Journal: Nature Communications

Article Title: Histamine-tuned subicular circuit mediates alert-driven accelerated locomotion in mice

doi: 10.1038/s41467-024-54347-2

Figure Lengend Snippet: HDC-CreERT2 mice were injected with AAV-CAG-FLEX-oChIEF-tdTomato into TMN and mixed AAV-EF1a-DIO-EYFP, AAV-ESARE-ERT2-Cre-ERT2 to label TMN-SUB activated c-fos+ neurons (TMN-SUB sti. activated) in SUB ( a ), and immunostaining of CaMKIIα/GABA ( b ), scale bar = 200 μm, n = 4 mice. c , d CaMKIIα-Cre mice were injected with AAV-hSyn-FLEX-mCherry-2A-TVA-2A-RvG and RV-EnvA-△G-EGFP into SUB, with starter cells in SUB (top, scale bar = 200 μm), SUB-projecting TMN neurons (bottom, scale bar = 100 μm), and quantification, n = 4 mice. e HDC-CreERT2 mice were injected with AAV-hSyn-FLEX-ChrimsonR-tdTomato into TMN and AAV-CaMKIIα-GCaMP6s into SUB, scale bar = 100 μm. Fluorescence values ( f ), heatmaps ( g ) and quantification ( h ) of calcium activity during TMN-SUB circuit activation. n = 6 mice, ** p = 0.0087. i WT mice were injected with AAV-CaMKIIα-GCaMP6s into SUB, scale bar = 200 µm. j Images and quantification of CaMKIIα staining, scale bar = 50 µm, n = 4 mice. k Representative speed and corresponding calcium activity during locomotion ( k1 ), acceleration ( k2 , left), and quantification ( k2 , right) of SUB glutamatergic neuronal activity. n = 16 mice, ** p = 0.0017. Fluorescence signal, heatmaps ( l ), and quantification ( m ) of SUB glutamatergic neuronal activity during rotarod test. n = 12 mice, * p = 0.0432 B.S vs. 5 rpm, * p = 0.0205 B.S vs. 6 rpm, * p = 0.0229 B.S vs. 7 rpm, ** p = 0.0017 B.S vs. 8 rpm. Fluorescence signal, heatmaps ( n ), and quantification ( o ) of SUB glutamatergic neuronal activity during treadmill test. n = 20 mice, *** p = 0.0003 Acc vs. B.S, p = 0.4823 Max vs. Acc, & & p = 0.0028 Dec vs. Max. Speed, fluorescence values ( p ), and quantification ( q ) of SUB glutamatergic neuronal activity during alert-escape test. n = 8 mice, * *p = 0.0051. Two-tailed Student’s t test for ( h , k2 , o , q ) and one-way ANOVA followed by the Dunnett post hoc test for ( m ). Schematics in a , c , e , i were drew by PowerPoint. Bar graphs represent mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: AAV-CAG-FLEX-ArchT-GFP (viral titers: 1.29 × 10 13 vg/mL, serotype: AAV2/9) and AAV-CaMKIIα-hM4D(Gi)-EGFP (viral titers: 7.04 × 10 12 vg/mL, serotype: AAV2/9) were purchased from OBiO Technology Co.,Ltd (Shanghai, China).

Techniques: Injection, Immunostaining, Fluorescence, Activity Assay, Activation Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Histamine-tuned subicular circuit mediates alert-driven accelerated locomotion in mice

doi: 10.1038/s41467-024-54347-2

Figure Lengend Snippet: a Schematic and image of injection of AAV-CaMKIIα-hM3D-mCherry into SUB of WT mice, scale bar = 200 μm. b CaMKIIα-hM3D-based glutamatergic neuron excitation in vivo resulted in c-fos expression in SUB. n = 6 mice. Effects of chemogenetic activation of SUB glutamatergic neurons on the distance traveled ( c ), and average speed ( d ) in open field test, n = 7 mice ( c : p = 0.05; d : * p = 0.0496 ) . e Representative velocity distribution of saline control group and the CNO group. Total time ( f ), and distance moved ( g ) in open field test in 5-speed subsections of saline control group and CNO group. n = 7 mice ( f : * p = 0.0100, * p = 0.0101, * p = 0.0381, p = 0.3358, * p = 0.0362; g : p = 0.1169, p = 0.0506, p = 0.7248, *p = 0.0116, *p = 0.0349). h Schematic diagram and representative image of viral injection of AAV-CAG-FLEX-oChIEF-tdTomato into the TMN, and AAV-CaMKIIα-hM4D-GFP into the SUB of HDC-CreERT2 mice, and the timeline schematic diagram. scale bar = 200 μm. Effects of optogenetic activation of TMN-SUB circuit terminals combined with chemogenetic inhibition of SUB glutamatergic neurons on distance traveled ( i ), average speed ( j ) in the open field test. Saline group, n = 10 mice; CNO group, n = 10 mice; saline + 470 nm group, n = 11 mice; CNO + 470 nm group, n = 12 mice ( i : ** p = 0.0060 saline + 470 nm vs. saline group, #### p < 0.0001 CNO + 470 nm vs. saline + 470 nm group; j : ** p = 0.0060 saline + 470 nm vs. saline group, #### p < 0.0001 CNO + 470 nm vs. saline + 470 nm group). Two-tailed Student’s t test for ( c , d , f , g ) and one-way ANOVA followed by the Turkey’s post hoc test for ( i , j ). Schematics in a , h were drew by PowerPoint. Bar graphs represent mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: AAV-CAG-FLEX-ArchT-GFP (viral titers: 1.29 × 10 13 vg/mL, serotype: AAV2/9) and AAV-CaMKIIα-hM4D(Gi)-EGFP (viral titers: 7.04 × 10 12 vg/mL, serotype: AAV2/9) were purchased from OBiO Technology Co.,Ltd (Shanghai, China).

Techniques: Injection, In Vivo, Expressing, Activation Assay, Saline, Control, Inhibition, Two Tailed Test

Journal: Nature Communications

Article Title: Histamine-tuned subicular circuit mediates alert-driven accelerated locomotion in mice

doi: 10.1038/s41467-024-54347-2

Figure Lengend Snippet: a WT mice were implanted cannula in SUB, scale bar = 400 µm. Effects of administration of H3R antagonist on distance ( b ), speed ( c ) and 5-speed subsections analysis for active time ( d ), distance ( e ) in open field test. n = 9 mice (**** p < 0.0001 for ( b ) and ( c ); d : * p = 0.0154, * p = 0.0195, ** p = 0.0073, p = 0.6811, *** p = 0.0003; e : p = 0.9120, p = 0.3507, p = 0.0719, p = 0.0535, **** p < 0.0001). Effects of Thio and H1R/H2R-agonist on distance ( f ), speed ( g ) in open field test, n = 13, 13, 13, 14 mice for PBS, Thio, 2-Pel, Amt group ( f : * p = 0.0148, ** p = 0.0028, * p = 0. 0193; g : * p = 0.0141, ** p = 0.0029, * p = 0. 0201). Effects of Thio combined with H1R/H2R-related drugs on distance ( h ), speed ( i ), n = 8, 9, 9, 9, 8, 10, 9 mice for PBS, Thio, Pyr + Thio, U73122 + Thio, Zol + Thio, ZD7288 + Thio, SQ22536 + Thio group ( h : ** p = 0.0035 vs. PBS ; ## p = 0.0049, #### p < 0.0001, ## p = 0.0019 vs. Thio; i : * p = 0.0120 vs. PBS; # p = 0.0172, #### p < 0.0001, ## p = 0.0067 vs. Thio), ns with respect to Thio group. j CaMKIIα-Cre were injected with knockdown virus ( j1 ), validation of H1R/H2R expression with CaMKIIα immunostaining, scale bar = 10 µm ( j2 ). Distance ( k ), and speed ( l ) in open field test of H1R/H2R knockdown mice, n = 9, 9 for AAV-GFP group; n = 9, 10 for AAV-shHrh1 group; n = 12, 12 for AAV-shHrh2 group ( k : *** p = 0.0004 vs. PBS, && p = 0.0017 vs. AAV-GFP + Thio; l : *** p = 0.0005 vs. PBS, && p = 0.0017 vs. AAV-GFP + Thio). Two-tailed Student’s t test for ( b – e , k , l ) and one-way ANOVA followed by post-Dunnett’s test for ( f – i ). Schematics in a , j1 were drew by PowerPoint. Bar graphs represent mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: AAV-CAG-FLEX-ArchT-GFP (viral titers: 1.29 × 10 13 vg/mL, serotype: AAV2/9) and AAV-CaMKIIα-hM4D(Gi)-EGFP (viral titers: 7.04 × 10 12 vg/mL, serotype: AAV2/9) were purchased from OBiO Technology Co.,Ltd (Shanghai, China).

Techniques: Injection, Knockdown, Virus, Expressing, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Histamine-tuned subicular circuit mediates alert-driven accelerated locomotion in mice

doi: 10.1038/s41467-024-54347-2

Figure Lengend Snippet: a WT mice were injected with mixed AAV-ESARE-ERT2-Cre-ERT2, AAV-hSyn-DIO-GCaMP6m, and AAV-hSyn-DIO-hM3D(Gq)-mCherry/AAV-hSyn-DIO-hM4D(Gi)-mCherry into SUB. b Expression of hM3D-expressed (top) and hM4D-expressed (bottom) SUB treadmill training-labeled c-fos+ neuron, scale bar = 200 µm. Effects of chemogenetic activation or inhibition of SUB treadmill training-labeled c-fos+ neuron on total distance ( c ), speed ( d ) in open field test. n = 7 mice ( c : * p = 0.0218, * p = 0.0165; d : * p = 0.0197, * p = 0.0160). e – g Fluorescence value with corresponding speed, and quantification ( g ) of calcium activity of hM3D-expressed SUB treadmill training-labeled c-fos+ neuron in two groups during acceleration. n = 12 mice (**** p < 0.0001). h – j Fluorescence value with corresponding speed, and quantification ( j ) of calcium activity of hM4D-expressed SUB treadmill training-labeled c-fos+ neuron in two groups during acceleration. n = 12 mice (*** p = 0.0002, p = 0.2473). Schematic of injection of mixed AAV-ESARE-ERT2-Cre-ERT2, AAV-EF1a-DIO-EYFP into SUB of WT mice ( k ), and expression ( l ) of SUB treadmill training-labeled c-fos+ neuron in SUB with fiber distribution in RSG, AV, MMB. Scale bar = 400 µm. m HDC-CreERT2 mice were injected with AAV-CAG-FLEX-oChIEF-tdTomato into TMN, mixed AAV-ESARE-ERT2-Cre-ERT2, AAV-EF1a-DIO-EYFP into SUB to label TMN-SUB circuit-activated c-fos+ neurons combined with injection of AAV2/2Retro-hSyn-tdTomato into RSG. Viral expression in TMN and RSG, scale bar = 400 um ( n ), image and quantification ( o ) of TMN-SUB circuit-activated c-fos+ SUB neurons overlapping with Retro-tdTomato in SUB, n = 3 mice, scale bar = 100 µm. p – s Schematic diagram ( p ), image ( q ) of injection of AAV-hSyn-FLEX-ChrimsonR-tdTomato into TMN and AAV2/2Retro-hSyn-GCaMP6s into RSG of the HDC-CreERT2 mice. Scale bar = 100 µm. Fluorescence values, heatmap ( r ), and quantification ( s ) of calcium activity of RSG-projecting SUB neurons during TMN-SUB circuit activation. n = 8 mice, ** p = 0.0072. Two-tailed Student’s t test for ( c , d , g , j , s ). Schematics in a , k , m , p were drew by PowerPoint. Bar graphs represent mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: AAV-CAG-FLEX-ArchT-GFP (viral titers: 1.29 × 10 13 vg/mL, serotype: AAV2/9) and AAV-CaMKIIα-hM4D(Gi)-EGFP (viral titers: 7.04 × 10 12 vg/mL, serotype: AAV2/9) were purchased from OBiO Technology Co.,Ltd (Shanghai, China).

Techniques: Injection, Expressing, Labeling, Activation Assay, Inhibition, Fluorescence, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Histamine-tuned subicular circuit mediates alert-driven accelerated locomotion in mice

doi: 10.1038/s41467-024-54347-2

Figure Lengend Snippet: a WT mice were injected with mixed AAV-ESARE-ERT2-Cre-ERT2, AAV-hSyn-DIO-GCaMP6m, and AAV-hSyn-DIO-hM3D(Gq)-mCherry/AAV-hSyn-DIO-hM4D(Gi)-mCherry into SUB. b Expression of hM3D-expressed (top) and hM4D-expressed (bottom) SUB treadmill training-labeled c-fos+ neuron, scale bar = 200 µm. Effects of chemogenetic activation or inhibition of SUB treadmill training-labeled c-fos+ neuron on total distance ( c ), speed ( d ) in open field test. n = 7 mice ( c : * p = 0.0218, * p = 0.0165; d : * p = 0.0197, * p = 0.0160). e – g Fluorescence value with corresponding speed, and quantification ( g ) of calcium activity of hM3D-expressed SUB treadmill training-labeled c-fos+ neuron in two groups during acceleration. n = 12 mice (**** p < 0.0001). h – j Fluorescence value with corresponding speed, and quantification ( j ) of calcium activity of hM4D-expressed SUB treadmill training-labeled c-fos+ neuron in two groups during acceleration. n = 12 mice (*** p = 0.0002, p = 0.2473). Schematic of injection of mixed AAV-ESARE-ERT2-Cre-ERT2, AAV-EF1a-DIO-EYFP into SUB of WT mice ( k ), and expression ( l ) of SUB treadmill training-labeled c-fos+ neuron in SUB with fiber distribution in RSG, AV, MMB. Scale bar = 400 µm. m HDC-CreERT2 mice were injected with AAV-CAG-FLEX-oChIEF-tdTomato into TMN, mixed AAV-ESARE-ERT2-Cre-ERT2, AAV-EF1a-DIO-EYFP into SUB to label TMN-SUB circuit-activated c-fos+ neurons combined with injection of AAV2/2Retro-hSyn-tdTomato into RSG. Viral expression in TMN and RSG, scale bar = 400 um ( n ), image and quantification ( o ) of TMN-SUB circuit-activated c-fos+ SUB neurons overlapping with Retro-tdTomato in SUB, n = 3 mice, scale bar = 100 µm. p – s Schematic diagram ( p ), image ( q ) of injection of AAV-hSyn-FLEX-ChrimsonR-tdTomato into TMN and AAV2/2Retro-hSyn-GCaMP6s into RSG of the HDC-CreERT2 mice. Scale bar = 100 µm. Fluorescence values, heatmap ( r ), and quantification ( s ) of calcium activity of RSG-projecting SUB neurons during TMN-SUB circuit activation. n = 8 mice, ** p = 0.0072. Two-tailed Student’s t test for ( c , d , g , j , s ). Schematics in a , k , m , p were drew by PowerPoint. Bar graphs represent mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: AAV-CAG-FLEX-ArchT-GFP (viral titers: 1.29 × 10 13 vg/mL, serotype: AAV2/9) and AAV-CaMKIIα-hM4D(Gi)-EGFP (viral titers: 7.04 × 10 12 vg/mL, serotype: AAV2/9) were purchased from OBiO Technology Co.,Ltd (Shanghai, China).

Techniques: Injection, Expressing, Labeling, Activation Assay, Inhibition, Fluorescence, Activity Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine dances with astrocytic Ca 2+ activity in the PZ during sleep‐wake cycles. A) Setup for bilateral fiber photometric recording of adenosine with GRAB Ado1.0 and astrocytic Ca 2+ activity within the PZ during sleep‐wake cycles. B) Representative image of GRAB Ado1.0 expression within the PZ. Scale bar, 100 µm; green, GRAB Ado1.0 . C) Schematic illustrating the principle of GRAB Ado1.0 sensors: adenosine binding induces a conformational change thereby elevating EGFP fluorescence. D) Top to bottom, EEG power spectrogram (0‐20 Hz), EEG traces, EMG traces, vigilant states (color coded), and representative astrocytic Ca 2+ activity and GRAB Ado1.0 fluorescence traces (z‐score) during sleep‐wake cycles. E) Heatmaps of GRAB Ado1.0 fluorescence traces during the 25 s before and after transitions between NREM sleep, REM sleep, and wakefulness (top). Line plots are mean ΔF/F (±s.e.m.) during state transitions under baseline conditions (bottom). Vertical, dashed lines indicate time of state transition. n = 7 mice. F, G) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB Ado1.0 (F) and GRAB Ado1.0mut (G) during each state. GRAB Ado1.0 , n = 8 mice; GRAB Ado1.0mut , n = 6 mice. F, a one‐way ANOVA test, Tukey's multiple comparisons test, * p = 0.0259, ** p = 0.0018, **** p < 0.0001. G, Kruskal‐Wallis test, Dunn's multiple comparisons test. n.s. indicates not statistically significant. H, I) Correlation analysis of GCaMP6f and GRAB Ado1.0 signals (H) and the shuffled control (I). n = 29 trials, recorded from 7 individuals. Pearson correlation analysis, *** p = 0.0002, r = 0.6352, red line shows linear regression line (Y = 0.6524*X + 0.7430), dot line indicates 95% regression range.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Activity Assay, Expressing, Binding Assay, Fluorescence, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine generated through ATP hydrolysis exerts its wakefulness‐promoting effects by binding to A 1 receptors. A) Setup for local drug delivery through bilateral canula on top of PZ in C57BL/6J mice (top). Representative image of canula location (bottom). Scale bar, 100 µm. B) Hourly percentage of time spent in wakefulness, REM sleep and NREM sleep in 5 h after the administration of NECA ( n = 6 mice) or vehicle ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. Wake: * p = 0.0496, **** p < 0.0001; REM: * p = 0.0433; NREM: **** p < 0.0001. C) Relative EEG power (0‐30 Hz) during wakefulness after the administration of NECA ( n = 4 mice) or vehicle ( n = 4 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. n.s., p = 0.4756. D) Representative image of A1 receptor expression in the PZ by immunohistochemistry staining of A 1 receptors (red) and DAPI (blue) and a zoom‐in graph (right). Scale bar, 100 µm. E) Setup for optogenetic activation of astrocytes in the PZ following the administration of CPT (A 1 receptor inhibitor). F) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of CPT or vehicle. From NREM to: vehicle group ( n = 8 rats), CPT group ( n = 5 rats); From REM or Wake to: vehicle group ( n = 6 rats), CPT group ( n = 4 rats). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. G) Setup for simultaneous bilateral fiber photometry recordings of ATP and adenosine in the PZ throughout sleep‐wake cycles by recording GRAB ATP1.0 and GRAB Ado1.0 signals. H) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, representative GRAB ATP1.0 (ATP) and GRAB Ado1.0 (Ado) fluorescence traces (z‐score) during sleep‐wake cycles; color code indicates NREM sleep, REM sleep, and wakefulness. I) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB ATP1.0 (ATP) signals during each state. n = 3 mice. A one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0080 (Wake vs REM), * p = 0.0390 (NREM vs REM). J) Correlation analysis of GRAB ATP1.0 and GRAB Ado1.0 signals (left) and the shuffled control (right). n = 31 trials, recorded from 3 individuals. Pearson correlation analysis, * p = 0.0152, r = 0.4323, red line shows linear regression line (Y = 0.2824*X+1.788), dot line indicates 95% regression range. K) Setup for optogenetic activation of astrocytes in the PZ following the administration of ARL67156 (CD73 inhibitor), NBTI (ENT inhibitor), or vehicle. L) Representative image of the expression of AAV‐GfaABC1D‐ChrimsonR‐mCherry in the PZ and the location of bilateral canula. Scale bar, 100 µm; red, ChrimsonR; blue, DAPI. M‐N) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of ARL67156 ( n = 5 mice), NBTI ( n = 5 mice), or vehicle ( n = 5 mice). Two‐way ANOVA test, Tukey's multiple comparisons test. From NREM to Wake: ** p = 0.0029 (Vehicle+OPTO vs ARL67156+OPTO), ** p = 0.0044 (ARL67156+OPTO vs NBTI+OPTO); From NREM to NREM, ** p = 0.0029 (Vehicle + OPTO vs ARL67156+OPTO), ** p = 0.0026 (ARL67156+OPTO vs NBTI+OPTO). **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Generated, Binding Assay, Expressing, Immunohistochemistry, Staining, Activation Assay, Fluorescence, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Astrocytes located in the PZ significantly contribute to the elevation of adenosine levels during wakefulness. A) Setup for fiber photometric recording of adenosine in the PZ while activating astrocytes optogenetically through a combined injection of GRAB Ado1.0 with ChrimsonR virus. B‐J) Heatmaps show GRAB Ado1.0 fluorescence traces during the 15 s before and 30 s after optogenetic stimulation in NREM sleep (B), REM sleep (E), and wakefulness (H). Line plots are mean ΔF/F (±s.e.m.) under optogenetic stimulation in NREM sleep (C), REM sleep (F), and wakefulness (I). Area under curve (AUC) of GRAB Ado1.0 signals in 15 s of pre‐, during, and post‐stimulation periods. D, NREM: n = 14 trials from 13 mice, Friedman test, Dunn's multiple comparisons test * p = 0.0245, **** p < 0.0001; G, REM: n = 10 trials from 9 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0013 (pre‐stim vs stim), *** p = 0.0002 (pre‐stim vs post‐stim), ** p = 0.0012 (stim vs post‐stim); J, Wake: n = 10 trials from 10 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0018, * p = 0.0120. K) Setup for bilateral fiber photometry recording of GRAB Ado1.0 signals in the PZ, while inhibiting unilateral astrocytic Ca 2+ activity through hPMCA2w/b expression and mCherry labeling contralaterally as control. L) Heatmaps show GRAB Ado1.0 fluorescence traces during the 10 s before and 20 s after transitions to wakefulness between hPMCA2w/b and mCherry group. M) Mean ΔF/F (±s.e.m.) during the 10 s before and 20 s after transitions to wakefulness of hPMCA2w/b ( n = 10 mice) and mCherry group ( n = 6 mice). N) Mean ΔF/F (±s.e.m.) of GRAB Ado1.0 signals between hPMCA2w/b ( n = 10 mice) and mCherry group ( n = 6 mice) during wakefulness. A two‐tailed Mann–Whitney test, * p = 0.0160.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Injection, Virus, Fluorescence, Activity Assay, Expressing, Labeling, Control, Two Tailed Test, MANN-WHITNEY

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine generated through ATP hydrolysis exerts its wakefulness‐promoting effects by binding to A 1 receptors. A) Setup for local drug delivery through bilateral canula on top of PZ in C57BL/6J mice (top). Representative image of canula location (bottom). Scale bar, 100 µm. B) Hourly percentage of time spent in wakefulness, REM sleep and NREM sleep in 5 h after the administration of NECA ( n = 6 mice) or vehicle ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. Wake: * p = 0.0496, **** p < 0.0001; REM: * p = 0.0433; NREM: **** p < 0.0001. C) Relative EEG power (0‐30 Hz) during wakefulness after the administration of NECA ( n = 4 mice) or vehicle ( n = 4 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. n.s., p = 0.4756. D) Representative image of A1 receptor expression in the PZ by immunohistochemistry staining of A 1 receptors (red) and DAPI (blue) and a zoom‐in graph (right). Scale bar, 100 µm. E) Setup for optogenetic activation of astrocytes in the PZ following the administration of CPT (A 1 receptor inhibitor). F) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of CPT or vehicle. From NREM to: vehicle group ( n = 8 rats), CPT group ( n = 5 rats); From REM or Wake to: vehicle group ( n = 6 rats), CPT group ( n = 4 rats). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. G) Setup for simultaneous bilateral fiber photometry recordings of ATP and adenosine in the PZ throughout sleep‐wake cycles by recording GRAB ATP1.0 and GRAB Ado1.0 signals. H) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, representative GRAB ATP1.0 (ATP) and GRAB Ado1.0 (Ado) fluorescence traces (z‐score) during sleep‐wake cycles; color code indicates NREM sleep, REM sleep, and wakefulness. I) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB ATP1.0 (ATP) signals during each state. n = 3 mice. A one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0080 (Wake vs REM), * p = 0.0390 (NREM vs REM). J) Correlation analysis of GRAB ATP1.0 and GRAB Ado1.0 signals (left) and the shuffled control (right). n = 31 trials, recorded from 3 individuals. Pearson correlation analysis, * p = 0.0152, r = 0.4323, red line shows linear regression line (Y = 0.2824*X+1.788), dot line indicates 95% regression range. K) Setup for optogenetic activation of astrocytes in the PZ following the administration of ARL67156 (CD73 inhibitor), NBTI (ENT inhibitor), or vehicle. L) Representative image of the expression of AAV‐GfaABC1D‐ChrimsonR‐mCherry in the PZ and the location of bilateral canula. Scale bar, 100 µm; red, ChrimsonR; blue, DAPI. M‐N) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of ARL67156 ( n = 5 mice), NBTI ( n = 5 mice), or vehicle ( n = 5 mice). Two‐way ANOVA test, Tukey's multiple comparisons test. From NREM to Wake: ** p = 0.0029 (Vehicle+OPTO vs ARL67156+OPTO), ** p = 0.0044 (ARL67156+OPTO vs NBTI+OPTO); From NREM to NREM, ** p = 0.0029 (Vehicle + OPTO vs ARL67156+OPTO), ** p = 0.0026 (ARL67156+OPTO vs NBTI+OPTO). **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Generated, Binding Assay, Expressing, Immunohistochemistry, Staining, Activation Assay, Fluorescence, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Elevated astrocytic Ca 2+ activity in the PZ during wakefulness. A) Setup for fiber photometric recording in the PZ astrocytes during sleep‐wake cycles in combination with EEG/EMG recordings. B) Representative images of AAV‐GfaABC1D‐GCaMP6f‐EYFP expression in the PZ (position of coronal section) co‐localized with GFAP, while not with NeuN. Scale bar, 50 µm; green, GCaMP6f ‐EYFP; red, GFAP; blue, NeuN. C) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, vigilant states (blue, NREM sleep; grey, REM sleep; red, wakefulness), and astrocytic Ca 2+ activity (z‐score). D) Mean ΔF/F (z‐score) of GCaMP6f during wakefulness, NREM sleep, and REM sleep. n = 14 mice, a Kruskal–Wallis test, Dunn's multiple comparisons test; Wake versus NREM * p = 0.0242, Wake versus REM **** p < 0.0001, NREM versus REM ** p = 0.0076. E) Mean ΔF/F (z‐score) of EGFP during wakefulness, NREM sleep, and REM sleep. n = 10 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, n.s. indicates not statistically significant. F) Heatmaps of Ca 2+ fluorescence traces during the 25 s before and after state transitions between NREM sleep, REM sleep, and wakefulness (top). Line plots are mean ΔF/F (±s.e.m.) during state transitions under baseline conditions (bottom). Vertical dashed lines indicate time of state transitions. n = 14 mice.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Activity Assay, Expressing, Fluorescence

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Chemogenetic and optogenetic activation of PZ astrocytes induces wakefulness. A) Setup for chemogenetic activation of astrocytes within the PZ via bilateral injection of AAV‐GfaABC1D‐hM3Dq‐mCherry virus. B) Representative images of AAV‐GfaABC1D‐hM3Dq‐mCherry expression within the PZ (position of coronal section indicated by dashed line in schematic) co‐localized with GFAP, while not with NeuN. Scale bar, 50 µm; red, hM3Dq‐mCherry; green, GFAP; blue, NeuN. C) Setup for bilateral fiber photometric recording of astrocytic Ca 2+ activity within the PZ by mixed injection of GCaMP6f with hM3Dq virus (upper left). Representative Ca 2+ fluorescent traces (z‐score) during the pre‐ and post‐injection of CNO (1 mg kg −1 ) or saline intraperitoneally (bottom). D) Area under curve (AUC) of astrocytic Ca 2+ activity (z‐score) between CNO and saline group. n = 9 trials recorded from 3 mice, an unpaired two‐tailed t ‐test, ** p = 0.0093. E–G) Hourly percentages (±s.e.m.) of wakefulness (E), NREM sleep (F), and REM sleep (G) of saline ( n = 6 mice) and CNO group ( n = 6 mice) during ZT0‐ZT24. Two‐way ANOVA test, Sidak's multiple comparisons test, Wake: * p = 0.0122 (ZT7), **** p < 0.0001 (ZT8), * p = 0.0107 (ZT9); NREM: ** p = 0.0088 (ZT7), *** p = 0.0001 (ZT8), ** p = 0.0090 (ZT9); REM: ** p = 0.0053 (ZT8), * p = 0.0203 (ZT9), ** p = 0.0041 (ZT10), * p = 0.0160 (ZT11). H,I) Representative graph of state changes in 5 h after saline (H) or CNO (I) injection. Top to bottom, EMG traces, vigilant states (color coded), EEG power spectrogram (0‐30 Hz), and delta wave power (µV 2 ). J) Total time spent in each state during ZT7‐ZT10 between saline ( n = 6 mice) and CNO group ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001 (Wake), *** p = 0.0004 (REM), **** p < 0.0001 (NREM). K) Mean episode duration of each state in 3 h after CNO or saline injection (ZT7‐ZT10). Each pair of dots, data from one mouse (saline, n = 6 mice; CNO, n = 6 mice). Wake: a two‐tailed Wilcoxon rank test, * p = 0.0313; NREM: a two‐tailed paired t test, *** p = 0.0003; REM: a two‐tailed Wilcoxon rank test, * p = 0.0313.L) Episode number of each state in 3 h after CNO or saline injection (ZT7‐ZT10). Saline, n = 6 mice; CNO, n = 6 mice. Wake: a two‐tailed paired t test, *** p = 0.0003; NREM: a two‐tailed paired t test, *** p = 0.0003; REM: a two‐tailed Wilcoxon rank test, * p = 0.0313. M) Setup for optogenetic activation of astrocytes by embedding optical fiber on top of PZ in GFAP‐ChR2‐EYFP rats. N) Representative images of ChR2‐EYFP expression in the PZ (position of coronal section indicated by dashed line in schematic) co‐localized with GFAP, while not with NeuN. Scale bar, 100 µm; green, ChR2‐EYFP; red, GFAP; blue, NeuN. O–Q) Left, example graphs of optogenetic activation of PZ astrocytes respectively in NREM sleep (O), REM sleep (P), and wakefulness (Q). Top to bottom, brain states (color coded), EEG power spectrogram (0–30 Hz), EEG, and EMG traces. Light blue shading indicates 15 s blue laser stimulation. Right, comparison of relative EEG power (±s.e.m.) of 0–30 Hz between 15 s pre‐ and during stimulation. Red line indicates statistically significant. Two‐way ANOVA test, Bonferroni's multiple comparisons test. R–T) Probability of state transitions with and without optogenetic activation of astrocytes. n = 5 rats. Two‐way ANOVA test, Sidak's multiple comparisons test, IP: *** p = 0.0006 (REM to Wake), *** p = 0.0006 (REM to REM); AP: *** p = 0.0005 (REM to Wake), *** p = 0.0005 (REM to REM), **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Activation Assay, Injection, Virus, Expressing, Activity Assay, Saline, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Inhibition of astrocytic Ca 2+ activities in the PZ with hPMCA2w/b reduced wakefulness. A) hPMCA2w/b bilateral injection to attenuate astrocytic Ca 2+ activity in the PZ mediated by continuous calcium pumping. B) Representative images of AAV‐GfaABC1D‐hPMCA2w/b‐mCherry expression in the PZ labeling with GFAP (upper) and NeuN (down). Scale bar, 20 µm; red, hPMCA2w/b; green, GFAP or NeuN. C, D) Mean (±s.e.m.) ΔF/F (z‐score) of astrocytic Ca 2+ activity during the 50 s before and after the transition from REM sleep (C) or NREM sleep (D) to wakefulness between hPMCA2w/b and mCherry group. n = 5 mice. E) Mean ΔF/F (z‐score) of astrocytic Ca 2+ activity between hPMCA2w/b and mCherry group during wakefulness. n = 5 mice, paired two‐tailed t‐test, * p = 0.0363. F–H) Hourly percentages (±s.e.m.) of time spent in wakefulness (F), NREM sleep (G), and REM sleep (H) of mice that received mCherry virus injections into PZ (control, n = 8 mice), and littermates that received hPMCA2w/b virus injections (hPMCA2w/b, n = 10 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, * p = 0.0238 (NREM), * p = 0.0287 (wake). I) Percentages of total time spent in each state during IP and AP. (control, n = 9 mice; hPMCA2w/b, n = 11 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, * p = 0.0418 (wake), * p = 0.0347 (NREM). J–L) Mean episode duration spent in wakefulness (J), NREM sleep (K), and REM sleep (L) between control and hPMCA2w/b group during IP, AP, and 24h. (control, n = 8 mice; hPMCA2w/b, n = 10 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. M–O) Episode number of wakefulness (M), NREM sleep (N), and REM sleep (O) between control and hPMCA2w/b group during IP, AP, and 24h. (control, n = 8 mice; hPMCA2w/b, n = 10 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, Wake, ** p = 0.0015, *** p = 0.0004; NREM, ** p = 0.0016, *** p = 0.0004. P,Q) Number of state transitions including NREM sleep to REM sleep (N‐R), REM sleep to wakefulness (R‐W), NREM sleep to wakefulness (N‐W), and wakefulness to NREM sleep (W‐N) between control and hPMCA2w/b group during IP (P) and AP (Q). (control, n = 6 mice; hPMCA2w/b, n = 8 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, *** p = 0.0005, **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Inhibition, Injection, Activity Assay, Expressing, Labeling, Two Tailed Test, Virus, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine generated through ATP hydrolysis exerts its wakefulness‐promoting effects by binding to A 1 receptors. A) Setup for local drug delivery through bilateral canula on top of PZ in C57BL/6J mice (top). Representative image of canula location (bottom). Scale bar, 100 µm. B) Hourly percentage of time spent in wakefulness, REM sleep and NREM sleep in 5 h after the administration of NECA ( n = 6 mice) or vehicle ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. Wake: * p = 0.0496, **** p < 0.0001; REM: * p = 0.0433; NREM: **** p < 0.0001. C) Relative EEG power (0‐30 Hz) during wakefulness after the administration of NECA ( n = 4 mice) or vehicle ( n = 4 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. n.s., p = 0.4756. D) Representative image of A1 receptor expression in the PZ by immunohistochemistry staining of A 1 receptors (red) and DAPI (blue) and a zoom‐in graph (right). Scale bar, 100 µm. E) Setup for optogenetic activation of astrocytes in the PZ following the administration of CPT (A 1 receptor inhibitor). F) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of CPT or vehicle. From NREM to: vehicle group ( n = 8 rats), CPT group ( n = 5 rats); From REM or Wake to: vehicle group ( n = 6 rats), CPT group ( n = 4 rats). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. G) Setup for simultaneous bilateral fiber photometry recordings of ATP and adenosine in the PZ throughout sleep‐wake cycles by recording GRAB ATP1.0 and GRAB Ado1.0 signals. H) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, representative GRAB ATP1.0 (ATP) and GRAB Ado1.0 (Ado) fluorescence traces (z‐score) during sleep‐wake cycles; color code indicates NREM sleep, REM sleep, and wakefulness. I) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB ATP1.0 (ATP) signals during each state. n = 3 mice. A one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0080 (Wake vs REM), * p = 0.0390 (NREM vs REM). J) Correlation analysis of GRAB ATP1.0 and GRAB Ado1.0 signals (left) and the shuffled control (right). n = 31 trials, recorded from 3 individuals. Pearson correlation analysis, * p = 0.0152, r = 0.4323, red line shows linear regression line (Y = 0.2824*X+1.788), dot line indicates 95% regression range. K) Setup for optogenetic activation of astrocytes in the PZ following the administration of ARL67156 (CD73 inhibitor), NBTI (ENT inhibitor), or vehicle. L) Representative image of the expression of AAV‐GfaABC1D‐ChrimsonR‐mCherry in the PZ and the location of bilateral canula. Scale bar, 100 µm; red, ChrimsonR; blue, DAPI. M‐N) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of ARL67156 ( n = 5 mice), NBTI ( n = 5 mice), or vehicle ( n = 5 mice). Two‐way ANOVA test, Tukey's multiple comparisons test. From NREM to Wake: ** p = 0.0029 (Vehicle+OPTO vs ARL67156+OPTO), ** p = 0.0044 (ARL67156+OPTO vs NBTI+OPTO); From NREM to NREM, ** p = 0.0029 (Vehicle + OPTO vs ARL67156+OPTO), ** p = 0.0026 (ARL67156+OPTO vs NBTI+OPTO). **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Generated, Binding Assay, Expressing, Immunohistochemistry, Staining, Activation Assay, Fluorescence, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine dances with astrocytic Ca 2+ activity in the PZ during sleep‐wake cycles. A) Setup for bilateral fiber photometric recording of adenosine with GRAB Ado1.0 and astrocytic Ca 2+ activity within the PZ during sleep‐wake cycles. B) Representative image of GRAB Ado1.0 expression within the PZ. Scale bar, 100 µm; green, GRAB Ado1.0 . C) Schematic illustrating the principle of GRAB Ado1.0 sensors: adenosine binding induces a conformational change thereby elevating EGFP fluorescence. D) Top to bottom, EEG power spectrogram (0‐20 Hz), EEG traces, EMG traces, vigilant states (color coded), and representative astrocytic Ca 2+ activity and GRAB Ado1.0 fluorescence traces (z‐score) during sleep‐wake cycles. E) Heatmaps of GRAB Ado1.0 fluorescence traces during the 25 s before and after transitions between NREM sleep, REM sleep, and wakefulness (top). Line plots are mean ΔF/F (±s.e.m.) during state transitions under baseline conditions (bottom). Vertical, dashed lines indicate time of state transition. n = 7 mice. F, G) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB Ado1.0 (F) and GRAB Ado1.0mut (G) during each state. GRAB Ado1.0 , n = 8 mice; GRAB Ado1.0mut , n = 6 mice. F, a one‐way ANOVA test, Tukey's multiple comparisons test, * p = 0.0259, ** p = 0.0018, **** p < 0.0001. G, Kruskal‐Wallis test, Dunn's multiple comparisons test. n.s. indicates not statistically significant. H, I) Correlation analysis of GCaMP6f and GRAB Ado1.0 signals (H) and the shuffled control (I). n = 29 trials, recorded from 7 individuals. Pearson correlation analysis, *** p = 0.0002, r = 0.6352, red line shows linear regression line (Y = 0.6524*X + 0.7430), dot line indicates 95% regression range.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Activity Assay, Expressing, Binding Assay, Fluorescence, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine generated through ATP hydrolysis exerts its wakefulness‐promoting effects by binding to A 1 receptors. A) Setup for local drug delivery through bilateral canula on top of PZ in C57BL/6J mice (top). Representative image of canula location (bottom). Scale bar, 100 µm. B) Hourly percentage of time spent in wakefulness, REM sleep and NREM sleep in 5 h after the administration of NECA ( n = 6 mice) or vehicle ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. Wake: * p = 0.0496, **** p < 0.0001; REM: * p = 0.0433; NREM: **** p < 0.0001. C) Relative EEG power (0‐30 Hz) during wakefulness after the administration of NECA ( n = 4 mice) or vehicle ( n = 4 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. n.s., p = 0.4756. D) Representative image of A1 receptor expression in the PZ by immunohistochemistry staining of A 1 receptors (red) and DAPI (blue) and a zoom‐in graph (right). Scale bar, 100 µm. E) Setup for optogenetic activation of astrocytes in the PZ following the administration of CPT (A 1 receptor inhibitor). F) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of CPT or vehicle. From NREM to: vehicle group ( n = 8 rats), CPT group ( n = 5 rats); From REM or Wake to: vehicle group ( n = 6 rats), CPT group ( n = 4 rats). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. G) Setup for simultaneous bilateral fiber photometry recordings of ATP and adenosine in the PZ throughout sleep‐wake cycles by recording GRAB ATP1.0 and GRAB Ado1.0 signals. H) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, representative GRAB ATP1.0 (ATP) and GRAB Ado1.0 (Ado) fluorescence traces (z‐score) during sleep‐wake cycles; color code indicates NREM sleep, REM sleep, and wakefulness. I) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB ATP1.0 (ATP) signals during each state. n = 3 mice. A one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0080 (Wake vs REM), * p = 0.0390 (NREM vs REM). J) Correlation analysis of GRAB ATP1.0 and GRAB Ado1.0 signals (left) and the shuffled control (right). n = 31 trials, recorded from 3 individuals. Pearson correlation analysis, * p = 0.0152, r = 0.4323, red line shows linear regression line (Y = 0.2824*X+1.788), dot line indicates 95% regression range. K) Setup for optogenetic activation of astrocytes in the PZ following the administration of ARL67156 (CD73 inhibitor), NBTI (ENT inhibitor), or vehicle. L) Representative image of the expression of AAV‐GfaABC1D‐ChrimsonR‐mCherry in the PZ and the location of bilateral canula. Scale bar, 100 µm; red, ChrimsonR; blue, DAPI. M‐N) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of ARL67156 ( n = 5 mice), NBTI ( n = 5 mice), or vehicle ( n = 5 mice). Two‐way ANOVA test, Tukey's multiple comparisons test. From NREM to Wake: ** p = 0.0029 (Vehicle+OPTO vs ARL67156+OPTO), ** p = 0.0044 (ARL67156+OPTO vs NBTI+OPTO); From NREM to NREM, ** p = 0.0029 (Vehicle + OPTO vs ARL67156+OPTO), ** p = 0.0026 (ARL67156+OPTO vs NBTI+OPTO). **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Generated, Binding Assay, Expressing, Immunohistochemistry, Staining, Activation Assay, Fluorescence, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Astrocytes located in the PZ significantly contribute to the elevation of adenosine levels during wakefulness. A) Setup for fiber photometric recording of adenosine in the PZ while activating astrocytes optogenetically through a combined injection of GRAB Ado1.0 with ChrimsonR virus. B‐J) Heatmaps show GRAB Ado1.0 fluorescence traces during the 15 s before and 30 s after optogenetic stimulation in NREM sleep (B), REM sleep (E), and wakefulness (H). Line plots are mean ΔF/F (±s.e.m.) under optogenetic stimulation in NREM sleep (C), REM sleep (F), and wakefulness (I). Area under curve (AUC) of GRAB Ado1.0 signals in 15 s of pre‐, during, and post‐stimulation periods. D, NREM: n = 14 trials from 13 mice, Friedman test, Dunn's multiple comparisons test * p = 0.0245, **** p < 0.0001; G, REM: n = 10 trials from 9 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0013 (pre‐stim vs stim), *** p = 0.0002 (pre‐stim vs post‐stim), ** p = 0.0012 (stim vs post‐stim); J, Wake: n = 10 trials from 10 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0018, * p = 0.0120. K) Setup for bilateral fiber photometry recording of GRAB Ado1.0 signals in the PZ, while inhibiting unilateral astrocytic Ca 2+ activity through hPMCA2w/b expression and mCherry labeling contralaterally as control. L) Heatmaps show GRAB Ado1.0 fluorescence traces during the 10 s before and 20 s after transitions to wakefulness between hPMCA2w/b and mCherry group. M) Mean ΔF/F (±s.e.m.) during the 10 s before and 20 s after transitions to wakefulness of hPMCA2w/b ( n = 10 mice) and mCherry group ( n = 6 mice). N) Mean ΔF/F (±s.e.m.) of GRAB Ado1.0 signals between hPMCA2w/b ( n = 10 mice) and mCherry group ( n = 6 mice) during wakefulness. A two‐tailed Mann–Whitney test, * p = 0.0160.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Injection, Virus, Fluorescence, Activity Assay, Expressing, Labeling, Control, Two Tailed Test, MANN-WHITNEY

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Elevated astrocytic Ca 2+ activity in the PZ during wakefulness. A) Setup for fiber photometric recording in the PZ astrocytes during sleep‐wake cycles in combination with EEG/EMG recordings. B) Representative images of AAV‐GfaABC1D‐GCaMP6f‐EYFP expression in the PZ (position of coronal section) co‐localized with GFAP, while not with NeuN. Scale bar, 50 µm; green, GCaMP6f ‐EYFP; red, GFAP; blue, NeuN. C) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, vigilant states (blue, NREM sleep; grey, REM sleep; red, wakefulness), and astrocytic Ca 2+ activity (z‐score). D) Mean ΔF/F (z‐score) of GCaMP6f during wakefulness, NREM sleep, and REM sleep. n = 14 mice, a Kruskal–Wallis test, Dunn's multiple comparisons test; Wake versus NREM * p = 0.0242, Wake versus REM **** p < 0.0001, NREM versus REM ** p = 0.0076. E) Mean ΔF/F (z‐score) of EGFP during wakefulness, NREM sleep, and REM sleep. n = 10 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, n.s. indicates not statistically significant. F) Heatmaps of Ca 2+ fluorescence traces during the 25 s before and after state transitions between NREM sleep, REM sleep, and wakefulness (top). Line plots are mean ΔF/F (±s.e.m.) during state transitions under baseline conditions (bottom). Vertical dashed lines indicate time of state transitions. n = 14 mice.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Activity Assay, Expressing, Fluorescence

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Chemogenetic and optogenetic activation of PZ astrocytes induces wakefulness. A) Setup for chemogenetic activation of astrocytes within the PZ via bilateral injection of AAV‐GfaABC1D‐hM3Dq‐mCherry virus. B) Representative images of AAV‐GfaABC1D‐hM3Dq‐mCherry expression within the PZ (position of coronal section indicated by dashed line in schematic) co‐localized with GFAP, while not with NeuN. Scale bar, 50 µm; red, hM3Dq‐mCherry; green, GFAP; blue, NeuN. C) Setup for bilateral fiber photometric recording of astrocytic Ca 2+ activity within the PZ by mixed injection of GCaMP6f with hM3Dq virus (upper left). Representative Ca 2+ fluorescent traces (z‐score) during the pre‐ and post‐injection of CNO (1 mg kg −1 ) or saline intraperitoneally (bottom). D) Area under curve (AUC) of astrocytic Ca 2+ activity (z‐score) between CNO and saline group. n = 9 trials recorded from 3 mice, an unpaired two‐tailed t ‐test, ** p = 0.0093. E–G) Hourly percentages (±s.e.m.) of wakefulness (E), NREM sleep (F), and REM sleep (G) of saline ( n = 6 mice) and CNO group ( n = 6 mice) during ZT0‐ZT24. Two‐way ANOVA test, Sidak's multiple comparisons test, Wake: * p = 0.0122 (ZT7), **** p < 0.0001 (ZT8), * p = 0.0107 (ZT9); NREM: ** p = 0.0088 (ZT7), *** p = 0.0001 (ZT8), ** p = 0.0090 (ZT9); REM: ** p = 0.0053 (ZT8), * p = 0.0203 (ZT9), ** p = 0.0041 (ZT10), * p = 0.0160 (ZT11). H,I) Representative graph of state changes in 5 h after saline (H) or CNO (I) injection. Top to bottom, EMG traces, vigilant states (color coded), EEG power spectrogram (0‐30 Hz), and delta wave power (µV 2 ). J) Total time spent in each state during ZT7‐ZT10 between saline ( n = 6 mice) and CNO group ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001 (Wake), *** p = 0.0004 (REM), **** p < 0.0001 (NREM). K) Mean episode duration of each state in 3 h after CNO or saline injection (ZT7‐ZT10). Each pair of dots, data from one mouse (saline, n = 6 mice; CNO, n = 6 mice). Wake: a two‐tailed Wilcoxon rank test, * p = 0.0313; NREM: a two‐tailed paired t test, *** p = 0.0003; REM: a two‐tailed Wilcoxon rank test, * p = 0.0313.L) Episode number of each state in 3 h after CNO or saline injection (ZT7‐ZT10). Saline, n = 6 mice; CNO, n = 6 mice. Wake: a two‐tailed paired t test, *** p = 0.0003; NREM: a two‐tailed paired t test, *** p = 0.0003; REM: a two‐tailed Wilcoxon rank test, * p = 0.0313. M) Setup for optogenetic activation of astrocytes by embedding optical fiber on top of PZ in GFAP‐ChR2‐EYFP rats. N) Representative images of ChR2‐EYFP expression in the PZ (position of coronal section indicated by dashed line in schematic) co‐localized with GFAP, while not with NeuN. Scale bar, 100 µm; green, ChR2‐EYFP; red, GFAP; blue, NeuN. O–Q) Left, example graphs of optogenetic activation of PZ astrocytes respectively in NREM sleep (O), REM sleep (P), and wakefulness (Q). Top to bottom, brain states (color coded), EEG power spectrogram (0–30 Hz), EEG, and EMG traces. Light blue shading indicates 15 s blue laser stimulation. Right, comparison of relative EEG power (±s.e.m.) of 0–30 Hz between 15 s pre‐ and during stimulation. Red line indicates statistically significant. Two‐way ANOVA test, Bonferroni's multiple comparisons test. R–T) Probability of state transitions with and without optogenetic activation of astrocytes. n = 5 rats. Two‐way ANOVA test, Sidak's multiple comparisons test, IP: *** p = 0.0006 (REM to Wake), *** p = 0.0006 (REM to REM); AP: *** p = 0.0005 (REM to Wake), *** p = 0.0005 (REM to REM), **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Activation Assay, Injection, Virus, Expressing, Activity Assay, Saline, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Inhibition of astrocytic Ca 2+ activities in the PZ with hPMCA2w/b reduced wakefulness. A) hPMCA2w/b bilateral injection to attenuate astrocytic Ca 2+ activity in the PZ mediated by continuous calcium pumping. B) Representative images of AAV‐GfaABC1D‐hPMCA2w/b‐mCherry expression in the PZ labeling with GFAP (upper) and NeuN (down). Scale bar, 20 µm; red, hPMCA2w/b; green, GFAP or NeuN. C, D) Mean (±s.e.m.) ΔF/F (z‐score) of astrocytic Ca 2+ activity during the 50 s before and after the transition from REM sleep (C) or NREM sleep (D) to wakefulness between hPMCA2w/b and mCherry group. n = 5 mice. E) Mean ΔF/F (z‐score) of astrocytic Ca 2+ activity between hPMCA2w/b and mCherry group during wakefulness. n = 5 mice, paired two‐tailed t‐test, * p = 0.0363. F–H) Hourly percentages (±s.e.m.) of time spent in wakefulness (F), NREM sleep (G), and REM sleep (H) of mice that received mCherry virus injections into PZ (control, n = 8 mice), and littermates that received hPMCA2w/b virus injections (hPMCA2w/b, n = 10 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, * p = 0.0238 (NREM), * p = 0.0287 (wake). I) Percentages of total time spent in each state during IP and AP. (control, n = 9 mice; hPMCA2w/b, n = 11 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, * p = 0.0418 (wake), * p = 0.0347 (NREM). J–L) Mean episode duration spent in wakefulness (J), NREM sleep (K), and REM sleep (L) between control and hPMCA2w/b group during IP, AP, and 24h. (control, n = 8 mice; hPMCA2w/b, n = 10 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. M–O) Episode number of wakefulness (M), NREM sleep (N), and REM sleep (O) between control and hPMCA2w/b group during IP, AP, and 24h. (control, n = 8 mice; hPMCA2w/b, n = 10 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, Wake, ** p = 0.0015, *** p = 0.0004; NREM, ** p = 0.0016, *** p = 0.0004. P,Q) Number of state transitions including NREM sleep to REM sleep (N‐R), REM sleep to wakefulness (R‐W), NREM sleep to wakefulness (N‐W), and wakefulness to NREM sleep (W‐N) between control and hPMCA2w/b group during IP (P) and AP (Q). (control, n = 6 mice; hPMCA2w/b, n = 8 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, *** p = 0.0005, **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Inhibition, Injection, Activity Assay, Expressing, Labeling, Two Tailed Test, Virus, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine generated through ATP hydrolysis exerts its wakefulness‐promoting effects by binding to A 1 receptors. A) Setup for local drug delivery through bilateral canula on top of PZ in C57BL/6J mice (top). Representative image of canula location (bottom). Scale bar, 100 µm. B) Hourly percentage of time spent in wakefulness, REM sleep and NREM sleep in 5 h after the administration of NECA ( n = 6 mice) or vehicle ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. Wake: * p = 0.0496, **** p < 0.0001; REM: * p = 0.0433; NREM: **** p < 0.0001. C) Relative EEG power (0‐30 Hz) during wakefulness after the administration of NECA ( n = 4 mice) or vehicle ( n = 4 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. n.s., p = 0.4756. D) Representative image of A1 receptor expression in the PZ by immunohistochemistry staining of A 1 receptors (red) and DAPI (blue) and a zoom‐in graph (right). Scale bar, 100 µm. E) Setup for optogenetic activation of astrocytes in the PZ following the administration of CPT (A 1 receptor inhibitor). F) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of CPT or vehicle. From NREM to: vehicle group ( n = 8 rats), CPT group ( n = 5 rats); From REM or Wake to: vehicle group ( n = 6 rats), CPT group ( n = 4 rats). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. G) Setup for simultaneous bilateral fiber photometry recordings of ATP and adenosine in the PZ throughout sleep‐wake cycles by recording GRAB ATP1.0 and GRAB Ado1.0 signals. H) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, representative GRAB ATP1.0 (ATP) and GRAB Ado1.0 (Ado) fluorescence traces (z‐score) during sleep‐wake cycles; color code indicates NREM sleep, REM sleep, and wakefulness. I) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB ATP1.0 (ATP) signals during each state. n = 3 mice. A one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0080 (Wake vs REM), * p = 0.0390 (NREM vs REM). J) Correlation analysis of GRAB ATP1.0 and GRAB Ado1.0 signals (left) and the shuffled control (right). n = 31 trials, recorded from 3 individuals. Pearson correlation analysis, * p = 0.0152, r = 0.4323, red line shows linear regression line (Y = 0.2824*X+1.788), dot line indicates 95% regression range. K) Setup for optogenetic activation of astrocytes in the PZ following the administration of ARL67156 (CD73 inhibitor), NBTI (ENT inhibitor), or vehicle. L) Representative image of the expression of AAV‐GfaABC1D‐ChrimsonR‐mCherry in the PZ and the location of bilateral canula. Scale bar, 100 µm; red, ChrimsonR; blue, DAPI. M‐N) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of ARL67156 ( n = 5 mice), NBTI ( n = 5 mice), or vehicle ( n = 5 mice). Two‐way ANOVA test, Tukey's multiple comparisons test. From NREM to Wake: ** p = 0.0029 (Vehicle+OPTO vs ARL67156+OPTO), ** p = 0.0044 (ARL67156+OPTO vs NBTI+OPTO); From NREM to NREM, ** p = 0.0029 (Vehicle + OPTO vs ARL67156+OPTO), ** p = 0.0026 (ARL67156+OPTO vs NBTI+OPTO). **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Generated, Binding Assay, Expressing, Immunohistochemistry, Staining, Activation Assay, Fluorescence, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Elevated astrocytic Ca 2+ activity in the PZ during wakefulness. A) Setup for fiber photometric recording in the PZ astrocytes during sleep‐wake cycles in combination with EEG/EMG recordings. B) Representative images of AAV‐GfaABC1D‐GCaMP6f‐EYFP expression in the PZ (position of coronal section) co‐localized with GFAP, while not with NeuN. Scale bar, 50 µm; green, GCaMP6f ‐EYFP; red, GFAP; blue, NeuN. C) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, vigilant states (blue, NREM sleep; grey, REM sleep; red, wakefulness), and astrocytic Ca 2+ activity (z‐score). D) Mean ΔF/F (z‐score) of GCaMP6f during wakefulness, NREM sleep, and REM sleep. n = 14 mice, a Kruskal–Wallis test, Dunn's multiple comparisons test; Wake versus NREM * p = 0.0242, Wake versus REM **** p < 0.0001, NREM versus REM ** p = 0.0076. E) Mean ΔF/F (z‐score) of EGFP during wakefulness, NREM sleep, and REM sleep. n = 10 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, n.s. indicates not statistically significant. F) Heatmaps of Ca 2+ fluorescence traces during the 25 s before and after state transitions between NREM sleep, REM sleep, and wakefulness (top). Line plots are mean ΔF/F (±s.e.m.) during state transitions under baseline conditions (bottom). Vertical dashed lines indicate time of state transitions. n = 14 mice.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Activity Assay, Expressing, Fluorescence

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Chemogenetic and optogenetic activation of PZ astrocytes induces wakefulness. A) Setup for chemogenetic activation of astrocytes within the PZ via bilateral injection of AAV‐GfaABC1D‐hM3Dq‐mCherry virus. B) Representative images of AAV‐GfaABC1D‐hM3Dq‐mCherry expression within the PZ (position of coronal section indicated by dashed line in schematic) co‐localized with GFAP, while not with NeuN. Scale bar, 50 µm; red, hM3Dq‐mCherry; green, GFAP; blue, NeuN. C) Setup for bilateral fiber photometric recording of astrocytic Ca 2+ activity within the PZ by mixed injection of GCaMP6f with hM3Dq virus (upper left). Representative Ca 2+ fluorescent traces (z‐score) during the pre‐ and post‐injection of CNO (1 mg kg −1 ) or saline intraperitoneally (bottom). D) Area under curve (AUC) of astrocytic Ca 2+ activity (z‐score) between CNO and saline group. n = 9 trials recorded from 3 mice, an unpaired two‐tailed t ‐test, ** p = 0.0093. E–G) Hourly percentages (±s.e.m.) of wakefulness (E), NREM sleep (F), and REM sleep (G) of saline ( n = 6 mice) and CNO group ( n = 6 mice) during ZT0‐ZT24. Two‐way ANOVA test, Sidak's multiple comparisons test, Wake: * p = 0.0122 (ZT7), **** p < 0.0001 (ZT8), * p = 0.0107 (ZT9); NREM: ** p = 0.0088 (ZT7), *** p = 0.0001 (ZT8), ** p = 0.0090 (ZT9); REM: ** p = 0.0053 (ZT8), * p = 0.0203 (ZT9), ** p = 0.0041 (ZT10), * p = 0.0160 (ZT11). H,I) Representative graph of state changes in 5 h after saline (H) or CNO (I) injection. Top to bottom, EMG traces, vigilant states (color coded), EEG power spectrogram (0‐30 Hz), and delta wave power (µV 2 ). J) Total time spent in each state during ZT7‐ZT10 between saline ( n = 6 mice) and CNO group ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001 (Wake), *** p = 0.0004 (REM), **** p < 0.0001 (NREM). K) Mean episode duration of each state in 3 h after CNO or saline injection (ZT7‐ZT10). Each pair of dots, data from one mouse (saline, n = 6 mice; CNO, n = 6 mice). Wake: a two‐tailed Wilcoxon rank test, * p = 0.0313; NREM: a two‐tailed paired t test, *** p = 0.0003; REM: a two‐tailed Wilcoxon rank test, * p = 0.0313.L) Episode number of each state in 3 h after CNO or saline injection (ZT7‐ZT10). Saline, n = 6 mice; CNO, n = 6 mice. Wake: a two‐tailed paired t test, *** p = 0.0003; NREM: a two‐tailed paired t test, *** p = 0.0003; REM: a two‐tailed Wilcoxon rank test, * p = 0.0313. M) Setup for optogenetic activation of astrocytes by embedding optical fiber on top of PZ in GFAP‐ChR2‐EYFP rats. N) Representative images of ChR2‐EYFP expression in the PZ (position of coronal section indicated by dashed line in schematic) co‐localized with GFAP, while not with NeuN. Scale bar, 100 µm; green, ChR2‐EYFP; red, GFAP; blue, NeuN. O–Q) Left, example graphs of optogenetic activation of PZ astrocytes respectively in NREM sleep (O), REM sleep (P), and wakefulness (Q). Top to bottom, brain states (color coded), EEG power spectrogram (0–30 Hz), EEG, and EMG traces. Light blue shading indicates 15 s blue laser stimulation. Right, comparison of relative EEG power (±s.e.m.) of 0–30 Hz between 15 s pre‐ and during stimulation. Red line indicates statistically significant. Two‐way ANOVA test, Bonferroni's multiple comparisons test. R–T) Probability of state transitions with and without optogenetic activation of astrocytes. n = 5 rats. Two‐way ANOVA test, Sidak's multiple comparisons test, IP: *** p = 0.0006 (REM to Wake), *** p = 0.0006 (REM to REM); AP: *** p = 0.0005 (REM to Wake), *** p = 0.0005 (REM to REM), **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Activation Assay, Injection, Virus, Expressing, Activity Assay, Saline, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Inhibition of astrocytic Ca 2+ activities in the PZ with hPMCA2w/b reduced wakefulness. A) hPMCA2w/b bilateral injection to attenuate astrocytic Ca 2+ activity in the PZ mediated by continuous calcium pumping. B) Representative images of AAV‐GfaABC1D‐hPMCA2w/b‐mCherry expression in the PZ labeling with GFAP (upper) and NeuN (down). Scale bar, 20 µm; red, hPMCA2w/b; green, GFAP or NeuN. C, D) Mean (±s.e.m.) ΔF/F (z‐score) of astrocytic Ca 2+ activity during the 50 s before and after the transition from REM sleep (C) or NREM sleep (D) to wakefulness between hPMCA2w/b and mCherry group. n = 5 mice. E) Mean ΔF/F (z‐score) of astrocytic Ca 2+ activity between hPMCA2w/b and mCherry group during wakefulness. n = 5 mice, paired two‐tailed t‐test, * p = 0.0363. F–H) Hourly percentages (±s.e.m.) of time spent in wakefulness (F), NREM sleep (G), and REM sleep (H) of mice that received mCherry virus injections into PZ (control, n = 8 mice), and littermates that received hPMCA2w/b virus injections (hPMCA2w/b, n = 10 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, * p = 0.0238 (NREM), * p = 0.0287 (wake). I) Percentages of total time spent in each state during IP and AP. (control, n = 9 mice; hPMCA2w/b, n = 11 mice). Two‐way ANOVA test, Sidak's multiple comparisons test, * p = 0.0418 (wake), * p = 0.0347 (NREM). J–L) Mean episode duration spent in wakefulness (J), NREM sleep (K), and REM sleep (L) between control and hPMCA2w/b group during IP, AP, and 24h. (control, n = 8 mice; hPMCA2w/b, n = 10 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. M–O) Episode number of wakefulness (M), NREM sleep (N), and REM sleep (O) between control and hPMCA2w/b group during IP, AP, and 24h. (control, n = 8 mice; hPMCA2w/b, n = 10 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, Wake, ** p = 0.0015, *** p = 0.0004; NREM, ** p = 0.0016, *** p = 0.0004. P,Q) Number of state transitions including NREM sleep to REM sleep (N‐R), REM sleep to wakefulness (R‐W), NREM sleep to wakefulness (N‐W), and wakefulness to NREM sleep (W‐N) between control and hPMCA2w/b group during IP (P) and AP (Q). (control, n = 6 mice; hPMCA2w/b, n = 8 mice) Two‐way ANOVA test, Sidak's multiple comparisons test, *** p = 0.0005, **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Inhibition, Injection, Activity Assay, Expressing, Labeling, Two Tailed Test, Virus, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Adenosine generated through ATP hydrolysis exerts its wakefulness‐promoting effects by binding to A 1 receptors. A) Setup for local drug delivery through bilateral canula on top of PZ in C57BL/6J mice (top). Representative image of canula location (bottom). Scale bar, 100 µm. B) Hourly percentage of time spent in wakefulness, REM sleep and NREM sleep in 5 h after the administration of NECA ( n = 6 mice) or vehicle ( n = 6 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. Wake: * p = 0.0496, **** p < 0.0001; REM: * p = 0.0433; NREM: **** p < 0.0001. C) Relative EEG power (0‐30 Hz) during wakefulness after the administration of NECA ( n = 4 mice) or vehicle ( n = 4 mice). Two‐way ANOVA test, Sidak's multiple comparisons test. n.s., p = 0.4756. D) Representative image of A1 receptor expression in the PZ by immunohistochemistry staining of A 1 receptors (red) and DAPI (blue) and a zoom‐in graph (right). Scale bar, 100 µm. E) Setup for optogenetic activation of astrocytes in the PZ following the administration of CPT (A 1 receptor inhibitor). F) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of CPT or vehicle. From NREM to: vehicle group ( n = 8 rats), CPT group ( n = 5 rats); From REM or Wake to: vehicle group ( n = 6 rats), CPT group ( n = 4 rats). Two‐way ANOVA test, Sidak's multiple comparisons test, **** p < 0.0001. G) Setup for simultaneous bilateral fiber photometry recordings of ATP and adenosine in the PZ throughout sleep‐wake cycles by recording GRAB ATP1.0 and GRAB Ado1.0 signals. H) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, EMG traces, representative GRAB ATP1.0 (ATP) and GRAB Ado1.0 (Ado) fluorescence traces (z‐score) during sleep‐wake cycles; color code indicates NREM sleep, REM sleep, and wakefulness. I) Mean (±s.e.m.) ΔF/F (z‐score) of GRAB ATP1.0 (ATP) signals during each state. n = 3 mice. A one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0080 (Wake vs REM), * p = 0.0390 (NREM vs REM). J) Correlation analysis of GRAB ATP1.0 and GRAB Ado1.0 signals (left) and the shuffled control (right). n = 31 trials, recorded from 3 individuals. Pearson correlation analysis, * p = 0.0152, r = 0.4323, red line shows linear regression line (Y = 0.2824*X+1.788), dot line indicates 95% regression range. K) Setup for optogenetic activation of astrocytes in the PZ following the administration of ARL67156 (CD73 inhibitor), NBTI (ENT inhibitor), or vehicle. L) Representative image of the expression of AAV‐GfaABC1D‐ChrimsonR‐mCherry in the PZ and the location of bilateral canula. Scale bar, 100 µm; red, ChrimsonR; blue, DAPI. M‐N) Probability of state transitions during optogenetic activation of PZ astrocytes following the administration of ARL67156 ( n = 5 mice), NBTI ( n = 5 mice), or vehicle ( n = 5 mice). Two‐way ANOVA test, Tukey's multiple comparisons test. From NREM to Wake: ** p = 0.0029 (Vehicle+OPTO vs ARL67156+OPTO), ** p = 0.0044 (ARL67156+OPTO vs NBTI+OPTO); From NREM to NREM, ** p = 0.0029 (Vehicle + OPTO vs ARL67156+OPTO), ** p = 0.0026 (ARL67156+OPTO vs NBTI+OPTO). **** p < 0.0001.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Generated, Binding Assay, Expressing, Immunohistochemistry, Staining, Activation Assay, Fluorescence, Control

Journal: Advanced Science

Article Title: Adenosine‐Dependent Arousal Induced by Astrocytes in a Brainstem Circuit

doi: 10.1002/advs.202407706

Figure Lengend Snippet: Astrocytes located in the PZ significantly contribute to the elevation of adenosine levels during wakefulness. A) Setup for fiber photometric recording of adenosine in the PZ while activating astrocytes optogenetically through a combined injection of GRAB Ado1.0 with ChrimsonR virus. B‐J) Heatmaps show GRAB Ado1.0 fluorescence traces during the 15 s before and 30 s after optogenetic stimulation in NREM sleep (B), REM sleep (E), and wakefulness (H). Line plots are mean ΔF/F (±s.e.m.) under optogenetic stimulation in NREM sleep (C), REM sleep (F), and wakefulness (I). Area under curve (AUC) of GRAB Ado1.0 signals in 15 s of pre‐, during, and post‐stimulation periods. D, NREM: n = 14 trials from 13 mice, Friedman test, Dunn's multiple comparisons test * p = 0.0245, **** p < 0.0001; G, REM: n = 10 trials from 9 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0013 (pre‐stim vs stim), *** p = 0.0002 (pre‐stim vs post‐stim), ** p = 0.0012 (stim vs post‐stim); J, Wake: n = 10 trials from 10 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0018, * p = 0.0120. K) Setup for bilateral fiber photometry recording of GRAB Ado1.0 signals in the PZ, while inhibiting unilateral astrocytic Ca 2+ activity through hPMCA2w/b expression and mCherry labeling contralaterally as control. L) Heatmaps show GRAB Ado1.0 fluorescence traces during the 10 s before and 20 s after transitions to wakefulness between hPMCA2w/b and mCherry group. M) Mean ΔF/F (±s.e.m.) during the 10 s before and 20 s after transitions to wakefulness of hPMCA2w/b ( n = 10 mice) and mCherry group ( n = 6 mice). N) Mean ΔF/F (±s.e.m.) of GRAB Ado1.0 signals between hPMCA2w/b ( n = 10 mice) and mCherry group ( n = 6 mice) during wakefulness. A two‐tailed Mann–Whitney test, * p = 0.0160.

Article Snippet: [ ] AAV‐GfaABC1D‐hPMCA2w/b‐mCherry (AAV2/5, 7.0 × 10 12 genomic copies mL −1 ) and AAV‐GfaABC1D‐mCherry (AAV2/5, 6.0 × 10 12 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0 (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB Ado1.0mut (AAV2/9, 1.52 × 10 13 genomic copies mL −1 ), AAV‐hsyn‐GRAB ATP1.0 (AAV2/9, 2 × 10 13 genomic copies mL −1 ), AAV‐GfaABC1D‐ChrimsonR‐mCherry (AAV2/5, 1.51 × 10 13 genomic copies mL −1 ) were constructed by Vigene Biosciences (Shandong, China).

Techniques: Injection, Virus, Fluorescence, Activity Assay, Expressing, Labeling, Control, Two Tailed Test, MANN-WHITNEY