Microglia facilitate and stabilize the response to general anesthesia via modulating the neuronal network in a brain region-specific manner
- Department of Neurosurgery, Huashan Hospital, Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, MOE Innovative Center for New Drug Development of Immune Inflammatory Diseases, Fudan University, China
- School of Basic Medical Sciences, Jinzhou Medical University, China
- Department of Neurology, Zhongshan Hospital, Department of Laboratory Animal Science, MOE Frontiers Center for Brain Science, Fudan University, China
- Co-Innovation Center of Neurodegeneration, Nantong University, China
Figures
Figure 1
Microglial depletion impedes anesthesia induction and accelerates emergence.
(A) Scheme of time points for microglial depletion and repopulation by PLX5622 and CD. (B) Mice exhibit delayed induction and early emergence in pentobarbital-, propofol-, chloral hydrate-, and ketamine-induced anesthesia. N = 11, 10, 10, and 12mice for pentobarbital, propofol, chloral hydrate, and ketamine, respectively. Repeated measures (paired) one-way ANOVA with Geisser–Greenhouse correction and Tukey’s multiple-comparison test. Data are presented as mean ± SD. PLX5622: PLX5622-formulated diet; CD: control diet; LORR: loss of righting reflex; RORR: recovery of righting reflex. All animals are male mice.
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Figure 1—source data 1
Raw data for LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig1-data1-v1.xlsx
Figure 2
Repetitive anesthetic treatment does not result in anesthesia tolerance.
(A) Scheme of time points for anesthetic treatments and righting reflex examination. (B) Repetitive treatment with pentobarbital, propofol, chloral hydrate or ketamine does not induce anesthesia tolerance in mice. N = 6, 7, 6, and 5mice are treated with pentobarbital, propofol, chloral hydrate, and ketamine, respectively. Repeated measures (paired) one-way ANOVA with Geisser–Greenhouse correction and Tukey’s multiple-comparison test. LORR: loss of righting reflex; RORR: recovery of righting reflex. All animals are male mice.
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Figure 2—source data 1
Raw data for LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig2-data1-v1.xlsx
Figure 3
CSF1R inhibition-induced general anesthesia regulation is not due to the depletion of peripheral macrophages.
(A) Scheme of time points for peripheral macrophage depletion by PLX73086. (B) CSF1R inhibition by PLX73086 dramatically ablates macrophages in the liver, lung, spleen, and kidney and does not ablate brain microglia. N = 4mice for each group. Two-tailed independent t-test. (C) Depletion of peripheral macrophages does not influence the anesthesia induction of pentobarbital, propofol, chloral hydrate, and ketamine or the emergence from propofol, chloral hydrate, and ketamine. However, it impedes anesthesia emergence from pentobarbital. N = 9, 8, 8, and 9mice for pentobarbital, propofol, chloral hydrate, and ketamine, respectively. Two-tailed paired t-test. Data are presented as mean ± SD. PLX73086: PLX73086-formulated diet; LORR: loss of righting reflex; RORR: recovery of righting reflex. All animals are male mice.
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Figure 3—source data 1
Assessment of PLX73086 influence on IBA1+ cell density in different organs.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig3-data1-v1.xlsx
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Figure 3—source data 2
Raw data for LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig3-data2-v1.xlsx
Figure 4
Electroencephalography (EEG) and electromyography (EMG) recordings reveal that mice with microglial depletion are resistant to general anesthesia by pentobarbital.
(A) Scheme of time points for animal surgery, microglial depletion, and EEG/EMG recording. (B–D) Microglial depletion shows no obvious change in EEG before the injection of pentobarbital. Instead, it influences the EEG in anesthesia induction and emergence. Two-tailed paired t-test. The gray area in (D) indicates p<0.05 between D7 and D21. (E) Microglial depletion does not change the EMG before the injection of pentobarbital. Instead, it influences the EMG in the anesthesia process. (F) Microglial depletion does not change the probability of consciousness before the injection of pentobarbital. Instead, it influences the consciousness probability in the anesthesia process. N = 9mice for each group. Data are presented as mean ± SD. RMS: root mean square; A.U.: arbitrary unit; PLX5622: PLX5622-formulated diet. All animals are male mice.
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Figure 4—source data 1
Raw data for induction and emergence times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig4-data1-v1.xlsx
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Figure 4—source data 2
Raw data for the normalized power during different stages.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig4-data2-v1.xlsx
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Figure 4—source data 3
Raw data for the RMS of EMG.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig4-data3-v1.xlsx
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Figure 4—source data 4
Raw data for the consciousness probability.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig4-data4-v1.xlsx
Figure 5
Electroencephalography (EEG) and electromyography (EMG) recordings reveal that mice with microglial depletion are resistant to general anesthesia by propofol.
(A) Scheme of time points for animal surgery, microglial depletion, and EEG/EMG recording. (B–D) Microglial depletion does not change the EEG before the injection of propofol. Instead, it influences the EEG in anesthesia induction and emergence. Two-tailed paired t-test. The gray area in (D) indicates p<0.05 between D7 and D21. (E) Microglial depletion does not change the EMG before the injection of propofol. Instead, it influences the EMG in the anesthesia process. (F) Microglial depletion does not change the probability of consciousness before the injection of propofol. Instead, it influences the consciousness probability in the anesthesia process. N = 5mice for each group. Data are presented as mean ± SD. RMS: root mean square; A.U.: arbitrary unit; PLX5622: PLX5622-formulated diet. All animals are male mice.
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Figure 5—source data 1
Raw data for induction and emergence times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig5-data1-v1.xlsx
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Figure 5—source data 2
Raw data for the normalized power during different stages.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig5-data2-v1.xlsx
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Figure 5—source data 3
Raw data for the RMS of EMG.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig5-data3-v1.xlsx
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Figure 5—source data 4
Raw data for the consciousness probability.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig5-data4-v1.xlsx
Figure 6
Electroencephalography (EEG) and electromyography (EMG) recordings reveal that mice with microglial depletion are mouse resistant to general anesthesia by ketamine.
(A) Scheme of time points for animal surgery, microglial depletion, and EEG/EMG recording. (B–D) Microglial depletion does not change the EEG before the injection of ketamine. Instead, it influences the EEG in anesthesia induction and emergence. Two-tailed paired t-test. The gray area in (D) indicates p<0.05 between D7 and D21. (E) Microglial depletion does not change the EMG before the injection of ketamine. Instead, it influences the EMG in the anesthesia process. (F) Microglial depletion does not change the probability of consciousness before the injection of ketamine. Instead, it influences the consciousness probability in the anesthesia process. N = 12mice for each group. Data are presented as mean ± SD. RMS: root mean square; A.U.: arbitrary unit; PLX5622: PLX5622-formulated diet. All animals are male mice.
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Figure 6—source data 1
Raw data for induction and emergence times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig6-data1-v1.xlsx
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Figure 6—source data 2
Raw data for the normalized power during different stages.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig6-data2-v1.xlsx
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Figure 6—source data 3
Raw data for the RMS of EMG.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig6-data3-v1.xlsx
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Figure 6—source data 4
Raw data for the consciousness probability.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig6-data4-v1.xlsx
Figure 7
Microglial depletion diversely influences neuronal activity in different anesthesia-related brain regions.
(A) Scheme of time points for microglial depletion and examination time points. (B) Influence of microglial depletion in anesthesia-activated brain regions. Microglial depletion reduces neuronal activity in the LHb (p=0.0356), SON (p=0.0203), and VLPO (p=0.1592) and does not influence neuronal activity in the TRN (p=0.9994). N = 12, 6, 9, and 12mice for LHb, SON, VLPO, and TRN in the CD group, respectively. N = 9, 5, 10, and 10mice for LHb, SON, VLPO, and TRN in the PLX5622 group, respectively. (C) Influence of microglial depletion in emergence-activated brain regions. Microglial depletion enhances neuronal activity in the PVT (p=0.0298), LC (p=0.0053), LH (p=0.0598), and VTA (p=0.1436). N = 12, 6, 12, and 12mice for PVT, LC, LH, and VTA in the CD group, respectively. N = 9, 5, 10, and 11mice for PVT, LC, LH, and VTA in the PLX5622 group, respectively. Two-tailed independent t-test. Data are presented as mean ± SD. PLX5622: PLX5622-formulated diet; CD: control diet; LHb: lateral habenula; SON: supraoptic nucleus; VLPO: ventrolateral preoptic nucleus; TRN: thalamic reticular nucleus; PVT: paraventricular thalamus; LC: locus coeruleus; LH: lateral hypothalamus; VTA: ventral tegmental area. All animals are male mice.
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Figure 7—source data 1
The influence of microglial depletion to the c-Fos+ cell density in the AABRs and EABRs.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig7-data1-v1.xlsx
Figure 8
Animal handling and intraperitoneal injection do not influence neuronal activity in anesthesia-related brain regions.
(A) Scheme of time points for microglial depletion and examination time points. (B) Animal handling and intraperitoneal injection do not influence neuronal activity in the LHb, SON, VLPO, or TRN. N = 5, 4, 5, and 4mice for LHb, SON, VLPO, and TRN in the w/o saline group, respectively. N = 5, 4, 5, and 4mice for LHb, SON, VLPO, and TRN in the w/ saline group, respectively. (C) Animal handling and intraperitoneal injection do not influence neuronal activity in the PVT, LC, LH, or VTA. N = 5, 4, 5, and 5mice for PVT, LC, LH, and VTA in the w/o saline group, respectively. N = 5, 5, 5, and 5mice for PVT, LC, LH, and VTA in the w/ saline group, respectively. Two-tailed independent t-test. Data are presented as mean ± SD. LHb: lateral habenula; SON: supraoptic nucleus; VLPO: ventrolateral preoptic nucleus; TRN: thalamic reticular nucleus; PVT: paraventricular thalamus; LC: locus coeruleus; LH: lateral hypothalamus; VTA: ventral tegmental area. All animals are male mice.
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Figure 8—source data 1
Influences of the animal handling and intraperitoneal injection to the c-Fos+ cell density in the AABRs and EABRs.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig8-data1-v1.xlsx
Figure 9
c-Fos protein and Fos mRNA dual staining dissects the influence of microglial depletion on consciousness and anesthesia states.
(A) Scheme of time points for microglial depletion and dual labeling. (B, C) The influence of microglial depletion on activated neurons in consciousness and anesthesia states in AABRs (LHb and SON) and EABRs (PVT and LC). N = 5 (LHb CD), 6 (LHb PLX5622), 5 (SON CD), 6 (SON PLX5622), 5 (PVT CD), 6 (PVT PLX5622), 5 (LC CD), and 5 (LC PLX5622) mice for each group. Two-tailed independent t-test. Data are presented as mean ± SD. PLX5622: PLX5622-formulated diet; AABRs; anesthesia-activated brain regions; EABRs; emergence-activated brain regions; CD: control diet; LHb: lateral habenula; SON: supraoptic nucleus; PVT: paraventricular thalamus; LC: locus coeruleus. All animals are male mice.
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Figure 9—source data 1
Influences of microglial depletion to the c-Fos+, Fos+, c-Fos+Fos+, c-Fos+Fos–, and c-Fos–Fos+ cells in the AABRs and EABRs.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig9-data1-v1.xlsx
Figure 10
Microglial depletion reduces the E/I ratio in SON but enhances the E/I ratio in LC.
(A) Scheme of time points for microglial depletion by PLX5622. (B) Representative traces for evoked postsynaptic currents in the SON to 10 increasing stimulation currents. (C) Amplitudes of evoked postsynaptic currents in the SON in response to increasing electrical stimulation intensities. Two-way ANOVA. Data are presented as mean ± SEM. (D) E/I ratios with different stimulation intensities in the SON. N = 21 (CD) and 19 (PLX5622) cells from fivemice for each group. Two-way ANOVA. Data are presented as mean ± SEM. (E) Representative traces (left) and quantitative results (right) show that PLX5622-treated mice exhibited a higher eEPSC PPR in the SON. N = 24 (CD) and 30 (PLX5622) cells from fivemice for each group. Two-tailed independent t-test. Data are presented as mean ± SD. (F) Representative traces (left) and quantitative results (right) show that PLX5622-treated mice exhibited a similar eIPSC PPR in the SON. N = 29 (CD) and 30 (PLX5622) cells from fivemice for each group. Two-tailed independent t-test. Data are presented as mean ± SD. (G) Representative traces for evoked postsynaptic currents in the LC in response to 10 increasing stimulation currents. (H) Amplitudes of evoked postsynaptic currents in the LC in response to increasing electrical stimulation intensities. in response to the electrical stimulation. N = 15 (EPSC CD), 18 (EPSC PLX5622), 15 (IPSC CD), and 18 (IPSC PLX5622) cells from fivemice for each group. Two-way ANOVA. Data are presented as mean ± SEM. (I) E/I ratios with different stimulation currents in the LC. N = 15 (EPSC CD), 18 (EPSC PLX5622), 15 (IPSC CD), and 18 (IPSC PLX5622) cells from fivemice for each group. Two-way ANOVA. Data are presented as mean ± SEM. (J) Representative traces (left) and quantitative results (right) show that PLX5622-treated mice exhibited a similar eEPSC PPR in the LC. N = 14 (CD) and 16 (PLX5622) cells from fivemice for each group. Two-tailed independent t-test. Data are presented as mean ± SD. (K) Representative traces (left) and quantitative results (right) show that PLX5622-treated mice exhibited a similar eIPSC PPR in the LC. N = 13 (CD) and 14 (PLX5622) cells from fivemice for each group. Two-tailed independent t-test. Data are presented as mean ± SD. PLX5622: PLX5622-formulated diet; SON: supraoptic nucleus; LC: locus coeruleus; PPR: paired-pulse ratio (PPR); CD: control diet. eEPSC: evoked excitatory postsynaptic current; eIPSC: evoked inhibitory postsynaptic current. All animals are male mice.
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Figure 10—source data 1
Raw data for eEPSC/eIPSC amplitudes and E/I ratios in the SON.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig10-data1-v1.xlsx
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Figure 10—source data 2
Raw data for eEPSC/eIPSC PPRs in the SON.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig10-data2-v1.xlsx
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Figure 10—source data 3
Raw data for eEPSC/eIPSC amplitudes and E/I ratios in the LC.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig10-data3-v1.xlsx
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Figure 10—source data 4
Raw data for eEPSC/eIPSC PPRs in the LC.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig10-data4-v1.xlsx
Figure 11
Interruption of the spine ‘eat me’ signal by C1qa–/– does not influence the anesthesia process and microglial depletion alters the proportion of spine categories.
(A) Scheme of time points for microglial depletion and examination time points. (B) CSF1R inhibition for 14d does not influence spine density but changes the proportion of spine subtypes. N = 18and 13cells from fivemice for each group of apical spines, N = 24and 19cells from fivemice for each group of basal spines. All animals are male mice. (C) Scheme of LORR and RORR tests in wild-type and C1qa–/– mice. (D) C1q knockout does not influence anesthesia induction and emergence in response to pentobarbital, propofol, and ketamine. N = 6 (pentobarbital WT; five male and one female mice), 9 (pentobarbital C1qa–/–; seven male and two female mice), 5 (propofol WT; four male and one female mice), 9 (propofol C1qa–/–; seven male and two female mice), 9 (ketamine WT; nine male mice), and 8 (ketamine C1qa–/–; eight male mice). Both sexes are used in this result. Two-tailed independent t-test. Data are presented as mean ± SD. PLX5622: PLX5622-formulated diet; CD: control diet; LORR: loss of righting reflex; RORR: recovery of righting reflex.
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Figure 11—source data 1
Raw data for the spine density and the proportion of each spine subtype.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig11-data1-v1.xlsx
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Figure 11—source data 2
Raw data for LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig11-data2-v1.xlsx
Figure 12
Microglial P2Y12 regulates the induction and emergence of anesthesia.
(A) Scheme of 2-MeSAMP administration and behavior tests for anesthesia. (B) P2Y12 inhibition by 2-MeSAMP drives microglia to a more reactive state. N = 4mice for each group. All animals are male mice. (C) P2Y12 inhibition by 2-MeSAMP results in delayed anesthesia induction and early emergence. N = 8mice for each group. All animals are male mice. (D) Scheme of animal treatment and examination time points for Cx3cr1+/CreER and Cx3cr1+/CreER::P2ry12fl/fl mice. (E) Tamoxifen induces efficient P2Y12 knockout in Cx3cr1+/CreER::P2ry12fl/fl mice. N = 7mice for the Cx3cr1+/CreER group and 4mice for the Cx3cr1+/CreER::P2ry12fl/fl group. All animals are male mice. (F) Efficient knockout of P2Y12 significantly elongates the LORR and shortens the RORR. N = 14mice (eleven male and three female mice) for the Cx3cr1+/CreER group and 15mice (eleven male and four female mice) for the Cx3cr1+/CreER::P2ry12fl/fl group. (G) Scheme of animal treatment and examination time points for Tmem119CreER/CreER and Tmem119CreER/CreER::P2ry12fl/fl mice. (H) Tamoxifen induces relatively low efficiency of P2Y12 knockout in Tmem119CreER/CreER::P2ry12fl/fl mice. N = 5mice for the Tmem119CreER/CreER group and 3mice for the Tmem119CreER/CreER::P2ry12fl/fl group. All animals are male mice. (I) Low-efficiency knockout of P2Y12 does not affect anesthesia induction but significantly shortens the emergence time. N = 9mice (six male and three female mice) for the Tmem119CreER/CreER group and 11mice (seven male and four female mice) for the Tmem119CreER/CreER::P2ry12fl/fl group. Two-tailed independent t-test. Data are presented as mean ± SD. ICV: intracerebroventricular; OG: oral gavage; LORR: loss of righting reflex; RORR: recovery of righting reflex.
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Figure 12—source data 1
Influences of 2-MeSAMP treatment to the microglial morphology and LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig12-data1-v1.xlsx
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Figure 12—source data 2
Knockout efficiency of Cx3cr1+/CreER::P2ry12fl/fl mice and the influence of high-efficiency P2Y12 knockout to LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig12-data2-v1.xlsx
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Figure 12—source data 3
Knockout efficiency of Tmem119CreER/CreER::P2ry12fl/fl mice and the influence of low-efficiency P2Y12 knockout to LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig12-data3-v1.xlsx
Figure 13
Mice with P2Y12– Mr BMT cells display delayed anesthesia induction and early emergence.
(A) Scheme of microglia replacement by Mr BMT and behavior tests for anesthesia. (B) Mr BMT cells exhibit a P2Y12– phenotype. N = 6mice for each group. (C) P2Y12– microglia lead to delayed anesthesia induction and early emergence. N = 6mice for the control group, 6mice for the Mr BMT (IRR) group, and 6mice for the Mr BMT (BU) group. Two-tailed independent t-test. Data are presented as mean ± SD. IP: intraperitoneal injection; Mr BMT: microglia replacement by bone marrow transplantation; BMT: bone marrow transplantation; Ctrl: control; IRR: irradiation; BU: busulfan; LORR: loss of righting reflex; RORR: recovery of righting reflex. All animals are female mice.
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Figure 13—source data 1
Percentages of P2Y12+ IBA1+ cells over IBA1+ cells.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig13-data1-v1.xlsx
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Figure 13—source data 2
Raw data for LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig13-data2-v1.xlsx
Figure 14
General anesthesia is regulated by intracellular calcium in microglia.
(A) Scheme of animal treatment and examination time points for Cx3cr1+/CreER and Cx3cr1+/CreER::hM3Dq-YFP+/– mice. (B) Tamoxifen induces high Cre-dependent recombination in Cx3cr1+/CreER::hM3Dq-YFP+/– mice. N = 5mice for the vehicle group and 6mice for the tamoxifen group. All animals are male mice. (C) Elevation of microglial intracellular Ca2+ results in a shorter anesthesia induction time and longer emergence time. N = 13 (LORR Cx3cr1+/CreER; eleven male and two female mice), 14 (LORR Cx3cr1+/CreER::hM3Dq-YFP+/–; ten male and four female mice), 12 (RORR Cx3cr1+/CreER; ten male and two female mice), and 11mice (RORR Cx3cr1+/CreER::hM3Dq-YFP+/–; seven male and 4 female mice) per group. (D) Scheme of animal treatment and examination time points for Cx3cr1+/CreER and Cx3cr1+/CreER::Stim1fl/fl mice. (E) qPCR results reveal decreased Stim1 transcription in Cx3cr1+/CreER::Stim1fl/fl mouse brains. N = 6mice (five male and one female mice) for the Cx3cr1+/CreER group and 5mice (three male and two female mice) for the Cx3cr1+/CreER::Stim1fl/fl group. (F) Downregulation of microglial intracellular Ca2+ results in longer anesthesia induction time and shorter emergence time. N = 7 Cx3cr1+/CreER (five male and two female mice) and 5 Cx3cr1+/CreER::Stim1fl/fl mice (three male and two female mice) per group. Two-tailed independent t-test. Data are presented as mean ± SD. OG: oral gavage; IP: intraperitoneal injection; TAM: tamoxifen; LORR: loss of righting reflex; RORR: recovery of righting reflex.
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Figure 14—source data 1
Efficiency of Cre-dependent recombination in Cx3cr1+/CreER::hM3Dq-YFP+/– mice and the influence of Ca2+-elevated microglia to LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig14-data1-v1.xlsx
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Figure 14—source data 2
Assessment of Stim1 knockout efficiency in Cx3cr1+/CreER::Stim1fl/fl mice and the influence of Ca2+-reduced microglia to LORR and RORR times.
- https://cdn.elifesciences.org/articles/92252/elife-92252-fig14-data2-v1.xlsx
Figure 15
Schematic summary of this study.
This figure summarizes the major findings of this study.
Videos
Video 1
PLX5622-treated mice displayed a longer time for LORR compared to the naïve mice.
Video 2
PLX5622-treated mice displayed a shorter time for RORR compared to the naïve mice.
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Microglia facilitate and stabilize the response to general anesthesia via modulating the neuronal network in a brain region-specific manner
eLife 12:RP92252.
https://doi.org/10.7554/eLife.92252.2
