thy1.2 promotor-chr2-venus transgenic rat lines  (BioResource International Inc)

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: For in vivo optogenetic astrocyte stimulations, astrocyte‐specific ChR2‐expressing rats (GFAP‐ChR2‐EYFP rats) were used with Sprague–Dawley background generated by the Institute of Neuroscience, Chinese Academy of Sciences.

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: 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: For in vivo optogenetic astrocyte stimulations, astrocyte‐specific ChR2‐expressing rats (GFAP‐ChR2‐EYFP rats) were used with Sprague–Dawley background generated by the Institute of Neuroscience, Chinese Academy of Sciences.

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: For in vivo optogenetic astrocyte stimulations, astrocyte‐specific ChR2‐expressing rats (GFAP‐ChR2‐EYFP rats) were used with Sprague–Dawley background generated by the Institute of Neuroscience, Chinese Academy of Sciences.

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: Astrocyte activation inhibits NREM‐promoting and PB‐projecting GABAergic neurons in the PZ. A) Setup for optogenetic activation of terminals from PZ GABA neurons projecting to the PB in combination with EEG/EMG recordings during sleep‐wake cycles. B) Top to bottom, EEG power spectrogram (0–20 Hz), EEG traces, and EMG traces throughout sleep‐wake cycles; color code indicates NREM sleep and wakefulness. Red shade indicates optogenetic activation (635 nm, 1 mW, 30 s, 40 Hz, 5 ms). C) Comparison of the probability of state transitions between the baseline condition and optogenetic activation. Two‐way ANOVA test, Sidak's multiple comparisons test, *** p = 0.0002. D) Relative EEG power (0–30 Hz) during natural NREM sleep ( n = 5 mice) and optogenetic activation ( n = 5 mice). Two‐way ANOVA test, Bonferroni's multiple comparisons test. E) Setup for fiber photometric recording of the terminal Ca 2+ activity from PZ GABA neurons projecting to the PB in response to the optogenetic activation of PZ astrocytes. F) Representative image of the terminal CCaMP6 m expression from PZ GABA neurons projecting to the PB (position of coronal section). Scale bar, 50 µm; green, CCaMP6 m; blue, DAPI. G) Heatmap shows CCaMP6 m fluorescence traces during the 50 s before and 100 s after optogenetic activation of PZ astrocytes. n = 3 mice. H) Mean (±s.e.m.) ΔF/F (z‐score) during the 50 s before and 100 s after optogenetic activation (red shade) of PZ astrocytes. n = 3 mice. I) Area under curve (AUC) of CCaMP6 m signals in 30 s of pre‐, during, and post‐stimulation periods. n = 3 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, * p = 0.0326 (pre‐stim vs stim). J) Setup for fiber photometric recording of the terminal EYFP fluorescence from PZ GABA neurons projecting to the PB in response to the optogenetic activation of PZ astrocytes. K) Heatmap shows EYFP fluorescence traces during the 50 s before and 100 s after optogenetic activation of PZ astrocytes. n = 3 mice. L) Mean (±s.e.m.) ΔF/F (z‐score) during the 50 s before and 100 s after optogenetic activation (red shade) of PZ astrocytes. n = 3 mice. M) AUC of EYFP fluorescence in 30 s of pre‐, during, and post‐stimulation periods. n = 3 mice, a one‐way ANOVA test, Tukey's multiple comparisons test. N) Setup for fiber photometric recording of extracellular GABA in the PB by recording iGABASnFR.st signals in combination with EEG/EMG recordings during sleep‐wake cycles. O) Representative image of iGABASnFR.st expression in the PB (position of coronal section). Scale bar, 100 µm; green, iGABASnFR.st; blue, DAPI. P) Top to bottom, EEG power spectrogram (0‐20 Hz), EEG traces, EMG traces, and representative iGABASnFR.st fluorescence traces (z‐score) during sleep‐wake cycles; color code indicates vigilant states. Q) Mean ΔF/F (z‐score) of iGABASnFR.st signals during NREM sleep, REM sleep, and wakefulness. n = 3 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, * p = 0.0357 (NREM vs Wake), * p = 0.0147 (NREM vs REM). R) Setup for fiber photometric recording of extracellular GABA in the PB in response to the optogenetic activation of terminals from PZ GABA neurons projecting to the PB. S) Heatmap shows iGABASnFR.st fluorescence traces during the 50 s before and 100 s after optogenetic activation of terminals from PZ GABA neurons projecting to the PB. n = 3 mice. T) Mean (±s.e.m.) ΔF/F (z‐score) during the 50 s before and 100 s after optogenetic activation (red shade) of terminals from PZ GABA neurons projecting to the PB. n = 3 mice. U) AUC of iGABASnFR.st signals in 30 s of pre‐, during and post‐stimulation periods. n = 3 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, * p = 0.0193 (pre‐stim vs stim), * p = 0.0201 (pre‐stim vs post‐stim). V) Setup for fiber photometric recording of extracellular GABA in the PB in response to the optogenetic activation of astrocytes in the PZ. W) Heatmap shows iGABASnFR.st fluorescence traces during the 50 s before and 100 s after optogenetic activation of astrocytes in the PZ. n = 3 mice. X) Mean (±s.e.m.) ΔF/F (z‐score) during the 50 s before and 100 s after optogenetic activation (red shade) of astrocytes in the PZ. n = 3 mice. Y) AUC of iGABASnFR.st signals in 30 s of pre‐, during and post‐stimulation periods. n = 4 mice, a one‐way ANOVA test, Tukey's multiple comparisons test, ** p = 0.0021 (pre‐stim vs stim), * p = 0.0340 (stim vs post‐stim).

Article Snippet: For in vivo optogenetic astrocyte stimulations, astrocyte‐specific ChR2‐expressing rats (GFAP‐ChR2‐EYFP rats) were used with Sprague–Dawley background generated by the Institute of Neuroscience, Chinese Academy of Sciences.

Techniques: Activation Assay, Comparison, Activity Assay, Expressing, Fluorescence

Journal: eLife

Article Title: Activation of astrocytes in hippocampus decreases fear memory through adenosine A 1 receptors

doi: 10.7554/eLife.57155

Figure Lengend Snippet: Example video of Ca 2+ imaging from a slice expressing ChR2-EYFP.

Article Snippet: GFAP-ChR2-EYFP rats (Sprague-Dawley background) were generated at the Institute of Neuroscience, Chinese Academy of Sciences.

Techniques:

Journal: eLife

Article Title: Activation of astrocytes in hippocampus decreases fear memory through adenosine A 1 receptors

doi: 10.7554/eLife.57155

Figure Lengend Snippet:

Article Snippet: GFAP-ChR2-EYFP rats (Sprague-Dawley background) were generated at the Institute of Neuroscience, Chinese Academy of Sciences.

Techniques: Virus, ATP Assay, Software