Journal:

Article Title: Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

doi: 10.1073/pnas.051631898

Figure Lengend Snippet: Generation of mutant mice with elevated GluR-B expression in GABAergic interneurons. (a) Schematic representation of gene segments of the wild-type GAD67 allele, the targeting vector, and the targeted GAD67 allele. Positions of EcoRI (E), NotI (N), and SalI (S) restriction sites are indicated. The PCR fragment used as probe for the Southern blot is indicated as a black bar. (Right) Southern blot analysis of tail DNA isolated from wild-type and heterozygous mice, digested with EcoRI to distinguish wild-type (7-kb) and targeted (5-kb) alleles. (b–e) GluR-B immunostaining in wild-type (b and d) and combined immunostaining and X-Gal staining in the mutant (c and e) hippocampus. Note the much higher GluR-B expression in LacZ-positive GABAergic interneurons compared with neighboring pyramidal cells in the mutant CA1 region (c) and hilus (e). This expression was never seen in the corresponding areas in control animals (b and d). (Scale bars, 100 μm.) so, Stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; h, hilus. For higher magnification see Fig. 6 in the supplemental data.

Article Snippet: After X-Gal staining, the free-floating sections were incubated overnight at 4°C with a rabbit anti-GluR-B antibody (PharMingen, dilution 1:25) in 2% normal goat serum diluted in Tris-buffered saline (TBS), pH 7.4.

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Southern Blot, Isolation, Immunostaining, Staining

Journal:

Article Title: Genetically altered AMPA-type glutamate receptor kinetics in interneurons disrupt long-range synchrony of gamma oscillation

doi: 10.1073/pnas.051631898

Figure Lengend Snippet: Firing properties of interneurons are altered by genetically manipulated overexpression of GluR-B. (a) Spontaneous EPSP rise time and decay time plots for stratum pyramidale fast-spiking interneurons, recorded from a membrane potential of −70 mV. Data are shown as cumulative probability for >4,000 EPSPs from five cells from five wild-type and five cells from five mutant mice. Model data show change in unitary EPSC for parameters that best fit the experimental data: “wild-type” unitary EPSC = t exp (−t/0.95) nS, “mutant” unitary EPSC = t exp(−t/1.05) nS. (Scale bars: 0.8 nS, 1 ms.) (b) Firing pattern in response to a single proximal stratum radiatum stimulation. Traces show response (from −70 mV) to increasing stimulus intensity (5–20 V) for interneurons from wild-type and mutant. Note increasing intensities generate double spikes in the cell from the mutant. (Scale bars: 50 mV, 40 ms.) Model data show voltage responses for single interneurons (holding current −0.165 nA) receiving identical EPSC onto single compartments on each of three dendrites. Each EPSC had the time course ct exp(−t/τ), where τ = 0.95 for control and 1.05 for mutant; c = 16.1, 47.4, 83.8 nS (top to bottom). Note the spike doublet in the mutant, but not in the wild type (as in experiment). (Scale bars: 50 mV, 30 ms.) (c) Pattern of pyramidal cell phasic inhibitory input (upper traces) and interneuron phasic excitatory input (lower traces) during posttetanic oscillations, in wild-type and in mutant mice. The interneuron was hyperpolarized by injection of −0.2 nA current. [Scale bars (experiment): 2 mV, 100 ms.] Model data show GABAA conductance to an e-cell (upper traces) and AMPA conductance to an i-cell. Note, in the “mutant,” the more variable amplitude of GABAA inputs, and the variable width of the AMPA inputs. [Scale bars (model): 150 nS, 100 ms.] (d) Example traces of interneuron firing patterns during gamma-frequency oscillations following paired tetanic stimulation, illustrating the increased incidence of doublet formation and the occurrence of occasional spike bursts. Model data show voltage of a selected interneuron from network simulations in the wild type and the mutant. The only difference in parameters for these simulations was in interneuron EPSC time course illustrated in a. [Scale bars (experiment and model): 20 mV, 100 ms.] Below are histograms illustrating the probability of a doublet of interval x relative to the sample mode. There are 45 doublets from wild-type data (n = three cells from three animals) and 130 doublets from mutant data (n = five cells from five animals). Model histograms were constructed by using ≈110 doublet intervals for simulations of “wild type” and “mutant” gamma oscillations, each pooled from seven interneurons.

Article Snippet: After X-Gal staining, the free-floating sections were incubated overnight at 4°C with a rabbit anti-GluR-B antibody (PharMingen, dilution 1:25) in 2% normal goat serum diluted in Tris-buffered saline (TBS), pH 7.4.

Techniques: Over Expression, Mutagenesis, Injection, Construct

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay