Ϫ NK cells produce abundant IFN-␥ in response to stimulation with interleukin (IL)-12 and proliferate robustly when cultured in IL-2, whereas CD56 dim CD16 ϩ NK cells are more cytolytic and produce significant amounts of cytokine when their activating receptors are engaged. 4 CD56 dim CD16 ϩ NK cells are considered mature NK cells and are differentiated from the immature CD56 bright CD16 -NK-cell subset. This is further supported by recent data demonstrating the dynamics of expression of the killer immunoglobulin-like receptors (KIR), CD57, CD94, and CD62L expression on the CD56 dim CD16 ϩ NK cells as they mature from CD56 bright CD16 -NK-cell precursors. [5][6][7][8][9] T-cell immunoglobulin-and mucin domain-containing (Tim)-3 is a member of Tim family of receptors of which there are 3 in humans (Tim-1, Tim-3, and Tim-4). 10 These molecules are involved in diverse metabolic and immunoregulatory processes. 11 Tim-3 is a type I transmembrane protein that contains no defined signaling motifs in its cytoplasmic domain, but it has been implicated both in activation and inhibition of immune responses 12,13 and in the induction of apoptosis of Tim-3-bearing cells through interactions with galectin-9. 14 Tim-3 is expressed on CD4 ϩ T cells, dendritic cells, monocytes, 15-17 CD8 ϩ T cells, 18,19 and NK cells. 20 In a comparison of lymphocyte populations in healthy human subjects, the highest transcription of the gene encoding Tim-3 was observed in NK cells. 21 There is evidence that engagement of Tim-3 on mouse T cells with the ligand galectin-9 promotes aggregation, leading to the death of T-helper 1 cells and the selective loss of interferon (IFN)-␥-producing T cells. 14 On human T cells, the expression of Tim-3 regulates cell proliferation and IFN-␥ secretion. 19,21,22 We and others have observed that increased amounts of Tim-3 on T cells during HIV, hepatitis C virus, and other chronic viral infections correlated with T-cell dysfunction, suggesting that Tim-3 is part of a negative regulatory pathway. 19,[23][24][25] In this study, we investigated the expression of Tim-3 on human NK cells and its regulation by cytokines, and we provide evidence for the role of Tim-3 in the restraint of NK cell-mediated cytotoxicity in healthy individuals. Methods Primary cells and cell linesPeripheral blood mononuclear cells (PBMCs) of healthy individuals were obtained from the Stanford Blood Bank. Cord blood PMBCs were obtained The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, ...

Summary Gene expression studies suggest that differential ion channel expression contributes to differences in rodent versus human neuronal physiology. We tested whether h-channels more prominently contribute to the physiological properties of human compared to mouse supragranular pyramidal neurons. Single cell/nucleus RNA sequencing revealed ubiquitous HCN1-subunit expression in excitatory neurons in human, but not mouse supragranular layers. Using patch-clamp recordings, we found stronger h-channel-related membrane properties in supragranular pyramidal neurons in human temporal cortex, compared to mouse supragranular pyramidal neurons in temporal association area. The magnitude of these differences depended upon cortical depth and was largest in pyramidal neurons in deep L3. Additionally, pharmacologically blocking h-channels produced a larger change in membrane properties in human compared to mouse neurons. Finally, using biophysical modeling, we provided evidence that h-channels promote the transfer of theta frequencies from dendriteto-soma in human L3 pyramidal neurons. Thus, h-channels contribute to between-species differences in a fundamental neuronal property.

The thalamus relays sensori-motor information to the cortex and is an integral part of cortical executive functions. The precise distribution of thalamic projections to the cortex is poorly characterized, particularly in mouse. We employed a systematic, high-throughput viral approach to visualize thalamocortical axons with high sensitivity. We then developed algorithms to directly compare injection and projection information across animals. By tiling the mouse thalamus with 254 overlapping injections, we constructed a comprehensive map of thalamocortical projections. We determined the projection origins of specific cortical subregions, and verified that the characterized projections formed functional synapses using optogenetic approaches. As an important application, we determined the optimal stereotaxic coordinates for targeting specific cortical sub-regions and expanded these analyses to localize layer-preferential projections. This dataset will serve as a foundation for functional investigations of thalamocortical circuits. Our approach and algorithms also provide an example for analyzing the projection patterns of other brain regions.

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A flashing ratchet transports diffusive particles using a time-dependent, asymmetric potential. The particle speed is predicted to increase when a feedback algorithm based on the particle position is used. We have experimentally realized such a feedback ratchet using an optical line trap, and observed that use of feedback increases velocity by up to an order of magnitude. We compare two different feedback algorithms for small particle numbers, and find good agreement with simulations. We also find that existing algorithms can be improved to be more tolerant to feedback delay times.