Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 1. A Pooled Approach for CRISPR Knockout and CRISPRi Screening in Human THP-1 Cells (A) Strategy for preparing CRISPR libraries and performing genetic screens. (B) THP-1-mediated phagocytosis of M. bovis BCG after three rounds of infection (MOI 10:1) with induced green fluorescence (map24::GFP) (Scale bar, 20 mm). (C) Viability of host cells after three rounds of M. bovis BCG infection. (D and E) Expression of Cas9 (D) and dCas9-KRAB (E) in 9 randomly selected monoclonal THP-1 cells. Wild-type THP-1 cells were used as negative control. Vinculin was used as a loading control. (F) An sgRNA for EGFP was introduced in both wild-type and Cas9-expressing THP-1 cells using a lentivirus (pXPR-011) that also contains EGFP as a target (Scale bar, 20 mm). (G) Cas9-expressing THP-1 cells were transduced with an sgRNA targeting AAVS1 at a low MOI. Mutations at the AAVS1 locus were detected by SURVEYOR assay. The size of the AAVS1 amplicon is 500 bp. The cleaved product sizes are 320 and 180 bp. (H) Growth measurement associated with sgRNAs targeting INTS9, MCM2, and non-targeting negative controls sgNC1 and sgNC13. (I and J) RT-qPCR analysis of INTS9 (I) and MCM2 (J) expression in dCas9-KRAB-expressing THP-1 cells. The values are normalized to GAPDH (glyceraldehyde- 3-phosphate dehydrogenase). Data represent the mean ± SD (n = 3) (two-tailed unpaired Student’s t test, *p < 0.05 **p < 0.01 ***p < 0.001). See also Figure S1; Table S13.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: CRISPR, Knock-Out, Infection, Expressing, Negative Control, Control, Transduction, Amplification, Quantitative RT-PCR, Two Tailed Test

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 2. Genome-wide Pooled CRISPR Knockout and CRISPRi Screens to Dissect Biological Pathways in Mycobacterial Infection (A and B) Volcano plots from CRISPR knockout (A) and CRISPRi (B) screens. For each sgRNA-targeted gene, the x axis shows its enrichment or depletion post- infection, and the y axis shows statistical significance measured by p value. Positive and negative screen hits are labeled as red and green dots, respectively. Gray dots represent non-targeting controls. For each screen, experiments were carried out in triplicate. (C) Enriched genes in the Venn diagram were filtered with a cut-off of FDR <0.1 and log2-fold change >1 in M. bovis BCG infection. The degree of significance of the overlap is given. (D) Gene-centric visualization of average fold change of CRISPR knockout and CRISPRi screens in infected versus non-infected host cells. Selected type I IFN and AHR/ARNT pathway components are highlighted in orange and blue. (E and F) Candidate genes identified by CRISPR knockout (E) and CRISPRi (F) screens were functionally categorized to understand the changes in biological functions involved in M. bovis BCG infection. Pathways shown in red are those identified by both screens. Color gradient of nodes represents the enrichment scores of gene sets. Node size represents the number of genes in the gene set. Edge width represents mutual overlap of genes. See also Figures S2 and S3; Tables S1, S2, S3, S4, S5, S11, and S12.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Labeling

Journal: Cell systems

Article Title: Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

doi: 10.1016/j.cels.2020.08.010

Figure Lengend Snippet: Figure 3. Secondary CRISPR Knockout and CRISPRi Screens Identify Host Genetic Hits in Mycobacterial Infection (A) Enriched genes were filtered with a cut-off of FDR <0.05 and log2-fold change >0.5 in M. bovis BCG infection. The degree of significance of the overlap is given. (B) Validation rate of genetic hits in secondary screens grouped by their p values in primary genome-wide screens in M. bovis BCG infection. Number of genes per category is indicated. (C) Genetic hits from both primary and secondary screens were ranked by their differential sgRNA abundance between M. bovis BCG-infected versus uninfected populations (log2 fold change). (D) Heatmap of screen hits (log2 fold change) clustered in different biological pathways in M. bovis BCG infection. See also Figures S4 and S5; Tables S6, S7, S8, S9, and S10.

Article Snippet: Human CRISPR knockout pooled library (Brunello) was obtained from Addgene (#73178).

Techniques: CRISPR, Knock-Out, Infection, Biomarker Discovery, Genome Wide

Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 1. A genome-wide CRISPR-Cas9 genetic screen identifies an essential requirement for CRAMP1 and histone H1.4 in PRC2-mediated reporter repression (A) Schematic representation of GFP reporter repression by the PRC2 complex. (B) The GFP reporter is derepressed upon CRISPR-Cas9-mediated gene disruption of any of the three core PRC2 subunits, as assayed by flow cytometry. (C) A genome-wide CRISPR-Cas9 screen to identify factors required for PRC2 function. Following Cas9 expression in KBM-7 cells harboring the PRC2-sensitive GFP reporter, genome-wide mutagenesis was carried out with the Sabatini/Lander single guide RNA (sgRNA) library, 36 and GFP + cells isolated through two sequential rounds of FACS. ‘‘Significance’’ on the y axis represents the negative log of the ‘‘pos|score’’ metric reported by Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK). 37

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: Genome Wide, CRISPR, Disruption, Flow Cytometry, Expressing, Mutagenesis, Isolation, Knock-Out

Journal: Molecular cell

Article Title: CRAMP1 drives linker histone expression to enable Polycomb repression.

doi: 10.1016/j.molcel.2025.05.031

Figure Lengend Snippet: Figure 5. Linker histones are not enriched at regions marked by H3K9me3 (A–D) Lack of linker histone enrichment at H3K9me3-marked genomic regions. (A) Tornado plots depicting linker histone CUT&Tag signal across H3K9me3 peaks from the ENCODE project; average signal intensity is shown in (B). (C) Heatmap depicting the lack of correlation between linker histone occupancy and H3K9me3. Cells are annotated with pairwise Spearman correlation coefficients. An example locus is shown in (D). (E) CUT&Tag faithfully profiles H3K9me3. Example loci comparing CUT&Tag versus H3K9me3 ChIP-seq data (ENCODE) are shown. (F and G) Linker histone insufficiency does not impair H3K9me3-dependent LINE-1 silencing by the HUSH complex. (F) Schematic representation of the dual- color reporter cell line designed to monitor both H3K9me3-dependent repression by the HUSH complex and linker histone-mediated PRC2-reporter repression. (G) HUSH-mediated LINE-1 silencing is unaffected upon CRAMP1 depletion. The indicated CRISPR sgRNAs were expressed in the dual-color reporter cell line, and GFP and iRFP fluorescence assayed by flow cytometry. See also Figure S5 and Table S2.

Article Snippet: Single guide RNA (sgRNA) sequences were selected from the Sabatini/Lander Human CRISPR Pooled Library (Addgene #1000000100, kindly deposited by David Sabatini and Eric Lander 81 ) or the Brunello Human CRISPR Knockout Pooled Library (Addgene #73178, kindly deposited by David Root and John Doench 82 ).

Techniques: ChIP-sequencing, CRISPR, Fluorescence, Flow Cytometry

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: Demographics, clinical information, and biomarker data in subjects recruited into the Parkinson’s disease biomarker program (PDBP)

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: Biomarker Discovery

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: Development of sensitive single-molecule array assays and standards for fluid measures of total LRRK2, total Rab10, and pT73-Rab10. ( A ) AlphaFold structure of Rab10 and ( B ) LRRK2. Specific epitopes selected for the quantification of total LRRK2, total Rab10, and pT73-Rab10. The assay for Rab10 and pT73-Rab10 share the same detector antibody on the C-terminus but differ in capture antibody epitopes. ( C ) Representative SDS-PAGE gels with Coomassie stain (blue) for the quantification of recombinant His-Rab10 (derived from E. coli cells) and FLAG-G2019S-LRRK2 (derived from HEK-293T cells). Recombinant bovine-serum albumin standards are shown. ( D ) Representative regression curves for the measurement of recombinant Rab10 and ( E ) recombinant LRRK2 in the assay buffer. Goodness-of-fit (r-squared), limit-of-detection (LOD), and lower-limit of quantification (LLOQ) are indicated and calculated from duplicate and triplicate values for each assay point. ( F ) Representative phos-tag immunoblot analysis showing the proportion of Rab10 protein phosphorylated in protein lysates. HEK-293T lysates were generated through transient transfection of plasmids expressing FLAG-R1441G-LRRK2 with human FLAG-Rab10. The ratio of pT73-Rab10 to total Rab is calculated from three independent preparations as 15.2% ± 0.9% SEM. Protein preparations analyzed by phos-tag analysis are utilized as standards for single-molecule arrays (SiMOA). ( G ) Representative regression curves for the analysis of LRRK2, ( H ) Rab10, and ( I ) pT73-Rab10, as measured in diluted (sample buffer) HEK-293T protein lysates. Prior to lysis, cells were treated with MLi2 (200 nM, 30 min, red dots and lines) or vehicle (blue dots and lines). Assay quality parameters (orange font) are indicated based on triplicate values. Error bars for experimental replicates are typically less than 5% and too short to be visualized graphically in most of the plots

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: SDS Page, Staining, Recombinant, Derivative Assay, Western Blot, Generated, Transfection, Expressing, Lysis

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: LRRK2 mutations increase the extracellular ratio of pT73-Rab10 to total Rab10 as measured with single-molecule array assays (SiMOA) in serum. ( A ) Levels of total LRRK2, ( B ) total Rab10 and ( C ) the ratio of pT73-Rab10 to total Rab10 as measured by SiMOA in venous mouse serum from Lrrk2 −/− (8 males and 3 females), non-transgenic C57BL6/J (12 males and 8 females), and knockin Lrrk2 R1441C/R1441C mice (9 males and 8 females). ( D-F ) A cohort of transgenic Tg-WT-BAC Lrrk2 (4 males and 9 females) and Tg-G2019S-BAC Lrrk2 mice (5 males and 3 females) were also analyzed for these markers. In these strains, no differences were noted between males and females. Ages in both cohorts of the mice ranged from 3–6 months. ( G ) Representative immunoblots of lysates from HEK-293T cells transfected with FLAG-LRRK2 plasmids 24-hours before harvesting lysates. ( H ) Quantification of endogenous pT73-Rab10 to total Rab10 ratio. ( I ) SiMOA assay analysis of the lysates for the ratio of pT73-Rab10 to total Rab10. Dots show mean values calculated from independent experiments. Columns show group means, error bars show SEM, and p values are determined with two-tailed t-tests, or from one-way ANOVA followed by Tukey’s post-hoc test in the graphs with more than two groups

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: Transgenic Assay, Knock-In, Western Blot, Transfection, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: Serum LRRK2 and pT73-Rab10 proteins originate primarily from bone marrow immune cells. ( A ) Representative cytographs and column graphs showing the analysis of live (7AAD- 7-Aminoactinomycin D negative) leukocytes in blood from outbred male CD-1 mice before and six-days after 11 grays of radiation. ( B ) SiMOA analysis of serum from these mice for (B) total LRRK2, ( C ) total Rab10 and ( D ) the ratio of pT73-Rab10 to total Rab10. ( E ) Cytographs show the analysis of host CD45.2 and donor CD45.1 mice before and after radiation, and the column graph shows the mean of the ratio of CD45.1 cells to total CD45 cells ( n = 3 male mice). Bone marrow transplantation of donor Tg-WT- Lrrk2 cells into host Lrrk2 −/− mice restores levels of ( F ) total LRRK2, ( G ) total Rab10 and ( H ) the ratio of pT73-Rab10 to total Rab10. Each dot shows mean values from the analysis of a single animal (all ~ 2 months of age), and p values are calculated from paired (before and after) sample t-tests

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: Transplantation Assay

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: Sepsis increases the extracellular ratio of pT73-Rab10 to total Rab10 in serum that primarily localizes to exosome-enriched serum fractions. ( A ) Microbe profile of cecal slurry generated from healthy CD-1 outbred male mice. Percentages of phylum-level taxonomy, identified by 16S rRNA amplicon sequencing, are depicted with the “Other” category including Actinobacteria, Cyanobacteria , and Verrocomicrobia. Slurries were injected intraperitoneally into a cohort of outbred male (n = 8) and female (n = 16) CD-1 mice, with serum procured from facial vein draws. At the 8 hr time point after injection, 4 female and 4 male mice were selected at random to measure serum ( B ) interferon-γ (IFNγ) and ( C ) TNF with ELISA analysis. ( D ) Before and after graphs show changes in serum LRRK2 levels, ( E ) total Rab10 and ( F ) the ratio of pT73-Rab10 to total Rab10 72 hrs post-injection. P values shown are from paired samples (before and after) t-tests. ( G ) Procedure for “Exo-Spin Serum mini columns’’ size-exclusion fractionation of 100 microliters of human serum spread into fractions 1–3. ( H ) Proteomic analysis of eluted fractions via mass spectrometry assessments of the relative abundance of unique peptides associated with characteristic serum exosome proteins CD9 (plasma-membrane protein enriched in exosomes and extracellular vesicles), myeloperoxidase (a soluble myeloid-cell produced enzyme enriched in exosomes and extracellular vesicles), and a small soluble cytokine HCC-1 (CCL14). ( I ) Column graphs show levels of LRRK2, ( J ) Rab10, ( K ) and pT73-Rab10 as measured in three different human biobanked serum samples (one male and one female with PD, and one healthy control). Green bars are measured from fraction one, yellow bars are fraction two, and purple are fraction three. Columns show group mean and error bars show SEM

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: Generated, Amplification, Sequencing, Injection, Enzyme-linked Immunosorbent Assay, Fractionation, Mass Spectrometry, Clinical Proteomics, Membrane, Produced, Control

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: The ratio of pT73-Rab10 to total Rab10 in serum is elevated in mouse models of late-onset PD. ( A ) Levels of total LRRK2, ( B ) total Rab10 and ( C ) the ratio of pT73-Rab10 to total Rab10 as measured by SiMOA in venous serum from VPS35 WT/WT (2 males and 4 females) mice, VPS35 WT/D620N (4 males and 4 females) mice, and VPS35 D620N/D620N (6 males) mice. ( D ) Graphs show numbers of neutrophils (CD11b + Ly6G+), ( E ) classical monocytes (CD11b + Ly6G- Ly6C+), and ( F ) CD4 T-cells measured by flow cytometry in VPS35 WT/WT and VPS35 D620N/D620N serum samples. ( G ) Representative immunoblots and quantification of cultured bone marrow derived macrophages demonstrating elevated pT73-Rab10 to total Rab10 levels with mutant VPS35 expression. ( H ) Levels of total LRRK2, ( I ) total Rab10 and ( J ) the ratio of pT73-Rab10 to total Rab10 from Snca −/− (3 males and 6 females) mice, and PAC -SNCA/Snca −/− (5 males and 4 females) mice. ( K ) Graphs show numbers of neutrophils (CD11b + Ly6G+), ( L ) classical monocytes (CD11b + Ly6G- Ly6C+), and ( M ) CD4 T-cells measured by flow cytometry in Snca −/− and PAC -SNCA/Snca −/− mice blood. ( N ) Representative immunoblots and quantification of cultured bone marrow-derived macrophages demonstrating low but equivalent pT73-Rab10 to total Rab10 between Snca −/− and PAC -SNCA/Snca −/− mice. Mouse ages ranged from 3–6 months. Columns depict group mean, dots represent the mean values from an animal or independent experiment, and error bars show SEM. P values are from two-tailed t-tests or one-way ANOVA analysis with Tukey’s multiple comparison when three groups are tested

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: Flow Cytometry, Western Blot, Cell Culture, Derivative Assay, Mutagenesis, Expressing, Two Tailed Test, Comparison

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: The ratio of pT73-Rab10 to total Rab10 in serum is pharmacodynamic. In mice and rats, the LRRK2 small molecule inhibitor PFE-360 was given as a single oral dose (10 mg per kg), serum (mouse facial vein or rat lateral saphenous vein) collected one-hour after a vehicle-only administration (Vehicle), and serum next collected at the indicated time post-drug administration, with a final collection at 48 h after drug (Washout). The ratio of pT73-Rab10 to total Rab10 diminishes relative to baseline in ( A ) Tg-G2019S-BAC- Lrrk2 (4 male mice) and ( B ) VPS35 D620N/D620N (7 male mice), as well as ( C ) nTg Long-Evans Lrrk2 +/+ rats (7 male rats). Rodent ages were 3–5 months, bars show group means, error bars show SEM, and dots show the mean values from individual rodents. In repetitive venous draws, some samples were not successfully collected due to insufficient volume collected or hemolysis, thereby excluding the sample for analysis (see Additional File for all raw data collected). Bars in panels A-C show group mean, error bars show SEM, and p values were calculated from one-way ANOVA analysis with Tukey’s post-hoc comparisons. ( D ) Graphs show levels of LRRK2, ( E ) Rab10, and ( F ) the ratio of pT73-Rab10 to total Rab10 in Long-Evans Lrrk2 −/− rats (3 males and 3 females) compared to non-transgenic Long-Evans Lrrk2 +/+ rats (4 males and 4 females). Columns in panels D-E show group mean, error bars show SEM, and p values are from two-tailed t-test

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: Transgenic Assay, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: LRRK2 protein levels predict pT73-Rab10 levels that are elevated in PD cases with worse MDS-UPDRS scores. ( A ) Scatter plots show relationships between measured serum levels of total Rab10 and LRRK2, ( B ) pT73-Rab10 and LRRK2, and ( C ) the ratio of pT73-Rab10 to Rab10 and LRRK2. N = 522 cases and controls (see Table ). R values show correlation coefficients and p values correspond to mean linear regression analysis of lines (red) with 95% confidence intervals shown (red dashed curves). ( D ) Box and whisker plot comparing the mean MDS-UPDRS part III scores in PD cases split to lower (< 0.5) and higher (> 0.5) ratios of pT73-Rab10/Rab10. P value indicated is from a Mann-Whitney U test for comparisons of groups with unequal sizes

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: Whisker Assay, MANN-WHITNEY

Journal: Molecular Neurodegeneration

Article Title: Single molecule array measures of LRRK2 kinase activity in serum link Parkinson’s disease severity to peripheral inflammation

doi: 10.1186/s13024-024-00738-4

Figure Lengend Snippet: The ratio of pT73-Rab10 to total Rab10 in serum, and MDS-UPDRS III scores, are associated with myeloid cell activation and antigenic responses. ( A ) A weighted gene co-expression network analysis (WGCNA) identifies 27 gene modules of densely co-expressed genes (see Supplemental Fig. for hierarchical clustering). Two modules, Purple and Light Cyan, are significantly correlated with the ratio of pT73-Rab10 to total Rab10. Pearson’s correlation coefficients and associated p values are additionally shown for correlation to LRRK2 protein levels in serum and MDS-UPDRS-III scores. ( B ) Top-scoring StringDB protein-protein interaction clusters from Purple and Light Cyan modules are shown. Differentially expressed genes (DEGs) in PD subjects vs. control are indicated from DESeq2 negative binomial fit, with level of significance (Benjamini-Hochberg adjusted p values) shown with a blue gradient. ( C ) All terms from the Reactome Pathway database (FDR-corrected p-value cutoff 1 × 10 − 4 ) are listed. Top process terms from Purple ( N = 290 searchable genes) and ( D ) Light Cyan ( N = 102 searchable genes) are shown. All terms with a p < 0.05 cutoff from multiple databases are listed in Supplemental Material

Article Snippet: Lrrk2 knock-out Long-Evans rats were obtained from Envigo.

Techniques: Activation Assay, Expressing, Control

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 1. Responses to acoustic stimuli from central auditory areas in rats. (A) BAEP from a single rat elicited by the presentation of click stimuli (90 dB SPL); averaged CAEP from a representative multiunit cluster elicited by the presentation of a 50 ms noise burst stimuli (90 dB SPL); cortical spiking activity from a representative multiunit cluster elicited by the presentation of a 50 ms noise burst stimulus or from a six-pulse train composed of 25 ms noise bursts presented at six different repetition rates (90 dB SPL). Dot raster plot (dot = 1 spike; row = 1 trial) and/or line peristimulus time histogram (PSTH; 2 ms bins) shown. (B) Representative recording penetration in the primary auditory cortex (A1) in the rat, accompanied by schematics of the locations of electrode penetrations reconstructed from histological sections for each of the rats that underwent in vivo electrophysiological recordings (wildtype rats, n = 7 and Cntnap2−/−rats, n = 8). Electrodes were advanced into the cortex falling between 3.72 and 4.56 mm caudal to bregma.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques: Activity Assay, In Vivo

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 2. Intact auditory brainstem responses in adult Cntnap2−/−rats. (A) Averaged BAEP waveforms from wildtype (n = 7) and Cntnap2−/−rats (n = 8), in response to a 90 dB SPL, 0.1 ms click stimulus. Solid line and shaded region denote mean ± standard error (SE). (B) Amplitude and (C) latencies of peaks I and IV representing activity from the auditory nerve and lateral lemniscus terminating at the inferior colliculus respectively, represented as mean ± SE.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques: Activity Assay

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 3. Cntnap2 −/−cortical auditory evoked potentials in adulthood reflect an immature-like profile. (A) Averaged CAEP waveforms from wildtype (n = 180 waveforms) and Cntnap2−/−(n = 161 waveforms) rats in response to a 90 dB SPL noise burst. Solid line and shaded region denote mean ± SE. (B) Increased amplitudes and (C) prolonged latencies of the N1 and P2 potentials reflect an immature-like response profile in the adult rats. Data represented as mean ± SE. ∗P < 0.05.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques:

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 4. Spiking profiles in Cntnap2−/−rat auditory cortex is hyper-responsive. (A) Multiunit cortical response dynamics in the primary auditory cortex to a 50 ms noise burst stimulus (90 dB SPL), represented as an averaged line PSTH from wildtype (n = 180 clusters) and Cntnap2−/−(n = 161 clusters) rats. Solid line and shaded region denote mean ± SE. Horizontal dashed line represents spontaneous activity. (B) Spontaneous activity, (C) response onset latency, (D) response duration, (E) average response firing rate, (F) peak firing rate, (G) peak latency, (H) the rebound response firing rate, and (I) the onset of the rebound response represented as mean ± SE. Longer response duration and increased firing rate are indicative of a hyperexcitable response. ∗P <0.05.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques: Activity Assay

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 5. Cntnap2 −/−rats have a reduced ability to consistently respond to a six-pulse noise burst train. (Left) responses of multiunit clusters in the primary auditory cortex to a six-pulse train of 25 ms noise burst stimuli (90 dB SPL), presented at repetition rates of 0.3, 0.9, 2.8, 5.2, 7.4, and 9.2 pps, represented as an averaged line PSTH from wildtype (n = 176 clusters) and Cntnap2−/−(n = 161 clusters) rats. Solid line and shaded region denote mean ± SE. Horizontal dashed line represents spontaneous activity. (Right) average firing rate in a 35 ms window (5–40 ms after stimulus onset) in response to each noise burst in the six-pulse train for each repetition rate, represented as mean ± SE. Horizontal dashed line represents spontaneous activity. As the repetition rate increases, the ability of multiunit clusters to respond to each noise burst stimulus in the six-pulse train decreases; this effect is more pronounced in the responses of Cntnap2−/−rats at rates of 5.2 pps and greater. ∗P <0.05.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques: Activity Assay

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 7. No deficit in the cortical phase-locking of multi-unit responses in Cntnap2−/−rats. (A) Cortical spike timing in relation to stimulus phase was determined using vector strength, measured at the various repetition rates for each multi-unit cluster. A higher value signifies more precise spike timing. (B) The significance of the phase locking is measured with the Rayleigh statistic, where >13.82 indicates P < 0.001. Data represented as mean ± SE. ∗P < 0.05.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques: Plasmid Preparation

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 6. Poor temporal processing is reflected in the decreased rate-following ability in Cntnap2−/−rats. (A) RRTF reveals that the subsequent capacity of Cntnap2−/−multiunit clusters is poorer than wildtypes at repetition rates of 7.4 and 9.2 pps despite knockout clusters having an increased firing rate as compared with the first noise burst in the six-pulse train. The average firing rate to noise bursts 2–6 in the six pulse train are represented by the solid line, with error bars denoting SE. Dashed line and shaded region represent the mean firing rate ± SE to the first noise burst. (B) The tMTF normalizes the RRTF to the firing rate in response to the first noise burst stimulus showing that in both wildtype and Cntnap2−/−rats, the ability of multiunit clusters to follow repeated stimuli begins to decrease at a repetition rate of 2.8 pps. (C) The fh1/2 confirms that Cntnap2−/−multiunit clusters have a poorer capacity for processing high-rate stimuli as the rate at which the tMTF of each cluster was at half its maximum is lower in Cntnap2−/−rats compared with wildtypes. Data represented as mean ± SE. ∗P < 0.05.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques: Knock-Out

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 8. Cntnap2 −/−neurons are synaptically comparable to wildtype neurons. (A) Sample recording trace of spontaneous EPSCs from the auditory cortex of an adult wildtype rat with the (B) mean frequency and (C) mean amplitudes. (D) Representative recording areas including the primary auditory cortex (A1) in the rat, accompanied by the positions of the recording and stimulating electrodes in layers 2/3 and layers 5/6, respectively. (E) Sample traces of paired pulse evoked EPSCs from a wildtype rat at various ISIs, vertical lines represent stimulation pulses, and (F) paired-pulse ratios (amplitude of the second EPSC divided by first EPSC). All data are represented as mean ± SE (B, C: wildtype n = 19 cells, Cntnap2−/−n = 13 cells; F: wildtype n = 12 cells, Cntnap2−/−n = 6 cells). ∗P <0.05.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques:

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 9. Cntnap2 −/−neurons are generally more excitable. (A) Sample voltage-clamp recordings from wildtype (top left) and Cntnap2−/−(bottom left) neurons and representative magnified fast inward currents from wildtype (grey) and Cntnap2−/−(black) voltage-clamp recordings. (B) No differences in resting membrane potential,but (C) Cntnap2−/−neurons have a lower cell capacitance.(D) Voltage-clamp recordings normalized to cell capacitance reveal Cntnap2−/−neurons have a higher current density of transient voltage-activated inward currents and activation at lower voltages. (E) Representative current-clamp recording of action potentials from wildtype (grey) and Cntnap2−/−(black) neurons with firing threshold and rheobase indicated. (F) The action potential firing threshold and (G) rheobase current at which the neurons fire the first action potential reveal Cntnap2−/−neurons are more excitable. All data are represented as mean ± SE (B: wildtype n = 16 cells, C, D: wildtype n = 17 cells, F: wildtype n = 15 cells, G: wildtype n = 16 cells; B–G: Cntnap2−/−n = 10 cells). ∗P <0.05.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques: Membrane, Activation Assay

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

doi: 10.1093/cercor/bhab517

Figure Lengend Snippet: Figure 10. Cntnap2 −/−neurons have delayed currents and fire more easily but elicit fewer maximum spikes. (A) Sample current clamp recordings of wildtype (grey) and Cntnap2−/−(black) neurons with labeled metrics of interest. Action potential features presented are (B) action potential peak voltage, (C) action potential half-width, (D) fast trough of after-hyperpolarization, (E) after-hyperpolarization slow trough, and (F) first interspike interval. (G) Firing frequency in response to increasing steady-state current injections as indicated. All data are represented as mean ± SE (B, G: wildtype n = 16 cells; C, F: wildtype n = 15 cells; D: wildtype n = 17 cells; E: wildtype n = 14 cells; B–G: Cntnap2−/−n = 10 cells). ∗P <0.05.

Article Snippet: Homozygous knockout breeders were obtained from Horizon Discovery (Boyertown, PA; originally created at SAGE Laboratories, Inc. in conjunction with Autism Speaks; the line is now maintained by Envigo) and bred to obtain Cntnap2−/− rats.

Techniques: Labeling