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.2012 Nov 13;109(46):18974-9.
doi: 10.1073/pnas.1209448109. Epub 2012 Oct 29.

General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex

Affiliations

General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex

Alexander Stanley Thrane et al. Proc Natl Acad Sci U S A..

Abstract

Calcium signaling represents the principle pathway by which astrocytes respond to neuronal activity. General anesthetics are routinely used in clinical practice to induce a sleep-like state, allowing otherwise painful procedures to be performed. Anesthetic drugs are thought to mainly target neurons in the brain and act by suppressing synaptic activity. However, the direct effect of general anesthesia on astrocyte signaling in awake animals has not previously been addressed. This is a critical issue, because calcium signaling may represent an essential mechanism through which astrocytes can modulate synaptic activity. In our study, we performed calcium imaging in awake head-restrained mice and found that three commonly used anesthetic combinations (ketamine/xylazine, isoflurane, and urethane) markedly suppressed calcium transients in neocortical astrocytes. Additionally, all three anesthetics masked potentially important features of the astrocyte calcium signals, such as synchronized widespread transients that appeared to be associated with arousal in awake animals. Notably, anesthesia affected calcium transients in both processes and soma and depressed spontaneous signals, as well as calcium responses, evoked by whisker stimulation or agonist application. We show that these calcium transients are inositol 1,4,5-triphosphate type 2 receptor (IP(3)R2)-dependent but resistant to a local blockade of glutamatergic or purinergic signaling. Finally, we found that doses of anesthesia insufficient to affect neuronal responses to whisker stimulation selectively suppressed astrocyte calcium signals. Taken together, these data suggest that general anesthesia may suppress astrocyte calcium signals independently of neuronal activity. We propose that these glial effects may constitute a nonneuronal mechanism for sedative action of anesthetic drugs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
General anesthesia depresses and desynchronizes spontaneous calcium transients in cortical astrocytes. (A) Two-photon laser scanning microscopy (2PLSM) was used to image spontaneous astrocyte calcium transients in awake and anesthetized head-restrained mice. Neuronal activity was recorded using an ECoG microelectrode. (B) Cortical astrocytes were identified by eGFP expressed under theGlt1 promoter and loaded with calcium indicator rhod-2. Representative images are shown before (Left) and during (Center) a spontaneous calcium transient. (Scale bar: 60 μm.) Rhod-2 signals were normalized to eGFP fluorescence to reduce movement artifacts (dotted line represents +2 SDs) (Right). (C) ECoG and ΔF/F0 traces of spontaneous calcium transients illustrate the effects of anesthetic induction (isoflurane 1.5%) on neuronal and astrocyte signaling, respectively. (D,Left) Representative ECoG recordings from awake and anesthetized mice. (Right) Representative ECoG power spectra illustrating the shift to slower frequency neuronal activity induced by anesthesia. (E) Bar graph summarizing the effects of different anesthetics on spontaneous calcium signals. *P < 0.0001 [n = 75 (awake),n = 30 (isoflurane),n = 25 (ketamine), andn = 20 (urethane) cells from 13 animals; pairedt test]. (F) Reciprocal changes in ECoG power ratio and astrocyte calcium-transient frequency following anesthesia induction (isoflurane 1.5%). *P < 0.01 (n = 13 animals; Kruskal–Wallis test); **P < 0.001 (n = 75 cells; pairedt test). (G) Pearson product–moment correlation for ΔF/F0 in different astrocytes (cells). *P < 0.0001 [n = 75 (awake),n = 30 (isoflurane),n = 25 (ketamine), andn = 20 (urethane) cells; pairedt test]. (H) Cell–cell correlation increases as a function of proximity. (Left) Representative astrocytes joined by lines whose thicknesses represent the strength of their ΔF/F0 correlation. (Scale bar: 30 μm.) (Right) Regression of cell–cell correlation of astrocyte pairs on distance [R2 = 0.34 (awake) andR2 = 0.0091 (anesthetized);n = 75 pairs for each group]. (I,Upper) False-color images illustrating calcium transient in an astrocyte process. (Lower) Rhod-2–intensity traces (ΔF/F0) for subcellular regions of interest (ROI) are shown. (Scale bar: 10 μm.) (J) Mean frequency of calcium transients in astrocyte processes is decreased in anesthetized mice. *P < 0.001 [n = 32 (awake),n = 12 (isoflurane),n = 9 (ketamine), andn = 11 (urethane) cells from 13 animals; pairedt test]. (K,Left) Representative ECoG recordings following drug application, anesthetic induction, or IP3R2 deletion. (Right) Spontaneous calcium activity following TTX (100 μM), CNQX (200 μM)/AP5 (500 μM), and PPADS (100 μM)/suramin (300 μM) application and in the same animals after isoflurane anesthesia (1.5%) and in IP3R2 KO mice. *P < 0.01, **P < 0.001 [n = 212 (control),n = 80 (TTX),n = 79 (CNQX/AP5),n = 66 (PPADS/suramin),n = 149 (anesthesia), andn = 88 (IP3R2 KO) cells from 17 animals; pairedt test (before vs. after drug) and unpairedt test (WT vs. KO)]. Data are shown as means ± SEM.
Fig. 2.
Fig. 2.
Astrocyte calcium responses evoked by whisker stimulation are diminished by general anesthesia. (A) Astrocyte calcium responses and ECoG neuronal responses to whisker stimulation (w.s.) were recorded before, during, and after general anesthesia. (B) False-color image illustrating a calcium response to whisker stimulation. (Scale bar: 60 μm.) (C) Representative ECoG and ΔF/F0 calcium responses to whisker stimulation before and after anesthetic induction (1.0% isoflurane). (D) Mean calcium-transient frequency and ECoG response sum induced by whisker stimulation when increasing doses of isoflurane are administered. *P < 0.001 [n = 53 (awake),n = 28 (0.5% isoflurane), andn = 25 (1.5% isoflurane) cells from 6 animals; one-way ANOVA]. (E) Effect of different anesthetics on the probability of finding an active astrocyte (≥1 transient in the first 3 min) following whisker stimulation. *P < 0.001 [n = 12 (awake),n = 12 (isoflurane),n = 4 (ketamine), andn = 4 (urethane) animals; Wilcoxon signed-rank test]. (F) Mean calcium-transient frequency induced by different types of whisker stimulation in awake (gray bars) and anesthetized mice (red bars), including air puffs to the C6 whisker (on target) or the incorrect whisker (off-target). *P < 0.001;P = 0.389 (off vs. on-target;n = 53 cells for each group; one-way ANOVA). (G) Astrocyte calcium response amplitude (ΔF/F0) regressed on the neural response sum in awake and anesthetized mice. (H) The calcium response to whisker stimulation partially recovers after anesthetic withdrawal. *P < 0.01, **P < 0.001 (n = 53 cells; pairedt test). (I) Representative ECoG and ΔF/F0 traces in awake WT mice exposed to TTX (100 μM), CNQX (200 μM)/AP5 (500 μM), and PPADS (100 μM)/suramin (300 μM) or isoflurane anesthesia (1.5%) and in IP3R2 KO mice. (J andK) Bar graphs summarizing the effect of drugs and IP3R2 KO on calcium-transient frequency and amplitude, respectively. *P < 0.01, **P < 0.001 [n = 212 (control),n = 80 (TTX),n = 79 (CNQX/AP5),n = 66 (PPADS/suramin),n = 149 (anesthesia), andn = 88 (IP3R2 KO) cells from 17 animals; pairedt test (before vs. after drug) and unpairedt test (WT vs. KO)]. Data are shown as means ± SEM.
Fig. 3.
Fig. 3.
Astrocyte calcium mobilization in response to ATP stimulation is impaired in anesthetized animals. (A) ATP was microinjected into the cortex in vivo using a fine glass electrode, and astrocyte calcium responses were recorded using 2PLSM before, during, and after general anesthesia. (B) Representative false-color images illustrate the calcium response to ATP application. To standardize the distance from ATP microinjection to the astrocytes chosen for analysis, two concentric circles were drawn (radius, 25 and 100 μm), and cells within this range were used. (Scale bar: 30 μm.) (C) Representative ΔF/F0 traces of ATP-evoked calcium transients in awake and anesthetized mice. (D) Bar graph summarizing the effect of anesthesia on ATP-evoked calcium signals. *P < 0. 01 (awake vs. anesthesia); **P < 0.001 (anesthesia vs. recovery) (n = 137 cells; pairedt test). (E) Representative individual ATP-responses illustrating the detailed effects of anesthesia. (F andG) Mean calcium-response amplitude and duration, respectively, before and after anesthesia induction. *P < 0.01 (n = 137 cells; pairedt test). Data are shown as means ± SEM.
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