Journal: Cell reports

Article Title: AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences

doi: 10.1016/j.celrep.2023.112903

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal Rabbbit anti-TTR (prealbumin) AbCam Cat # ab75815; RRID:AB_1310604 Monoclonal Rabbit anti-Insulin Cell Signaling Cat # 3014; RRID:AB_2126503 Polyclonal Rabbit anti-mCherry Rockland Cat # 600-401-P16; RRID:AB_2614470 Monoclonal Rabbit anti- p -Akt (Ser473) Cell Signaling Cat # 4060; RRID:AB_2315049 Monoclonal Mouse anti-Alpha1 Na+/K+ ATPase AbCam Cat # ab7671; RRID:AB_306023 Bacterial and virus strains AAV5-CMV-CRE Vector Biolabs Cat # 7012 AAV5-CMV-GFP Vector Biolabs Cat # 7006 AAV5-CMV-Ins2 Vector Biolabs Cat # AAV-262269 AAV5-CMV-GCaMP6f Vector Biolabs Built to order AAV5-TTR-hM3Dq-mCherry Vector Biolabs Built to order AAV5-CMV-Psck1 Vector Biolabs Cat # AAV-268239 AAV5-CMV-Pcsk2 Vector Biolabs Cat # AAV-268242 Chemicals, peptides, and recombinant proteins Compound 21 TOCRIS Cat # 5548 Critical commercial assays PicoPure RNA Isolation Kit ThermoFisher Cat # KIT0204 Ultra Sensitive Insulin ELISA Kit CrystalChem Cat # 90080 Deposited data Raw and analyzed data of RNA-seq This paper GEO: GSE218188 Experimental models: Cell lines iPS(IMR90)-4 WiCell WISCi004-B Experimental models: Organisms/strains C57BL/6J The Jackson Laboratory Strain # 000664 Ai14 The Jackson Laboratory Strain #007914 Ins1 −/− Ins2 fl/fl This paper N/A Software and algorithms ImageJ NIH https://imagej.nih.gov/ij/ GraphPad Prism GraphPad https://graphpad.com R R Foundation for Statistical Computing https://www.r-project.org Other Normal chow diet Dyets Inc Cat# 101845 High fat/high sugar diet Dyets Inc Cat# 103806 Open in a separate window KEY RESOURCES TABLE.

Techniques: Virus, Plasmid Preparation, Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: eLife

Article Title: Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice

doi: 10.7554/eLife.84740

Figure Lengend Snippet: ( a ) Frontal EEG spectra during NREM sleep following CNO injections relative to saline injections for the acute (first 2 hr, left column) and prolonged (6 hr, right column) observation period. Note the sustained reduction of power in frequency bands >6–8 Hz in the 5 and 10 mg/kg CNO conditions. ( b ) Transitions between vigilance states in the 6 hr period following saline and CNO injections. Note the increased stability of REM and NREM sleep for the 5 mg/kg CNO (REM>REM: p=0.0192, Cohen’s d =0.73681; NREM>NREM: p=0.0132, Cohen’s d =0.71052) and 10 mg/kg CNO (REM>REM: p=0.0492, Cohen’s d =0.65815; NREM>NREM: p=0.0214, Cohen’s d =0.77396) condition. Solid olive lines indicate significantly increased transitions/continuations of vigilance states in the respective CNO condition compared to the saline condition, dashed grey lines indicate all possible vigilance state transitions/continuations. ( c ) Cumulative amount of NREM sleep before the first occurrence of REM sleep. ( d ) Frequency of brief awakenings (4–16 s) per hour of sleep for the first 2 hr after injections. ( e ) summary of effects of 5 and 10 mg/kg CNO on sleep in DREADD-free mice. n=10 for saline, n=6 for 1 mg/kg, n=10 for 5 mg/kg, n=8 for 10 mg/kg for spectral analysis. n=16 for saline, n=11 for 1 mg/kg, n=15 for 5 mg/kg, n=14 for 10 mg/kg for vigilance state analysis. n=15 for saline, n=11 for 1 mg/kg, n=15 for 5 mg/kg, n=13 for 10 mg/kg for analysis of brief awakenings. Asterisks in panels c and d indicate post hoc comparisons with significant differences (*p<0.05, **p<0.01, ***p<0.001,, ****p<0.001). Asterisks in panel a indicate frequency bins with significant differences in post hoc comparisons using uncorrected paired t-tests (p<0.05) following a significant interaction effect between ‘frequency’ and ‘condition’ in two-way ANOVAs. Data in panel a are presented as the mean ± s.e.m. (shaded areas). ANOVA: analysis of variance. CNO: clozapine-N-oxide. EEG: electroencephalogram. NREM: non-rapid eye movement sleep.

Article Snippet: For C21 injections we used the water-soluble version of DREADD agonist C21 (C21 dihydrochloride, Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: HB6124).

Techniques: Saline

Journal: eLife

Article Title: Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice

doi: 10.7554/eLife.84740

Figure Lengend Snippet: ( a ) Time course of wakefulness, NREM, and REM sleep in the 6 hr following injection of C21 or saline at light onset (ZT 0). ( b ) Percentage of time spent in the three vigilance states during the first 2 hr (left column) and over the entire 6 hr observation period after saline and C21 injections. Note that REM sleep is presented both as proportion of the recording time (third row) and of the total sleep time (fourth row). n=7. Asterisks indicate t-tests with significant differences (*p<0.05, **p<0.01, ***p<0.001). C21: compound 21. NREM: non-rapid eye movement sleep. REM: rapid eye movement sleep. TST: total sleep time. ZT: zeitgeber time.

Article Snippet: For C21 injections we used the water-soluble version of DREADD agonist C21 (C21 dihydrochloride, Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: HB6124).

Techniques: Injection, Saline

Journal: eLife

Article Title: Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice

doi: 10.7554/eLife.84740

Figure Lengend Snippet: ( a ) NREM sleep architecture and ( b ) REM sleep architecture for the 6 hr observation period following C21 injections. ( c ) Frontal EEG spectra during NREM sleep relative to saline injections for the acute (first 2 hr, dark blue) and full (6 hr, light blue) observation period following C21 injections. Asterisks indicate frequency bins with significant differences in post hoc comparisons using uncorrected paired t-tests (p<0.05; acute: dark blue, full: light blue) following a significant interaction effect between ‘frequency’ and ‘condition’ in two-way ANOVAs. ( d ) Transitions between vigilance states in the 6 hr period following saline and C21 injections. Note the increased stability of REM and NREM sleep (REM>REM: p=0.0144, Cohen’s d =1.0325; NREM>NREM: p=0.0384, Cohen’s d =0.81527). Solid olive lines indicate significantly increased transitions/continuations of vigilance states in the C21 condition compared to the saline condition, dashed grey lines indicate all possible vigilance state transitions/continuations. ( e ) Cumulative amount of NREM sleep before the first occurrence of REM sleep. ( f ) Frequency of brief awakenings (4–16 s) per hour of sleep for the first 2 hr after injections. Number of animals n=7 mice for vigilance state analysis in panels a, b, d, and e. For analysis of EEG NREM spectra in panel c and brief awakenings in panel f: n=6 mice. Asterisks in panels a, b, e, and f indicate t-tests with significant differences (*p<0.05, **p<0.01, ***p<0.001). Data in c are presented as the mean ± s.e.m. (shaded areas). ANOVA: analysis of variance. C21: compound 21. EEG: electroencephalogram. NREM: non-rapid eye movement sleep. REM: rapid eye movement sleep.

Article Snippet: For C21 injections we used the water-soluble version of DREADD agonist C21 (C21 dihydrochloride, Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: HB6124).

Techniques: Saline

Journal: eLife

Article Title: Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice

doi: 10.7554/eLife.84740

Figure Lengend Snippet: ( a ) Time course of REM sleep expressed as the ratio between REM and NREM sleep over the 6 hr following intraperitoneal injections of C21 or saline at light onset (ZT 0). ( b ) REM/NREM ratio during the first 2 hr (left column) and over the entire 6 hr observation period (right column) after saline and C21 injections. n=7. Asterisks indicate t-tests with significant differences (*p<0.05, **p<0.01, ***p<0.001). C21: compound 21. NREM: non-rapid eye movement sleep. REM: rapid eye movement sleep. TST: total sleep time. ZT: zeitgeber time.

Article Snippet: For C21 injections we used the water-soluble version of DREADD agonist C21 (C21 dihydrochloride, Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: HB6124).

Techniques: Saline

Journal: eLife

Article Title: Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice

doi: 10.7554/eLife.84740

Figure Lengend Snippet: ( a ) Frontal EEG spectra in 0.25 Hz bins between 0.5 and 30 Hz for the acute (first 2 hr, left) and full (first 6 hr, right) observation time window. For the 2 hr time window, the two-way ANOVA revealed an interaction effect between ‘condition’ and ‘frequency’ ( F (118,590) =2.961, p<0.0001), but the main effect of ‘condition’ did not reach statistical significance ( F (1,5) = 6.560, p=0.0506). In the 6 hr time window, there was an interaction effect between ‘condition’ and ‘frequency’ ( F (118, 590) =4.726, p<0.0001) as well as a main effect of ‘condition’ ( F (1,5) = 10.32, p=0.0237). Both ANOVAs also revealed a main effect of ‘frequency’. Frequency bins with significant differences in post hoc tests using uncorrected paired t-tests for α-error probability of p=0.05 are indicated with grey asterisks, for α-error probability of p=0.01 with black asterisks, and using Benjamini-Hochberg (BH) correction for multiple testing with light blue asterisks. ( b ) Frontal EEG spectra during NREM sleep following C21 injections relative to saline injections for the acute (first 2 hr, left) and prolonged (6 hr, right) observation period. Frequency bins with significant differences in uncorrected paired t-tests for α-error probability of p=0.05 are indicated with grey asterisks. n=6 for saline (grey) vs. 3 mg/kg C21 (blue). Data are presented as the mean ± s.e.m. (shaded areas). ANOVA: analysis of variance. C21: compound 21. EEG: electroencephalogram. NREM: non-rapid eye movement sleep. REM: rapid eye movement sleep.

Article Snippet: For C21 injections we used the water-soluble version of DREADD agonist C21 (C21 dihydrochloride, Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: HB6124).

Techniques: Saline

Journal: eLife

Article Title: Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice

doi: 10.7554/eLife.84740

Figure Lengend Snippet: Frontal EEG spectra in 0.25 Hz bins between 0.5 and 30 Hz arranged by vigilance state and time window. Note that due to the low number of suitable animals n=3 for the wake condition (because of movement artefacts in the EEG) no null-hypothesis testing was performed and the data are only presented for qualitative assessment. Data for all three vigilance states are only presented for n=3 to maintain the within-subject comparison. Data are presented as the mean ± s.e.m. (shaded areas). C21: clozapine-N-oxide. EEG: electroencephalogram. NREM: non-rapid eye movement sleep. REM: rapid eye movement sleep.

Article Snippet: For C21 injections we used the water-soluble version of DREADD agonist C21 (C21 dihydrochloride, Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: HB6124).

Techniques: Comparison

Journal: Cell Reports

Article Title: Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture

doi: 10.1016/j.celrep.2021.109133

Figure Lengend Snippet:

Article Snippet: GC-376 , Selleckchem Inc. , Cat# S0457.

Techniques: Virus, Recombinant, Purification, Fluorescence, Protease Inhibitor, Transduction, Plasmid Preparation, Expressing, Software

Journal: eLife

Article Title: Entorhinal-retrosplenial circuits for allocentric-egocentric transformation of boundary coding

doi: 10.7554/eLife.59816

Figure Lengend Snippet: ( A ) An AAV encoding inhibitory DREADDs hM4Di was injected into MEC while tetrodes were positioned in RSC. Scale bar, 1 mm. ( B ) Four tetrodes were placed locally near the virus injection site, showing a subset of affected MEC neurons that decreased their activity 10–15 min after subcutaneous administration of agonist-21 (DREADDs agonist). ( C ) Two example RSC border cells that were affected by MEC inhibition and lost their spatial tuning. ( D-E ) Border cells in RSC exhibited increased EMD scores as well as lower firing rates after inhibition of MEC. Gray lines indicate individual cells. ( F ) Reversed experiment, with electrophysiological recordings in MEC while the AAV was injected into RSC. Scale bar, 1 mm. ( G ) A subset of RSC neurons decreased their activity after the administration of agonist-21. ( H ) Two example MEC border cells that were unaffected by inhibition of RSC. ( I-J ) Border cells in MEC did not show any significant qualitative changes in border tuning or firing rates after RSC inhibition. Gray lines indicate individual cells. ( K ) An AAV encoding inhibitory Halorhodopsin channels was injected into MEC, while an optrode with tetrodes and optic fiber was implanted in RSC to inhibit MEC axons. Scale bar, 1 mm. ( L ) Two example border cells in RSC that showed disrupted border tuning during inhibition of MEC terminals near the recording site. ( M ) A retroAAV encoding red-shifted inhibitory Cruxhalorhodopsin (Jaws) channels was injected into RSC together with the implantation of a 64-channel silicon-probe, while an optic fiber was placed at the dorsal edge of MEC for the silencing of cells that project to RSC. Histology shows the expression of the virus in neurons located in deep layers of MEC. Scale bar, 1 mm. ( N ) Two examples of RSC border cells that were disrupted due to optogenetic silencing of RSC-projecting neurons in MEC. ( O ) RSC border cells showed disrupted border tuning on a population-level as a direct result of MEC inhibition, both when silencing RSC-projecting neurons in MEC as well as during local inhibition of their axon terminals near the recording sites in RSC. *p<0.05, **p<0.01, ***p<0.001, Wilcoxon signed-rank test.

Article Snippet: For the DREADDs-mediated manipulation experiments, animals were injected with agonist-21 (DREADDs agonist 21 dihydrochloride, 3.52 mg/mL [10 mM]; Hellobio) subcutaneously after the first recording session, followed by at least 30 min waiting time to allow the drug to reach the brain and take effect before starting the next recording session.

Techniques: Injection, Activity Assay, Inhibition, Expressing

Journal: eLife

Article Title: Entorhinal-retrosplenial circuits for allocentric-egocentric transformation of boundary coding

doi: 10.7554/eLife.59816

Figure Lengend Snippet: Left: Nissl-stained sections and fluorescent images from individual animals used for the DREADDs experiments. In rat #169 and #170, recordings were performed from bilateral MEC and AAV (AAV8-hSyn-hM4Di-mCherry) was injected into the right RSC. Sagittal sections are shown for both Nissl-stained and fluorescent images. Positions of tetrode tracks are indicated by red circles. In rat #171 and #217, recordings were performed from the right RSC, and the AAV was injected into bilateral MEC. Coronal sections are shown for Nissl-stained images, and sagittal sections are shown for fluorescent images. Right two columns: the left plots show normalized firing rates of cells recorded from the virus injected site. The DREADDs agonist-21 was injected at the beginning of the recording sessions. Two red traces show representative cells that exhibited a significant reduction of firing rates after the injection (p<0.05, Wilcoxon ranksum test for rate changes between 0–10 min and 30–40 min), and blue traces are cells that were not significantly affected by the drug. The activity of cells was partly disrupted by the agonist-21 administration, as 70% (14/20) of the recorded cells near the injection site in RSC significantly reduced their firing rates to 53.0 ± 6.4% of their baseline firing, whereas 59% (26/44) of the MEC cells decreased their spiking rate to 47.2 ± 5.5% of baseline. The right plots show the probability density of the animal’s running speed during random foraging in the open field arena, before and after the drug injection. DREADDs-mediated inactivation did not significantly affect the animal’s running speed (p>0.05 in Friedman test). Each plot shows mean (solid lines) and SEM (shaded).

Article Snippet: For the DREADDs-mediated manipulation experiments, animals were injected with agonist-21 (DREADDs agonist 21 dihydrochloride, 3.52 mg/mL [10 mM]; Hellobio) subcutaneously after the first recording session, followed by at least 30 min waiting time to allow the drug to reach the brain and take effect before starting the next recording session.

Techniques: Staining, Injection, Activity Assay