A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation.

TL;DR: This study reveals sex-specific GABAergic microcircuits in the brain that switch vulnerability to stress into resilience, with SST neurons in the prelimbic cortex promoting stress resilience in males and the ventral hippocampus in females, and reversing chronic stress-induced behavioral alterations.

Abstract: Additional file 5: Fig. S2. Genomic organization at the Abi3 locus of Mus musculus. a. RNAseq based confirmation of Abi3, Gngt2 and APP levels from three Abi3 genotypes in APP transgenic (APP, Tg) mice or nontransgenic (APP, nTg) mice. Abi3, WT: Abi3+/+; Abi3, het: Abi3+/−; Abi3, hom: Abi3−/−. x axis denotes the genotypes and y axis denotes FPKM values of the corresponding RNA indicated on top of the graph. N=4 mice/genotype. b. The Abi3 knockout mice (Abi3tm1.1(KOMP)Vlcg) was generated by cre-mediated excision of the parental Abi3tm1(KOMP)Vlcg allele resulting in the removal of the neomycin selection cassette, leaving behind the inserted lacZ reporter sequence. The sequence that was excised out on chromosome 11 was located between 95842143 and 95832627 (indicated by black bar). This fragment encompasses the Abi3 coding region and 5’ region of a Gngt2 isoform.

Abstract: Additional file 2: Supplementary Figure 1. Chemical structure and pharmacological characterisation of THIK-1 inhibitor C100814. A Chemical structure of small molecule THIK-1 inhibitor C100814. B C100814 modulation of constitutive activity of hKCNK13 (THIK-1), mKCNK13, hKCNK6 (TWIK2), hKCNK2 (TREK-1), and potassium sulphate-induced hKv2.1 activity upon expression in HEK293 cells examined by a FLIPR thallium flux assay. Data show mean ± SD normalised to controls using the nonselective THIK-1 antagonist tetrapentylammonium (TPA) from 2 independent experiments performed in duplicates. IC50 values for human and murine THIK-1 were 0.071 µM and 0.061 µM, respectively. C Inhibition of THIK-1-mediated current in hTHIK-1 expressing HEK cells by C100814 using the Qpatch 48 platform, with an IC50 value of 0.143 µM. Current amplitudes were determined at the end of each depolarizing voltage step and are shown normalised to TPA controls. Data indicate mean ± SD from 2 independent experiments (yielding 2–3 data points/concentration). D C100814 displacement of radioligand binding to THIK-1 using membranes from HEK-hKCNK13 cells. Responses show the average percent inhibition of specific binding of the tool compound (C101505) (± SD) from one experimental run (performed in duplicates). E Left: Specimen current profiles of a patch-clamped microglia in human neocortical slices to voltage steps from − 150 to + 60 mV that expresses inwardly rectifying K+ currents (Kir) in the absence (1) and presence of 5 µM C100814 (2). Right: Current–voltage relationship of the currents before (1) and during (2) C100814 application and after subtracting (2) − (1), showing a lack of effect of C100814. Supplementary Figure 2. Extracellular ATP increases K+ efflux from murine microglia via THIK-1 and P2X7 receptors similar than their human counterparts. A Bidirectional changes in membrane potential of patch-clamped murine microglia held in the voltage follower configuration in response to locally applied 100 µM and 1 mM ATP via a puffing pipette causing hyperpolarization to ~ − 60 mV and depolarization to ~ 0 mV, respectively. B Example recording showing the concentration dependence of C100814-mediated blockade of THIK-1 current evoked by repeated local applications of 100 µM ATP (left). Quantification of C100814-sensitive THIK-1 current normalized to the mean ATP-evoked response prior to drug application of the same cell (right). C, D Single traces showing the change in voltage (C) and current (D) in microglia on application of 5 mM ATP. Note the lack of desensitization. E Left: Specimen current traces to voltage steps from − 150 to + 60 mV in the presence of 5 mM ATP and blockade of currents upon co-application of 10 µM of the P2X7 blocker A740003. Right: Voltage-dependence of P2X7-mediated currents before and after A74003 application, revealing a reversal potential of ~ 0 mV characteristic of a nonselective cation channel (P2X7). F Time course of inhibition upon application of 20 µM A740003 on repeatedly triggered 5 mM ATP-evoked P2X7 current (left), and analysis of P2X7 inhibition normalised to the mean current response before blocker application in the same cell (right). G Left: Lack of membrane current in response to 200 µM locally applied ATP in the presence of 20 µM Ivermectin added to the perfusate to facilitate P2X4 receptor activation. Right: Quantification of responses, normalised to the membrane current before agonist application in the same cell. To avoid simultaneously triggered THIK-1 K+ currents and improve the signal-to-noise ratio, a CsCl-based intracellular patch solution was used for this experiment. Data information: data indicate mean ± SEM. Numbers on bars show tested cells. Data are from 3 (A, B) and 4 wild type (C–E) mice. P-values are from paired Student’s t tests. Supplementary Figure 3. Expression of human THIK-1 and pharmacology of THIK-1-dependent IL-1β release. A In addition to NETSseq, RNA expression data from www.humanproteinatlas.com demonstrate specific expression of THIK-1 across different human cell types. B Analysis of IL-1β levels upon activation of microglia with 100 µM ATP added to murine slices for 3 h. Prior to purinergic activation, microglia were primed for 3 h by exposure of slices to 1 µg/ml LPS. Data are normalised to the control condition (priming only). C, D Lack of effect of C100814 on P2X7-evoked current (C), and A740003 on ATP-gated THIK-1 current (D) in microglia in murine slices, normalised to internal control responses prior to application of respective antagonist. E Release of IL-1β upon activation of murine microglia with 5 mM ATP in the presence (black bars) and absence (grey bars) of LPS priming, and blockade by 5 µM C100814 in slices from wild-type mice. Note different scales on the left and right y-axes. F Concentration-dependent blockade of IL-1β release by C100814 from LPS-primed primary microglia exposed to K+-free medium for NLRP3 activation. Data show percent inhibition relative to DMSO-treated microglia (control) and are from 4 independent experiments carried out in duplicates. G, H IL-1β release upon activation of murine microglia with 1 mM ATP (3 h) and concentration dependent blockade by C100814 and 10 µM MCC950 from LPS-treated slices from wild-type mice (G) that is abolished in THIK-1 KO mice (H). Data are normalised to the control condition (priming only). I Application of 50 µM tetrapentylammonium (TPA) to slices from THIK-1 KO mice further reduces IL-1β levels, indicating unspecific, THIK-1 independent effects of TPA. Data are normalised to the ATP condition. Data information: Data indicate mean ± SEM. Numbers on bars show tested cells (C, D) or number of slices (B, F–H). Data are from 3 (C, D) and 4 (B, E, G–I) mice. P-values are from paired (C, D) and unpaired (B, E, G–I) Student’s t tests. Supplementary Figure 4. Apart from the plasma membrane, THIK-1 may also play a role intracellularly. A Confocal images showing z-projections of Iba1-labeled primary murine microglia from WT and THIK-1 KO mice (green) co-stained with the endosomal marker Rab5 (red). Scale bar, 25 µm. B Analysis of mean Rab5 signal intensity per microglia. C Lysotracker-labeled primary microglia from WT and THIK-1 KO mice (red) live imaged by 2-photon microscopy. Scale bar, 50 µm. D Analysis of Lysotracker signal intensity per microglia. E Confocal image of HEK293 cells recombinantly expressing THIK-1-GFP fusion protein (green). Arrow indicates intracellular localisation of THIK-1 near the apical corner of the cell. Scale bar, 25 µm. F Confocal images showing Iba-1-labeled microglia (red) in human neocortical slices and THIK-1 immunoreactivity (green). Arrow points toward the intracellular localisation of THIK-1 predominantly at the apical corner of the cell as shown in orthogonal projections (right). Scale bar, 10 µm. Data information: data indicate mean ± SEM. Numbers on bars show analysed cells. Data from primary microglia were obtained from 4 WT or KO mice. P-values are from Mann–Whitney tests.

TL;DR: GHSR in a subset of GABA neurons controls food deprivation-induced hyperphagia in male mice. Ghrelin primarily targets a subset of Gad2-expressing neurons in the arcuate nucleus to induce food intake.

TL;DR: A strategy to "anchor" diverse datasets together, enabling us to integrate single-cell measurements not only across scRNA-seq technologies, but also across different modalities.

TL;DR: An analytical strategy for integrating scRNA-seq data sets based on common sources of variation is introduced, enabling the identification of shared populations across data sets and downstream comparative analysis.

TL;DR: A summary of the layout of cortical areas associated with vision and with other modalities, a computerized database for storing and representing large amounts of information on connectivity patterns, and the application of these data to the analysis of hierarchical organization of the cerebral cortex are reported on.

TL;DR: Uniform Manifold Approximation and Projection (UMAP) is a dimension reduction technique that can be used for visualisation similarly to t-SNE, but also for general non-linear dimension reduction.