Cerebral blood flow is modulated by astrocytic cAMP elevation independently of IP3R2-mediated Ca2+ signaling in mice

Abstract

Local neural activation drives regional increase of cerebral blood flow (CBF), in a phenomenon known as functional hyperemia. Astrocytes, which enwrap cerebral blood vessels and respond to neuronal activity through their G protein-coupled receptors (GPCRs), play a vital role in brain energy metabolism. Although astrocytic calcium (Ca²⁺) signaling has been widely studied in relation to neurovascular coupling, the role of cyclic adenosine monophosphate (cAMP), another key second messenger of GPCRs, on CBF has not been established. In this study, we explored the effects of optogenetically induced astrocytic cAMP elevation on CBF. We engineered adeno-associated viral vectors (AAVs) to express a bacterial photoactivated adenylyl cyclase in astrocytes, which triggers an increase in cAMP upon blue light stimulation. Opto-stimulation also elevated astrocytic Ca²⁺, albeit with a delayed onset under mild stimulation. In vivo imaging of anesthetized and awake wild-type mice through a thinned skull preparation revealed that optogenetically induced astrocytic cAMP elevation led to pronounced arteriole dilation, with a latency of 1.8 s and maximal dilation reached within 10 s in the awake state and slower response under anesthesia. Mild opto-stimulation causing sensory-level cAMP elevations was sufficient to induce arteriole dilation. This effect was preserved in IP₃ receptor type 2-knockout (IP₃R2^-/-) mice, indicating a mechanism independent of GPCR-induced intracellular Ca²⁺ elevations. These findings highlight astrocytic cAMP as a key modulator of cerebral vasodilation, contributing to our understanding of local CBF regulation. This study opens broad avenues for understanding astrocyte-mediated control of CBF and its implications in neurological diseases characterized by dysregulated blood flow.

Keywords: astrocytes; blood flow; cAMP elevation; neurovascular coupling; optogenetic GPCR.

Fig. 1.

Opto-stimulation of bPAC induces astrocytic cAMP increase in anesthetized mice. (A) Experiment timeline. (B) Corrected normalized Pink Flamindo signal following 1-s, 0.2-s, and 3-s bPAC stimulation (n = 4 mice). (C) Representative images of raw (Upper) and normalized Pink Flamindo fluorescence signal (Lower) following 1-s bPAC stimulation. (Scale bar, 500 µm.) (D) Representative raw Pink Flamindo fluorescence intensity following 1-s bPAC stimulation. (E) A representative trace for background- and photobleaching-corrected normalized Pink Flamindo signal following 1-s bPAC stimulation. Peak is the maximal amplitude. Rise time (highlighted in green) is the time interval for Pink Flamindo to peak, 80% decay time (highlighted in pink) is the time interval for Pink Flamindo to decay to 80% of its peak value and response duration is the sum of rise time and decay time. (F) Differentials of Pink Flamindo signal following 1-s, 0.2-s, and 3-s bPAC stimulation (n = 4 mice). (G−J) Peak (G), rise time (H), decay time (I), and response duration (J) for Pink Flamindo following 1-s (recording #1), 0.2-s, 3-s, and 1-s (recording #4) bPAC stimulation (n = 4 mice for recordings #1−3, 3 mice for #4). Two-sample paired t tests followed by correction for false discovery rate (alpha = 0.05) with the Benjamini–Hochberg procedure: *P < 0.05. (K) Example images of endogenous fluorescence signals from cortical brain slices of mice injected with PHP.eB/mGFAP(ABC1D)-2pabPAC(F198Y) and PHP.eB/mGFAP(ABC1D)-Lck-PinkFlamindo. bPAC and Pink Flamindo are both expressed in astrocytes. (Scale bar, 100 µm.) Graphs plot means ± SEM and individual values.

Fig. 2.

Opto-stimulation of bPAC induces arteriole dilation in anesthetized mice. (A) Experiment timeline. (B) Relative arteriole diameter changes following 1-s, 0.2-s, and 3-s bPAC stimulation (n = 3 arterioles from 2 mice; 1-2 arterioles/mouse). (C) Representative images of Pink Flamindo and Alb-mScarlet fluorescence signals in astrocytes and vasculature, respectively. A rectangular ROI was defined around the arteriole of interest, and the mean pixel intensity was plotted over time to create a kymograph. The distance between the arteriole borders (highlighted in green in the kymograph) was measured to compute the arteriole diameter changes following 1-s bPAC stimulation. (Scale bar, 100 µm.) (D) Area under the curve for the relative arteriole diameter changes measured in the first 38.6 s post-LED stimulation, i.e., half cAMP response duration (n = 3 arterioles from 2 mice; 1-2 arterioles/mouse). One-way ANOVA (F = 0.0041, P = 0.0041) followed by post hoc Tukey’s multiple comparisons test: *P < 0.05 and **P < 0.01. (E) Representative differential of relative arteriole diameter changes following 1-s bPAC stimulation. Positive differential values indicate dilation, whereas negative values indicate constriction. Arteriole dilation duration (highlighted in green) is defined as the time interval where the arteriole dilates for the first time. Maximum dilation rate is the maximal amplitude of this dilation event. (F−H) Differentials of relative arteriole diameter changes following 1-s, 0.2-s, and 3-s bPAC stimulation. Arteriole dilation duration (G) and maximum dilation rate (H) for the differentials measured in the first 38.6 s post-LED stimulation (n = 3 arterioles from 2 mice; 1-2 arterioles/mouse). One-way ANOVA (F = 9.904, P = 0.0126 for dilation duration and F = 41.91, P = 0.0003 for maximum dilation rate) followed by post hoc Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01 and ***P < 0.001. (I) Arteriole dilation duration plotted versus cAMP response duration (Left) maximum dilation rate plotted versus cAMP peak (Right). (J) cAMP response and arteriole dilation following 1-s bPAC stimulation in anesthetized mice (Left) and their differentials (Right). The black dotted lines indicate the time-to-peak (t) for the arteriole dilation (Left) and the delay (d) between cAMP response and arteriole dilation (Right). Graphs plot means ± SEM and individual values.

Fig. 3.

Opto-stimulation of bPAC induces astrocytic cAMP increase and arteriole dilation in awake mice. (A) Experiment timeline. (B−D) cAMP imaging. Corrected normalized Pink Flamindo signal (B), peak (C), and response duration (D) following 1-s bPAC stimulation in awake WT mice (n = 5 mice). Mixed-effects analysis using a REML model: F_{1.823, 6.685} = 0.7991, P = 0.4775 for peak (C); F_{0.6271, 2.299} = 3.251, P = 0.1797 for response duration (D). (E−I) Arteriole imaging. Relative arteriole diameter changes (E) following 1-s bPAC stimulation in bPAC and control WT mice. Area under the curve (F) measured in the first 38.6 s post-LED stimulation, i.e., half cAMP response duration. Differentials of relative arteriole diameter changes (G). Arteriole dilation duration (H) and maximum dilation rate (I) for the differentials measured in the first 38.6 s post-LED stimulation. N = 33 arterioles from 5 bPAC mice and 31 arterioles from 5 ctrl mice; 5 to 8 arterioles/mouse. Unpaired t test: t = 3.445, df = 62, **P < 0.01 (F), t = 1.171, df = 62, P = 0.2462 (H); t = 2.754, df = 62, **P < 0.01 (I). (J) Arteriole dilation duration plotted versus cAMP response duration (Left) and maximum dilation rate plotted versus cAMP peak (Right). (K) cAMP response and arteriole dilation following 1-s bPAC stimulation in awake mice (Left) and their differentials (Right). The black dotted lines indicate the time-to-peak (t) for the arteriole dilation (Left) and the delay (d) between cAMP response and arteriole dilation (Right). Graphs plot means ± SEM and individual values.

Fig. 4.

bPAC-induced arteriole dilation is independent from large astrocytic Ca²⁺ signals. (A−E) cAMP imaging. Corrected normalized Pink Flamindo signal (A), peak (B) rise time (C), decay time (D), and response duration (E) following 1-s bPAC stimulation in awake IP₃R2^−/− and IP₃R2^+/+ mice (n = 6 mice per group). Two-way ANOVA for peak (B): F_{1, 10} = 0.5064, P = 0.4930 for genotype; F_{2.640, 26.40} = 0.5748, P = 0.6156 for recording; F_{3, 30} = 1.626, P = 0.2042 for genotype × recording. Two-way ANOVA for rise time (C): F_{1, 10} = 0.6943, P = 0.4242 for genotype; F_{1.996, 19.96} = 2.297, P = 0.1266 for recording; F_{3, 30} = 1.143, P = 0.3478 for genotype × recording. Two-way ANOVA for decay time (D): F_{1, 10} = 0.9511, P = 0.3524 for genotype; F_{1.622, 16.22} = 2.411, P = 0.1285 for recording; F_{3, 30} = 0.3636, P = 0.7797 for genotype × recording. Two-way ANOVA for response duration (E): F_{1, 10} = 1.139, P = 0.3109 for genotype; F_{1.620, 16.20} = 2.262, P = 0.1427 for recording; F_{3, 30} = 0.3115, P = 0.8169 for genotype × recording. (F−I) Arteriole imaging. Relative arteriole diameter changes (F) following 1-s bPAC stimulation in IP₃R2^−/− and IP₃R2^+/+ mice. Area under the curve (G) measured in the first 38.6 s post-LED stimulation. Arteriole dilation duration (H) and maximum dilation rate (I) for the differentials measured in the first 38.6 s post-LED stimulation. N = 41 arterioles from 6 IP₃R2^−/− mice, 34 arterioles from 5 IP₃R2^+/+ mice; 5 to 8 arterioles/mouse. Unpaired t test: t = 0.0048, df = 34, P = 0.9962 (G), t = 0.7425, df = 34, P = 0.4629 (H); t = 0.5705, df = 34, P = 0.5721 (I). (J) Arteriole dilation duration plotted versus cAMP response duration (Left) and maximum dilation rate plotted versus cAMP peak (Right). (K) For both IP₃R2^−/− (green header) and IP₃R2^+/+ mice (gray header), cAMP response and arteriole dilation following 1-s bPAC stimulation (Left) and their differentials (Right). The black dotted lines indicate the time-to-peak (t) for the arteriole dilation (Left) and the delay (d) between cAMP response and arteriole dilation (Right). Graphs plot means ± SEM and individual values.

Fig. 5.

bPAC opto-stimulation induces astrocytic Ca²⁺ increase. (A) Experiment timeline. (B−D) Imaging in ketamine–xylazine anesthetized WT mice. Normalized RCaMP3 signal (B), peak (C), and response duration (D) following 1-s, 0.2-s, and 3-s bPAC stimulation (n = 5 mice). Repeated measures one-way ANOVA for recording: F_{1.464, 5.857} = 7.181, P = 0.0311 for peak (C); F_{1.327, 5.307} = 13.51, P = 0.0108 for response duration (D). (E−G) Imaging in awake habituated WT mice. Normalized RCaMP3 signal (E), peak (F), and response duration (G) following 1-s bPAC stimulation (n = 5 mice). Repeated measures one-way ANOVA for recording: F_{1.843, 7.373} = 5.083, P = 0.0428 for peak (F); F_{1.702, 6.810} = 2.069, P = 0.1989 for response duration (G). (H−J) Imaging in awake habituated IP₃R2^−/− and IP₃R2^+/+ mice. Normalized RCaMP3 signal (H), peak (I), and response duration (J) following 1-s bPAC stimulation (n = 4 IP₃R2^−/− and 3 IP₃R2^+/+ mice). Mixed-effects analysis using a REML model for peak (I): F_{1, 3} = 4.477, P = 0.1247 for genotype; F_{3, 9} = 13.37, P = 0.0012 for recording; F_{3, 4} = 6.162, P = 0.0557 for genotype × recording. Two-way ANOVA for response duration (J): F_{1, 5} = 324.4, P < 0.0001 for genotype; F_{1.563, 7.815} = 6.509, P = 0.0259 for recording; F_{3, 15} = 6.856, P = 0.0040 for genotype × recording. Graphs plot means ± SEM and individual values.

Fig. 6.

Mild bPAC activation increases astrocytic cAMP and Ca²⁺ to promote arteriole dilation. (A) Experiment timeline. (B) Calcium response, cAMP response, and arteriole dilation following 25-ms bPAC stimulation in anesthetized mice. The black dotted lines indicate the time-to-peak for cAMP response (t₂), calcium response (t₃), and arteriole dilation (t₁) from stimulation onset. (C−E) cAMP imaging. Corrected normalized Pink Flamindo signal (C), peak (D), and response duration (E) following 25-ms bPAC stimulation (n = 4 mice). Paired t test: t = 1.141, df = 3, P = 0.3368 (D); t = 1.214, df = 3, P = 0.3117 (E). (F−H) Ca²⁺ imaging. Normalized RCaMP3 signal (F), peak (G), and response duration (H) following 25-ms bPAC stimulation (n = 4 mice). Repeated measures one-way ANOVA for recording: F_{1.188, 3.563} = 29.66, P = 0.0072 for peak (G); F_{1.193, 3.580} = 41.22, P = 0.0041 for response duration (H). (I−L) Arteriole imaging. Relative arteriole diameter changes (I) following 25-ms bPAC stimulation, with zoomed-in area used for quantification. Area under the curve (J) measured in the first 14 s post-LED stimulation. Arteriole dilation duration (K) and maximum dilation rate (L) for the differentials measured in the first 14 s post-LED stimulation. N = 13 arterioles from 6 bPAC mice and 14 arterioles from 4 ctrl mice (I and J) or 12 arterioles from 5 bPAC mice and 10 arterioles from 4 ctrl mice (K and L); 1 to 5 arterioles/mouse. Unpaired t test: t = 2.925, df = 25, **P < 0.01 (J); t = 1.389, df = 20, P = 0.1802 (K); t = 2.045, df = 20, P = 0.0543 (L). Graphs plot means ± SEM and individual values.