Hippocampal Network Dynamics during Rearing Episodes

Animals build a model of their surroundings on the basis of information gathered during exploration. Rearing on the hindlimbs changes the vantage point of the animal, increasing the sampled area of the environment. This environmental knowledge is suggested to be integrated into a cognitive map stored by the hippocampus. Previous studies have found that damage to the hippocampus impairs rearing. Here, we characterize the operational state of the hippocampus during rearing episodes. We observe an increase of theta frequency paralleled by a sink in the dentate gyrus and a prominent theta-modulated fast gamma transient in the middle molecular layer. On the descending phase of rearing, a decrease of theta power is detected. Place cells stop firing during rearing, while a different subset of putative pyramidal cells is activated. Our results suggest that the hippocampus switches to a different operational state during rearing, possibly to update spatial representation with information from distant sources

IL-33 Receptor-Expressing Regulatory T Cells Are Highly Activated, Th2 Biased and Suppress CD4 T Cell Proliferation through IL-10 and TGFβ Release

Immunomodulatory Foxp3+ regulatory T cells (Tregs) form a heterogeneous population consisting of subsets with different activation states, migratory properties and suppressive functions. Recently, expression of the IL-33 receptor ST2 was shown on Tregs in inflammatory settings. Here we report that ST2 expression identifies highly activated Tregs in mice even under homeostatic conditions. ST2+ Tregs preferentially accumulate at non-lymphoid sites, likely mediated by their high expression of several chemokine receptors facilitating tissue homing. ST2+ Tregs exhibit a Th2-biased character, expressing GATA-3 and producing the Th2 cytokines IL-5 and IL-13 –especially in response to IL-33. Yet, IL-33 is dispensable for the generation and maintenance of these cells in vivo. Furthermore, ST2+ Tregs are superior to ST2− Tregs in suppressing CD4+ T cell proliferation in vitro independent of IL-33. This higher suppressive capacity is partially mediated by enhanced production and activation of the anti-inflammatory cytokines IL-10 and TGFβ. Thus, ST2 expression identifies a highly activated, strongly suppressive Treg subset preferentially located in non-lymphoid tissues. Here ST2+ Tregs may be well positioned to immediately react to IL-33 alarm signals. Their specific properties may render ST2+ Tregs useful targets for immunomodulatory therapies

Do not waste your electrodes – principles of optimal electrode geometry for spike sorting

Objective: This study examines how the geometrical arrangement of electrodes influences spike sorting efficiency, and attempts to formalise principles for the design of electrode systems enabling optimal spike sorting performance. Approach: The clustering performance of KlustaKwik, a popular toolbox, was evaluated using semi-artificial multi-channel data, generated from a library of real spike waveforms recorded in the CA1 region of mouse Hippocampus in vivo. Main results: Based on spike sorting results under various channel configurations and signal levels, a simple model was established to describe the efficiency of different electrode geometries. Model parameters can be inferred from existing spike recordings, which allowed quantifying both the cooperative effect between channels and the noise dependence of clustering performance. Significance: Based on the model, analytical and numerical results can be derived for the optimal spacing and arrangement of electrodes for one- and two-dimensional probe systems, targeting specific brain areas

ST2⁺ Tregs preferentially home outside of secondary lymphoid organs and exhibit a highly activated phenotype.

<p>Flow cytometric analysis of the phenotype and frequency of WT ST2⁺ and ST2⁻ Foxp3⁺ Tregs in spleen, pLN, blood, lung, lamina propria of the small intestine (siLP) and colon (coLP): <i>(A)</i> Frequency of ST2⁺ Tregs <i>(left)</i> and MFI of the ST2 staining on the ST2⁺ Treg fraction <i>(right)</i>. <i>(B)</i> MFI of chemokine receptor and α4β7 staining on ST2⁺ and ST2⁻ Tregs. <i>(C)</i> KLRG1 and CD103 expression in ST2⁺ <i>(top)</i> and ST2⁻ <i>(bottom)</i> Tregs from spleen; quantified frequencies from indicated organs <i>(right)</i>. <i>(D)</i> Frequency of CD44^hi, CD62L^lo and CTLA-4⁺ T cells within ST2⁺ and ST2⁻ Treg populations. <i>(E)</i> MFI of the Foxp3 staining <i>(left)</i> and geometric mean index of GATA-3 <i>(right)</i> in ST2⁺ and ST2⁻ Tregs. <i>(F)</i> Quantification of mRNA expression of the indicated genes from FACS-sorted ST2⁺ and ST2⁻ CD25⁺ Tregs from spleen and pLN <i>ex vivo</i>. mRNA expression normalized to <i>Hprt</i> endogenous control. <i>(G)</i> Frequency of ST2⁺ and ST2⁻ Tregs with IL-10 production capability as detected by GFP expression from <i>B6</i>.<i>Foxp3</i>^<i>hCD2</i> <i>xIl10</i>^<i>gfp</i> reporter mice. Fig <i>2A</i>: Data are representative of at least 2 independent experiments. Bar graphs show the mean ± SD of at least 5 biological replicates. Fig <i>2B</i>: pooled data from 2 independent experiments with 3–5 biological replicates each. Bar graphs show the mean ± SD. Fig <i>2C–2E</i> and <i>2G</i>: Data are representative of at least 2 independent experiments. Scatter plots depict one mouse as individual dot with mean ± SD. Fig <i>2F</i>: pooled data from 2 independent experiments. Significance was tested using unpaired Student’s t test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; non-significant (ns) p > 0.05.</p

ST2⁺ Tregs express Th2 cytokines and suppress CD4⁺ T cell proliferation via IL-10 and TGFβ.

<p><i>(A-D)</i> ST2⁺ and ST2⁻ Tregs from spleen and lymph nodes of WT mice activated <i>in vitro</i> by plate-bound anti-CD3/anti-CD28 antibodies in the presence of IL-2 with or without recombinant IL-33 for 60–70 hours: <i>(A)</i> Fold change in the number of viable Tregs upon IL-33 treatment. <i>(B) Tgfb1</i> mRNA expression normalized to <i>Hprt</i> endogenous control. <i>(C)</i> Cytokine concentration in the supernatants as determined by cytometric bead array. <i>(D)</i> Geometric mean index of GATA-3 in stable ST2⁺ and ST2⁻ Tregs at the end of culture. <i>(E) In vitro</i> suppression assay with ST2⁺ and ST2⁻ Tregs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g003" target="_blank">Fig 3</a> (Treg:Tresp ratio 1:5) with addition of blocking anti-IL-10R antibody or TGFβRI inhibitor. The relative division index indicates the fold increase in division of Tresp upon treatment. Division index of untreated Tresp was set to 1 in each group. <i>(F)</i> Quantification of mRNA expression of the indicated genes from sorted ST2⁺ and ST2⁻ CD25⁺ Tregs <i>ex vivo</i>. mRNA expression normalized to <i>Hprt</i> endogenous control. Fig <i>4A</i>–<i>4C</i>, <i>4E</i> and <i>4F</i>, data pooled from 2–3 independent experiments each performed with 2 replicates per condition. Fig <i>4D</i> is representative of 2 independent experiments with at least 2 replicates per condition each. Bar graphs show the mean ± SD. Significance was tested using unpaired Student’s t test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; non-significant (ns) p > 0.05.</p

ST2⁺ Tregs suppress CD4⁺ T cell proliferation more effectively than ST2⁻ Tregs <i>in vitro</i>.

<p><i>(A)</i> Proliferation profiles of CellTrace-labelled WT CD25^- CD62L^hi CD4⁺ responder T cells (Tresp) co-cultured with WT ST2⁺ (black) and ST2⁻ (grey) CD25⁺ Tregs during an <i>in vitro</i> suppression assay at day 4 of culture. T cells were stimulated by APCs and anti-CD3 antibody with <i>(right column)</i> or without <i>(left column)</i> the addition of recombinant IL-33. Treg:Tresp ratios are indicated <i>(left)</i>. Percentage of divided cells and the division index (number in brackets) are shown in each histogram in the respective color. <i>(B)</i> Proliferation profile of Tresp cultured under the same conditions as in 3A but without Tregs, either with (grey) or without (black) the addition of anti-CD3 antibody. <i>(C)</i> MFI of the ST2 staining on all Tregs recovered from the cultures described in 3A. Data are representative of 2–3 independent experiments.</p

ST2⁺ Tregs arise independently of IL-33 signals.

<p>Phenotype of Tregs in naive WT, <i>Il1rl1</i>^<i>-/-</i> and <i>Il33</i>^<i>-/-</i> mice: <i>(A)</i> ST2 and Foxp3 expression by splenic CD4⁺ T cells of one representative naive WT mouse; quadrant numbers indicate the average frequency ± SD in 4 mice. <i>(B)</i> Frequencies and total numbers of FoxP3⁺ Tregs in spleen (Spl), peripheral lymph nodes (pLN) and lung. <i>(C)</i> Frequency of ST2 expression in Tregs of spleen and lung. <i>(D)</i> Total number of ST2⁺ Tregs in spleen, pLN and lung. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g002" target="_blank">Fig <i>2A</i>, <i>2C</i> and <i>2D</i></a>: Data are representative of at least 2 independent experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g002" target="_blank">Fig <i>2B</i></a>: Pooled data from 2 independent experiments, each with 4 mice per genotype. Bar graphs show the mean ± SD of at least 4 individual mice. Significance was tested using unpaired Student’s t test. Asterisks indicate significance; all others non-significant. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.</p