27056 aav5 aav1 cag dio mcherrymbdnf shrnamir vector biolabs shaav 253926 chemicals  (Vector Biolabs)

Journal: PLOS Biology

Article Title: Cortical astrocyte histamine-1-receptors regulate intracellular calcium and extracellular adenosine dynamics across sleep and wake

doi: 10.1371/journal.pbio.3003376

Figure Lengend Snippet: (A) Experimental schematic. Acute V1 slices from adult mice (≥P28) expressing cortical astrocytic GCaMP6f are 2P-imaged before and after 50 μM HA addition. (B) Y-axis position and duration of Ca 2+ events before and after 50 μM HA addition without (left, gray) or with 50 μM H1R antagonist chlorpheniramine (right, orange); lower bar plots show number of active events per second. Purple bar = time HA is recirculating. (C) Change in % active pixels relative to mean 60 s pre-HA ± H1R antagonist. Traces = mean across slices. Shaded error bars = SEM. (D) Summary of data in C. Maximum Δ pixels active during 2-min post-HA for each slice, with mean ± SEM at right (no antagonist: 3.7 ± 1.4, +H1R antagonist: 0.09 ± 0.04). p -values via one-sided Wilcoxon rank-sum test. (E) Amplitude (left), duration (middle), and area (right) of Ca 2+ events pre- and post-HA ± H1R antagonist. Mean ± 95% CI estimated via bootstrapping with replacement. p -values via permutation test on data before bootstrapping. Underlying data available on Dryad ( https://doi.org/10.5061/dryad.2280gb64x ); panels B–E: Fig2.zip, Fig2_H1Rpharm_data.mat.

Article Snippet: For H1R cKO experiments, transgenic H1R fl/fl pups were injected with 60–80 nl of AAV5-GFAP(0.7)-RFP-T2A-iCre (5.0E12 vg/mL; Vector Biolabs, VB1133) and AAV5-GfaABC1D-cyto-GCaMP6f-SV40 at a 1:1 ratio.

Techniques: Expressing

Journal: PLOS Biology

Article Title: Cortical astrocyte histamine-1-receptors regulate intracellular calcium and extracellular adenosine dynamics across sleep and wake

doi: 10.1371/journal.pbio.3003376

Figure Lengend Snippet: (A) Schematic illustrates H1R conditional knockout procedure. Astrocytic Cre-RFP expression is driven via neonatal virus injections. Cre-mediated recombination excises exon 3, which encodes H1R. Proceeding experiments are conducted after 4 weeks of viral transduction. (B) Example confocal micrograph showing localized Cre-RFP expression in the cortex ~4 weeks after neonatal virus injection. Cre-RFP + cells are morphologically consistent with astrocytes. White line shows pia; scale bar = 100 μm. (C) Example confocal micrograph showing astrocyte expression of S100β (green), Cre-RFP (magenta), and H1R mRNA (cyan). Left: summed z-projection of all three confocal channels. Middle: RNAscope detected H1R mRNA with white and yellow arrows indicating example WT (S100β + /RFP − /H1R + ) and cKO (RFP + /H1R − ) soma regions of interest (ROIs), respectively. Right: Cre-RFP expression (top, magenta) and S100β expression (bottom, green). Scale bars = 10 μm. (D) Quantification of RNAscope detected H1R mRNA puncta in RFP+ and S100β + /RFP − astrocytes in H1R fl/fl cortex. Histogram shows distribution of 28 cKO soma and 28 randomly sampled data points from a total of 48 WT soma. cKO soma (RFP + ; n = 28 soma from 8 sections; N = 3 mice) had a mean of 0.46 puncta and a 36% probability of >0 H1R puncta. WT soma (S100β + /RFP − ; n = 28 soma from 8 sections; N = 3 mice) had a mean of 2.52 puncta and a 77% probability of >0 H1R puncta. (E) Example confocal micrograph showing astrocytic Cre-RFP (magenta), neuronal NeuN (green), and nuclear marker DAPI (blue). Left: summed z-projection of all three confocal channels. Middle: astrocytic Cre-RFP signal with astrocyte soma ROIs overlaid. Right: NeuN signal with astrocyte soma ROIs overlaid. Scale bars = 50 μm. (F) Quantification of Cre-RFP expression in cortical neurons. Histograms show percentage of astrocyte soma pixels that are RFP + (top, pink) and NeuN + (bottom, green). Mean percentage of soma pixels that are RFP + and NeuN + is 96% and 7%, respectively ( n = 57 soma from 6 sections, N = 3 mice). Underlying data available on Dryad ( https://doi.org/10.5061/dryad.2280gb64x ); panels C–F: Fig3.zip, panels C–D: Fig3_H1R_mRNA_quantification.xlsx, panels E–F: Fig3_CreRFP_NeuN_colocalization.xlsx.

Article Snippet: For H1R cKO experiments, transgenic H1R fl/fl pups were injected with 60–80 nl of AAV5-GFAP(0.7)-RFP-T2A-iCre (5.0E12 vg/mL; Vector Biolabs, VB1133) and AAV5-GfaABC1D-cyto-GCaMP6f-SV40 at a 1:1 ratio.

Techniques: Knock-Out, Expressing, Virus, Transduction, Injection, RNAscope, Marker

Journal: PLOS Biology

Article Title: Cortical astrocyte histamine-1-receptors regulate intracellular calcium and extracellular adenosine dynamics across sleep and wake

doi: 10.1371/journal.pbio.3003376

Figure Lengend Snippet: (A) Experimental schematic. Acute V1 slices from H1R fl/fl mice expressing cortical astrocytic GCaMP6f ± Cre-RFP are 2P-imaged before and after neuromodulator addition. Example images: four summed frames of 2P time-series with overlaid AQuA events after 50 μM HA (left) and 10 μM norepinephrine (NE) (right) addition. Regions of interest (ROIs) and arrows indicate example H1R cKO (yellow arrow) and WT (white arrow) astrocytes (see Methods for identification). Scale bars = 50 μm. (B) Mean change in percent of WT or cKO pixels active post-ligand. Purple bar = 50 μM HA addition; yellow bar = 10 μM NE addition. Shaded error bars = SEM. (C) Mean change in percent of WT or cKO pixels active after 10 μM NE addition (yellow bar) to untreated slices. Shaded error bars = SEM. (D) Summary of data in B. Left: WT vs. cKO mean maximum change in percent-ROI-active post-HA for each slice. Mean across slices ± SEM (WT: 21.4 ± 4.8%; cKO: 3.2 ± 0.4%). Right: post-NE condition (WT: 24.0 ± 3.6%; cKO: 59.1 ± 5.0%). For D and F, p -values calculated via one-sided, paired Wilcoxon rank-sum. (E) Linear regression analysis showing correlation between WT/cKO NE-response ratio and the amount of time separating HA and NE addition to recirculating ACSF bath. Each data point shows mean WT/cKO response ratio per slice. Displayed R 2 - and p -values indicate no correlation. (F) Summary of data in C. Mean maximum change in percent-ROI-active post-NE for each slice. Mean across slices ± SEM (WT: 38.0 ± 9.9; cKO: 43.8 ± 8.8). Data in panels B, D, E collected from 9 slices and 3 mice; data in panels C, F collected from 5 slices and 2 animals. Underlying data available on Dryad ( https://doi.org/10.5061/dryad.2280gb64x ); panels A–F: Fig 4.zip, panels A–B, D: Fig 4_H1RKO_HA_data.mat, Fig 4_H1RKO_NE_postHA_data.mat, panels C–F: Fig 4_H1RKO_NE_data.mat.

Article Snippet: For H1R cKO experiments, transgenic H1R fl/fl pups were injected with 60–80 nl of AAV5-GFAP(0.7)-RFP-T2A-iCre (5.0E12 vg/mL; Vector Biolabs, VB1133) and AAV5-GfaABC1D-cyto-GCaMP6f-SV40 at a 1:1 ratio.

Techniques: Expressing

Journal: PLOS Biology

Article Title: Cortical astrocyte histamine-1-receptors regulate intracellular calcium and extracellular adenosine dynamics across sleep and wake

doi: 10.1371/journal.pbio.3003376

Figure Lengend Snippet: (A) Conditional H1R knockout mouse surgery schematic shows astrocytic jRGECO and Cre-GFP viral injections into V1, photometry fiber placement, and contralateral EEG screw placement in H1R fl/fl mice. Wild-type surgeries omitted Cre virus. (B) Example z-scored jRGECO photometry traces from WT (left, gray) and H1R cKO (right, cyan) mice aligned to color-coded wake (yellow), NREM (blue), and REM (purple) periods with detected jRGECO peaks indicated by magenta arrows. Peaks detected using Python’s signal.find_peaks function with prominence level = 2, distance = 5 s, width = 1 s. White blocks in sleep/wake scoring indicate periods that were not successfully scored by HMM. Horizontal scale bar = 25 min. (C) Per bout analysis showing jRGECO peak frequency plotted against theta/delta ratio to distinguish NREM (blue), wake (yellow), and REM (purple) bouts in WT (left) and cKO (right). Very few REM bouts exhibit detectable jRGECO peaks. (D) Median jRGECO peaks per minute during wake, NREM, and REM in WT and H1R cKO recordings. Average median ± SEM at right. p -values calculated via one-sided Wilcoxon rank-sum test. (E) Median jRGECO peak amplitudes during wake and NREM in WT and H1R cKO recordings. Average median ± SEM at right. p -values calculated via one-sided Wilcoxon rank-sum test. (F–I) Event-triggered averages of change in jRGECO relative to mean of pre-transition period. Traces = mean jRGECO activity. Shaded error bar = SEM. Righthand plots show mean area under the curve (AUC) following state transition for WT and cKO. AUC is integration of signal during 60 s post-transition. Data points show mean per recording with overall mean ± SEM. p -values calculated via one-sided Wilcoxon rank-sum test. (F) Wake-NREM transitions spanning 400 s. (G) NREM-wake transitions spanning 400 s. (H) NREM-REM transitions spanning 120 s. Inset shows zoom-in around y-axis. (I) REM-wake transitions spanning 120 s. Underlying data available on Dryad ( https://doi.org/10.5061/dryad.2280gb64x ); panels B–I: Fig 6_WT_grabHA.pkl, Fig 6_KO_jRGECO.pkl, Fig 6_Fig 7_WT_grabAd.pkl, Fig 6_Fig 7_KO_grabAd.pkl.

Article Snippet: For H1R cKO experiments, transgenic H1R fl/fl pups were injected with 60–80 nl of AAV5-GFAP(0.7)-RFP-T2A-iCre (5.0E12 vg/mL; Vector Biolabs, VB1133) and AAV5-GfaABC1D-cyto-GCaMP6f-SV40 at a 1:1 ratio.

Techniques: Knock-Out, Virus, Activity Assay

Journal: PLOS Biology

Article Title: Cortical astrocyte histamine-1-receptors regulate intracellular calcium and extracellular adenosine dynamics across sleep and wake

doi: 10.1371/journal.pbio.3003376

Figure Lengend Snippet: (A) Conditional H1R knockout mouse surgery schematic shows extracellular GRAB-Ado and astrocytic Cre viral injections into V1, photometry fiber placement, and contralateral EEG screw placement in H1R fl/fl mice. WT surgeries omitted Cre virus. H1R cKO N = 4; WT N = 5. (B) Example z-scored GRAB-Ado photometry traces from WT (gray) and H1R cKO (cyan) recordings aligned to color-coded wake (yellow), NREM (blue), and REM (purple) periods. White blocks in sleep/wake scoring indicate periods that were not successfully scored by HMM. Horizontal scale bar = 25 min. (C) Distribution of GRAB-Ado z-score values from wake, NREM, or REM in WT and cKO recordings. Each box spans interquartile range (25th–75th percentile), with horizontal line indicating median, whiskers extending to most extreme values within 1.5× the IQR, and outliers plotted individually. p -values via one-sided Wilcoxon rank-sum test. (D–G) Event-triggered averages of change in GRAB-Ado relative to mean of pre-transition period. Trace = mean across recordings. Shaded error bars = SEM. Right: mean area under the curve (AUC) following state transition for WT and cKO. AUC is integration of GRAB-Ado signal during post-transition period. Data points show mean per recording with overall mean ± SEM. p -values calculated via one-sided Wilcoxon rank-sum test. (D) NREM-wake transitions spanning 400 s. (E) Wake-NREM transitions spanning 400 s. (F) NREM-REM transitions spanning 120 s. (G) REM-wake transitions spanning 60 s. Underlying data available on Dryad ( https://doi.org/10.5061/dryad.2280gb64x ); panels B–G: Fig 6_Fig 7_WT_grabAd.pkl, Fig 6_Fig 7_KO_grabAd.pkl.

Article Snippet: For H1R cKO experiments, transgenic H1R fl/fl pups were injected with 60–80 nl of AAV5-GFAP(0.7)-RFP-T2A-iCre (5.0E12 vg/mL; Vector Biolabs, VB1133) and AAV5-GfaABC1D-cyto-GCaMP6f-SV40 at a 1:1 ratio.

Techniques: Knock-Out, Virus

Journal: PLOS Biology

Article Title: Cortical astrocyte histamine-1-receptors regulate intracellular calcium and extracellular adenosine dynamics across sleep and wake

doi: 10.1371/journal.pbio.3003376

Figure Lengend Snippet: (A) Surgery schematics show V1 astrocytic Cre or sham virus injection and EEG screw location in transgenic H1R fl/fl mice. Mouse number in each cohort listed below schematics. Color code for experimental cohorts used in B–F. (B–D) WT and cKO sleep/wake architecture. For each plot, overlaid data points show mean per mouse. Error bars = SEM. p -values via one-sided Wilcoxon rank-sum test. (B) Left: % individual recordings scored as wake, NREM, or REM (WT: 96.0 ± 1.5%, cKO: 96.2 ± 0.7%). Right: % time in wake, NREM, and REM (mean for WT: 59.3 ± 3.2 wake, 37.2 ± 2.7 NREM, 3.6 ± 0.6 REM; cKO: 67.3 ± 1.9 wake, 31.0 ± 2.4 NREM, 1.7 ± 0.6 REM). cKO mice spend significantly more time awake and less time in REM sleep than WT. (C) Bouts per hour for each behavioral state (mean for WT: 4.6 ± 0.5 wake, 3.4 ± 0.2 NREM, 1.4 ± 0.2 REM; cKO: 4.3 ± 0.5 wake, 3.1 ± 0.2 NREM, 0.9 ± 0.3 REM). REM bout frequency significantly lower in cKO relative to WT. (D) Bout duration in minutes for each behavioral state (mean for WT: 8.4 ± 1.0 wake, 6.6 ± 0.2 NREM, 1.6 ± 0.2 REM; cKO: 9.8 ± 1.0 wake, 6.1 ± 0.6 NREM, 1.2 ± 0.2 REM). (E) Power spectral density (PSD) plots show WT and cKO % power relative to total power across 1–20 Hz during wake, NREM, and REM. PSDs calculated via multitaper spectrogram. Traces = mean across mice; shaded error bar = SEM. (F) Bar plots show WT and cKO % band power (relative to total power in each state) for δ (1–4 Hz), θ (6–10 Hz), and σ (10–15 Hz) during wake (top), NREM (middle), and REM (bottom). Bars show overall mean and overlaid data points show mean per mouse. Error bars = SEM. One-sided Wilcoxon rank-sum test with Benjamini–Hochberg multiple comparisons correction does not detect any significant differences between WT and cKO. Underlying data available on Dryad ( https://doi.org/10.5061/dryad.2280gb64x ); panels B–F: Fig 8_ipsi_SHAM.pkl, Fig 8_ipsi_KO.pkl.

Article Snippet: For H1R cKO experiments, transgenic H1R fl/fl pups were injected with 60–80 nl of AAV5-GFAP(0.7)-RFP-T2A-iCre (5.0E12 vg/mL; Vector Biolabs, VB1133) and AAV5-GfaABC1D-cyto-GCaMP6f-SV40 at a 1:1 ratio.

Techniques: Virus, Injection, Transgenic Assay