Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance methods
Aim. Evidence-backed execution summary for Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance methods from Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Histology
reagent used in the protocol.
- Use
- Virus injection of AAV-DJ/8-EF1a-DIO-EYFP in the LC was performed as described above. Two weeks after virus injection, mice were sacrificed by transcardiac perfusion. Briefly, 25 mL saline (0.9% NaCl) was perfused for 5 min, followed by 50 mL of fixative (4% paraformaldehyde in 0.1M phosphate buffe...
Imaging and head-fixed fear conditioning
reagent used in the protocol.
- Use
- In a subset of nLight imaging experiments, a selective NA reuptake inhibitor desipramine (10 mg/kg; Sigma-Aldrich) was injected intraperitoneally 30 min before imaging and compared with images taken before the drug application. Images were acquired at 2 Hz.
Histology
reagent used in the protocol.
- Use
- For glycogen staining, AAV9-GFAP-GFP or rAAV8-GFAP-Ha-rM3D-IRES-mCitrine-injected mice were sacrificed by focused microwave irradiation (5 kW for 1 s) 2 h after CNO (1 mg/kg, i.p.) injection. Briefly, after incubating the brain in 4% PFA overnight, sections containing the hippocampus was slic...
Temporally distinct astrocytic Ca 2+ and cAMP surges by NA
To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm wavelength laser which while optimized towards Pink Flamindo permits the observation of GCaMP responses with a comparable dynamic range. We...
- Use
- To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm wavelength laser which while optimized towards Pink Flamindo permits the observation of GCaMP responses with a comparable dynamic range. We...
Temporally distinct astrocytic Ca 2+ and cAMP surges by NA
To gain more insight into the differential time course of Ca 2+ and cAMP dynamics, we examined Ca 2+ and cAMP responses during longer PS (120 s), mimicking a saturated extracellular NA environment. To accommodate two-photon image acquisition we introduced brief photostimulus-free periods during the PS (Fig....
- Use
- To gain more insight into the differential time course of Ca 2+ and cAMP dynamics, we examined Ca 2+ and cAMP responses during longer PS (120 s), mimicking a saturated extracellular NA environment. To accommodate two-photon image acquisition we introduced brief photostimulus-free periods during the PS (Fig....
Prolonged vigilance induces astrocytic cAMP increases
To place our observations into a behavioral context, we imaged astrocytic Ca 2+ and cAMP dynamics in the auditory cortex of unanesthetized mice. Previous studies have shown that startle causes large Ca 2+ response in cortical astrocytes. Therefore, we first tested whether a startle response drives not only Ca 2+ bu...
- Use
- To place our observations into a behavioral context, we imaged astrocytic Ca 2+ and cAMP dynamics in the auditory cortex of unanesthetized mice. Previous studies have shown that startle causes large Ca 2+ response in cortical astrocytes. Therefore, we first tested whether a startle response drives not only Ca 2+ bu...
Prolonged vigilance induces astrocytic cAMP increases
We employed a cued fear conditioning paradigm in head-fixed mice to permit imaging of the cortex throughout the course of experiment (Fig. ). For fear conditioning, a foot shock (FS, 0.7 mA, 1 s) was delivered to the mouse during the last one second of the sound cue (Sound), whereas only Sound was...
- Use
- We employed a cued fear conditioning paradigm in head-fixed mice to permit imaging of the cortex throughout the course of experiment (Fig. ). For fear conditioning, a foot shock (FS, 0.7 mA, 1 s) was delivered to the mouse during the last one second of the sound cue (Sound), whereas only Sound was...
Methods
Adult mice (postnatal 2-4 months) were anesthetized with ketamine and xylazine (70 and 10 mg/kg, respectively, i.p.) for introduction and kept under stable anesthesia with isoflurane (0.5-1.0%) until the end of surgery. Surgery was performed using a stereotaxic apparatus. After a small cranial hole...
- Use
- Adult mice (postnatal 2-4 months) were anesthetized with ketamine and xylazine (70 and 10 mg/kg, respectively, i.p.) for introduction and kept under stable anesthesia with isoflurane (0.5-1.0%) until the end of surgery. Surgery was performed using a stereotaxic apparatus. After a small cranial hole...
Methods
For head-fixed fear conditioning experiments, additional surgery for electromyography (EMG) electrode implantation was performed. One week after the preparation of cranial window, tungsten wires (50 µm in diameter) were inserted in neck muscles and fixed with dental cement. After surgery, mice were kept o...
- Use
- For head-fixed fear conditioning experiments, additional surgery for electromyography (EMG) electrode implantation was performed. One week after the preparation of cranial window, tungsten wires (50 µm in diameter) were inserted in neck muscles and fixed with dental cement. After surgery, mice were kept o...
Histology
Virus injection of AAV-DJ/8-EF1a-DIO-EYFP in the LC was performed as described above. Two weeks after virus injection, mice were sacrificed by transcardiac perfusion. Briefly, 25 mL saline (0.9% NaCl) was perfused for 5 min, followed by 50 mL of fixative (4% paraformaldehyde in 0.1M phosphate buffe...
- Use
- Virus injection of AAV-DJ/8-EF1a-DIO-EYFP in the LC was performed as described above. Two weeks after virus injection, mice were sacrificed by transcardiac perfusion. Briefly, 25 mL saline (0.9% NaCl) was perfused for 5 min, followed by 50 mL of fixative (4% paraformaldehyde in 0.1M phosphate buffe...
Imaging and head-fixed fear conditioning
Two-photon imaging was performed using a B-scope (Thorlabs) with a Chameleon Vision 2 laser (Coherent) or a Bergamo (Thorlabs) with a Chameleon Ultra 2 laser (Coherent). For imaging of GCaMP or nLight alone, an excitation wavelength of 920 nm was used. For single or dual imaging involving Pink Flamindo or RCaM...
- Use
- Two-photon imaging was performed using a B-scope (Thorlabs) with a Chameleon Vision 2 laser (Coherent) or a Bergamo (Thorlabs) with a Chameleon Ultra 2 laser (Coherent). For imaging of GCaMP or nLight alone, an excitation wavelength of 920 nm was used. For single or dual imaging involving Pink Flamindo or RCaM...
Temporally distinct astrocytic Ca 2+ and cAMP surges by NA
Software used for acquisition, scoring, statistics, or reporting.
- Use
- To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm wavelength laser which while optimized towards Pink Flamindo permits the observation of GCaMP responses with a comparable dynamic range. We...
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All statistical analyses were done in origin. Comparisons between two groups were analyzed with Student's t -test with Welch's correction or paired t -test. For comparisons of data between before and after a manipulation, paired t -test was applied. For other comparisons of two sample means, Student̵...
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High vigilance induced prolonged NAergic activity
The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning. LC/NA axon Ca 2+ activity was imaged with the same paradigm as in Fig.. We found that MP signals were prevalent during the first ten seconds after the initial FS compared to the pre-FS period (Fig. ) (before: 2.82 ± 0.21 s, FS: 3.9 ± 0.54 s). However, this effect disappeared in later sessions (Fig. ) (before: 3.03 ± 0.28 s, FS: 3.07 ± 1.20 s), consistent with the reduced astrocytic Ca 2+ response we observed. We further analyzed LC/NA axonal Ca 2+ activity during the initial FS as illustrated in Fig.. Whereas the mean of individual MP signal durations was similar in all phases (Fig. ), MP signals were observed more frequently during post-FS1 co...
Temporally distinct astrocytic Ca 2+ and cAMP surges by NA
To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm wavelength laser which while optimized towards Pink Flamindo permits the observation of GCaMP responses with a comparable dynamic range. We employed two patterns of PS, which provided equal stimulation duration but with distinct temporal patterns. In the "repetitive" stimulation regime, 3-s PS, which induced Ca 2+ elevations but not cAMP elevations in astrocytes, was repeated 10 × with 7 s inter-stimulus intervals, whereas in the "single" continuous stimulation regime, 30 s of continuous PS was applied. The repetitive stimulation induced significant Ca 2+ elevations in astrocytes (Δ F / F 120.0 ± 6.6%) as well as a small elevation of cAMP (104.4̴...
Temporally distinct astrocytic Ca 2+ and cAMP surges by NA
To gain more insight into the differential time course of Ca 2+ and cAMP dynamics, we examined Ca 2+ and cAMP responses during longer PS (120 s), mimicking a saturated extracellular NA environment. To accommodate two-photon image acquisition we introduced brief photostimulus-free periods during the PS (Fig., see Methods). Under these conditions, we obtained distinct time courses for Ca 2+ and cAMP signals during activation of NAergic fibers. Ca 2+ activity showed an immediate increase after the start of PS (time to peak: 11.3 ± 1.3 s) and gradually decreased to basal levels after 70 s despite the presence of additional PS. Ca 2+ increase was not observed thereafter (Fig. ). On the other hand, cAMP signals took 30-40 s (32.9 ± 4.2 s) to reach the peak and did not return to basal level during the PS (Fig....
Bursting NAergic activity induces astrocytic Ca 2+ surges
To understand the mechanism behind the temporally distinct astrocytic second messenger dynamics, we imaged cortical NAergic axonal activities with a faster responsive Ca 2+ probe GCaMP6.f (Fig., Supplementary Fig. ). In awake conditions, cortical NAergic axons exhibited continual Ca 2+ activities in awake mice in a range of approximately 0.5-0.6 Hz (Fig. ) (0.56 ± 0.04 Hz), whereas such activities were not observable under deep isoflurane anesthesia (Supplementary Fig. ). To reveal how this NAergic activity influences astrocytes, we simultaneously imaged astrocytic and NAergic axonal Ca 2+ activities with R-CaMP1.07 and GCaMP6.f, respectively. We observed two types of NAergic Ca 2+ activity patterns: single peak (SP) signals whereby Δ F / F returns to the basal level before next firing initiation and multipeak (MP) signa...
Prolonged vigilance induces astrocytic cAMP increases
To place our observations into a behavioral context, we imaged astrocytic Ca 2+ and cAMP dynamics in the auditory cortex of unanesthetized mice. Previous studies have shown that startle causes large Ca 2+ response in cortical astrocytes. Therefore, we first tested whether a startle response drives not only Ca 2+ but also cAMP increases. In this experiment, mice received unpredictable air puffs onto the right side of the face for 10 s while the auditory cortex was imaged (Fig. ). In stark contrast to the expected astrocytic Ca 2+ elevations ( F / F 0 162.2 ± 15.6%) (Fig. ), we observed that air puffs did not lead to significant cAMP increases ( F / F 0 104.0 ± 1.7%) (Fig. ). These responses were qualitatively similar to the short stimulation of NA axons demonstrated in Fig. (e.g. 3- or 5-s PS), suggesting that transient a...
High vigilance induced prolonged NAergic activity
Finally, to investigate functional importance related to astrocytic cAMP signaling, we induced activated Gs signaling using a Gs-type DREADD (designer receptors exclusively activated by designer drugs) expressed in astrocytes via AAV8-GFAP-HA-rM3D-mCitrine (Fig. ). A clear sustained cAMP elevation in astrocytes was observed by Pink Flamindo after CNO (Clozapine-N-Oxide) injection, lasting more than 2 h (Fig. ) (0 min: 99.2 ± 0.6%, 60 min: 180.6 ± 21.4%, 160 min: 174.9 ± 16.6%). Considering that one of the downstream functions of cAMP signaling in astrocytes is glycogenolysis, we visualized glycogen after CNO injection by immunohistochemistry. We confirmed that mice with rM3D-mCitrine expression in cortical astrocytes had distinctively lower levels of glycogen compared to control animals (Fig....
Methods
Adult mice (postnatal 2-4 months) were anesthetized with ketamine and xylazine (70 and 10 mg/kg, respectively, i.p.) for introduction and kept under stable anesthesia with isoflurane (0.5-1.0%) until the end of surgery. Surgery was performed using a stereotaxic apparatus. After a small cranial hole was made above the cerebellum at the stereotaxic coordinate of AP -5.5 mm, ML +0.9 mm by a dental drill. Intracranial microinjection of AAV (AAV-DJ/8-EF1a-DIO-ChR2-EYFP, AAV-DJ/8-EF1a-DIO-GCaMP6.f, or AAV-DJ/8-EF1a-DIO-EYFP (1-3 × 10 12 vg/mL) was conducted using a glass micropipette connected to a Femtojet microinjector (Eppendorf) at three depths (2.5, 3.0, and 3.5 mm from the surface, 300 nL at each location). Microinjection of AAV to the cerebral cortex was performed after attachment of a stainless headplate t...
Methods
For head-fixed fear conditioning experiments, additional surgery for electromyography (EMG) electrode implantation was performed. One week after the preparation of cranial window, tungsten wires (50 µm in diameter) were inserted in neck muscles and fixed with dental cement. After surgery, mice were kept on a heat pad for recovery (37 °C, 2 days).
Measurement outputs
What raw and processed outputs should exist?
Similar to the air puff, FS induced an elevation in Ca 2+; however, the response attenuated with repeated shocks, with the amplitude decreasing to about 10% Δ F / F after...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning. LC/NA axon Ca 2+ activity was imaged with the same parad...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
To gain more insight into the differential time course of Ca 2+ and cAMP dynamics, we examined Ca 2+ and cAMP responses during longer PS (120 s), mimicking a saturated ext...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Similar to the air puff, FS induced an elevation in Ca 2+; however, the response attenuated with repeated shocks, with the amplitude decreasing to about 10% Δ F / F after...; The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning. LC/NA axon Ca 2+ activity was imaged with the same parad...; To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm...; To gain more insight into the differential time course of Ca 2+ and cAMP dynamics, we examined Ca 2+ and cAMP responses during longer PS (120 s), mimicking a saturated ext....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning. LC/NA axon Ca 2+ activity was imaged with the same parad...; To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm...; To place our observations into a behavioral context, we imaged astrocytic Ca 2+ and cAMP dynamics in the auditory cortex of unanesthetized mice. Previous studies have shown that...; We employed a cued fear conditioning paradigm in head-fixed mice to permit imaging of the cortex throughout the course of experiment (Fig. ). For fear conditioning, a foot...
from paperReporting output
Report representative outputs alongside summary comparisons for Similar to the air puff, FS induced an elevation in Ca 2+; however, the response attenuated with repeated shocks, with the amplitude decreasing to about 10% Δ F / F after..., The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning. LC/NA axon Ca 2+ activity was imaged with the same parad..., To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm..., To gain more insight into the differential time course of Ca 2+ and cAMP dynamics, we examined Ca 2+ and cAMP responses during longer PS (120 s), mimicking a saturated ext....
inferred from protocolStructured statistical methods
The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning. LC/NA axon Ca 2+ activity was imaged with the same parad...; To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm...; To place our observations into a behavioral context, we imaged astrocytic Ca 2+ and cAMP dynamics in the auditory cortex of unanesthetized mice. Previous studies have shown that...; We employed a cued fear conditioning paradigm in head-fixed mice to permit imaging of the cortex throughout the course of experiment (Fig. ). For fear conditioning, a foot...
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Evidence quotes (8)
The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning. LC/NA axon Ca 2+ activity was imaged with the same paradigm as in Fig.. We found that MP signals were prevalent during the first ten seconds after the initial FS compared to the pre-FS period (Fig. ) (before: 2.82 ± 0.21 s, FS: 3.9 ± 0.54 s). However, this effect disappeared in later sessions (Fig. ) (before: 3.03 ± 0.28 s, FS: 3.07 ± 1.20 s), consistent with the reduced astrocytic Ca 2+ response we observed. We further analyzed LC/NA axonal Ca 2+ activity during the initial FS as illustrated in Fig.. Whereas the mean of individual MP signal durations was similar in all phases (Fig. ), MP signals were observed more frequently during post-FS1 compared to the Control or Sound phase (Fig. ) (Control, Sound, post-FS, post-FS1, post-FS2: 3.6 ± 0.75, 3.6 ± 1.2, 7.4 ± 0.9, 9.0 ± 1.4, 5.8 ± 0.6 counts/min). This increase was not seen during post-FS2. Accord...
To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm wavelength laser which while optimized towards Pink Flamindo permits the observation of GCaMP responses with a comparable dynamic range. We employed two patterns of PS, which provided equal stimulation duration but with distinct temporal patterns. In the "repetitive" stimulation regime, 3-s PS, which induced Ca 2+ elevations but not cAMP elevations in astrocytes, was repeated 10 × with 7 s inter-stimulus intervals, whereas in the "single" continuous stimulation regime, 30 s of continuous PS was applied. The repetitive stimulation induced significant Ca 2+ elevations in astrocytes (Δ F / F 120.0 ± 6.6%) as well as a small elevation of cAMP (104.4 ± 1.6%) (Fig. ). By contrast, the continuous stimulation reliably and robustly induced both Ca 2+ and cAMP elevations (Fig. ) (Δ F / F GCaMP: 115.7 ± 4.0%, Pink Flamindo: 117.7 ± 4.8%). These results suggest that the two second messenge...
To gain more insight into the differential time course of Ca 2+ and cAMP dynamics, we examined Ca 2+ and cAMP responses during longer PS (120 s), mimicking a saturated extracellular NA environment. To accommodate two-photon image acquisition we introduced brief photostimulus-free periods during the PS (Fig., see Methods). Under these conditions, we obtained distinct time courses for Ca 2+ and cAMP signals during activation of NAergic fibers. Ca 2+ activity showed an immediate increase after the start of PS (time to peak: 11.3 ± 1.3 s) and gradually decreased to basal levels after 70 s despite the presence of additional PS. Ca 2+ increase was not observed thereafter (Fig. ). On the other hand, cAMP signals took 30-40 s (32.9 ± 4.2 s) to reach the peak and did not return to basal level during the PS (Fig. ). Such slow dynamics of cAMP cannot be explained by the characteristics of Pink Flamindo which reaches a peak within 5 s even at low cAMP concentrations. Therefore, these results indicate innate properties of individual astrocytic second messengers. Regarding duration, cAMP (118.5R...
To understand the mechanism behind the temporally distinct astrocytic second messenger dynamics, we imaged cortical NAergic axonal activities with a faster responsive Ca 2+ probe GCaMP6.f (Fig., Supplementary Fig. ). In awake conditions, cortical NAergic axons exhibited continual Ca 2+ activities in awake mice in a range of approximately 0.5-0.6 Hz (Fig. ) (0.56 ± 0.04 Hz), whereas such activities were not observable under deep isoflurane anesthesia (Supplementary Fig. ). To reveal how this NAergic activity influences astrocytes, we simultaneously imaged astrocytic and NAergic axonal Ca 2+ activities with R-CaMP1.07 and GCaMP6.f, respectively. We observed two types of NAergic Ca 2+ activity patterns: single peak (SP) signals whereby Δ F / F returns to the basal level before next firing initiation and multipeak (MP) signals whereby Ca 2+ events occur before reaching to the base level forming multiplet bursts. Interestingly, astrocytic Ca 2+ elevations occur reliably with MP signals (Fig., Supplementary Movie; coincidence: 51.4 ± 5.5%), particularly those with longer durations (Fig....
To place our observations into a behavioral context, we imaged astrocytic Ca 2+ and cAMP dynamics in the auditory cortex of unanesthetized mice. Previous studies have shown that startle causes large Ca 2+ response in cortical astrocytes. Therefore, we first tested whether a startle response drives not only Ca 2+ but also cAMP increases. In this experiment, mice received unpredictable air puffs onto the right side of the face for 10 s while the auditory cortex was imaged (Fig. ). In stark contrast to the expected astrocytic Ca 2+ elevations ( F / F 0 162.2 ± 15.6%) (Fig. ), we observed that air puffs did not lead to significant cAMP increases ( F / F 0 104.0 ± 1.7%) (Fig. ). These responses were qualitatively similar to the short stimulation of NA axons demonstrated in Fig. (e.g. 3- or 5-s PS), suggesting that transient activation does not result in a long-lasting elevation of cAMP. To substantiate this premise, we imaged NAergic axon Ca 2+ activity during the course of the facial air puff experiment, which showed clear MP signals occur during abrupt air puff (Fig. ). In addition, we found that MP Ca 2+ activi...
Finally, to investigate functional importance related to astrocytic cAMP signaling, we induced activated Gs signaling using a Gs-type DREADD (designer receptors exclusively activated by designer drugs) expressed in astrocytes via AAV8-GFAP-HA-rM3D-mCitrine (Fig. ). A clear sustained cAMP elevation in astrocytes was observed by Pink Flamindo after CNO (Clozapine-N-Oxide) injection, lasting more than 2 h (Fig. ) (0 min: 99.2 ± 0.6%, 60 min: 180.6 ± 21.4%, 160 min: 174.9 ± 16.6%). Considering that one of the downstream functions of cAMP signaling in astrocytes is glycogenolysis, we visualized glycogen after CNO injection by immunohistochemistry. We confirmed that mice with rM3D-mCitrine expression in cortical astrocytes had distinctively lower levels of glycogen compared to control animals (Fig. ). These results suggest a salient function of astrocytic cAMP is a boosting of energy metabolism mediated by glycogenolysis. Fig. 8 Chemogenetic elevation of astrocytic cAMP induces glycogen breakdown. a Representative images of Gs-DREADD with mCitrine (yellow) and Pink Flamindo (pseudocolor) with...
Adult mice (postnatal 2-4 months) were anesthetized with ketamine and xylazine (70 and 10 mg/kg, respectively, i.p.) for introduction and kept under stable anesthesia with isoflurane (0.5-1.0%) until the end of surgery. Surgery was performed using a stereotaxic apparatus. After a small cranial hole was made above the cerebellum at the stereotaxic coordinate of AP -5.5 mm, ML +0.9 mm by a dental drill. Intracranial microinjection of AAV (AAV-DJ/8-EF1a-DIO-ChR2-EYFP, AAV-DJ/8-EF1a-DIO-GCaMP6.f, or AAV-DJ/8-EF1a-DIO-EYFP (1-3 × 10 12 vg/mL) was conducted using a glass micropipette connected to a Femtojet microinjector (Eppendorf) at three depths (2.5, 3.0, and 3.5 mm from the surface, 300 nL at each location). Microinjection of AAV to the cerebral cortex was performed after attachment of a stainless headplate to the skull with dental cement. Microinjection (300 nL) of AAV9-GFAP-GCaMP7.09 (3.0 × 10 12 vg/mL), AAV9-GFAP-Pink Flamindo (6.6 × 10 12 vg/mL), AAV9-GFAP-RCaMP1.07 (3.0 × 10 12 vg/mL), or AAV9-hSynI-nLight (1.0 ...
For head-fixed fear conditioning experiments, additional surgery for electromyography (EMG) electrode implantation was performed. One week after the preparation of cranial window, tungsten wires (50 µm in diameter) were inserted in neck muscles and fixed with dental cement. After surgery, mice were kept on a heat pad for recovery (37 °C, 2 days).
Machine-readable layer
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"text": "The results in Fig. prompted us to examine the temporal organization of NAergic activity during fear conditioning. LC/NA axon Ca 2+ activity was imaged with the same paradigm as in Fig.. We found that MP signals were prevalent during the first ten seconds after the initial FS compared to the pre-FS period (Fig. ) (before: 2.82 ± 0.21 s, FS: 3.9 ± 0.54 s). However, this effect disappeared in later sessions (Fig. ) (before: 3.03 ± 0.28 s, FS: 3.07 ± 1.20 s), consistent with the reduced astrocytic Ca 2+ response we observed. We further analyzed LC/NA axonal Ca 2+ activity during the initial FS as illustrated in Fig.. Whereas the mean of individual MP signal durations was similar in all phases (Fig. ), MP signals were observed more frequently during post-FS1 co..."
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"name": "Temporally distinct astrocytic Ca 2+ and cAMP surges by NA",
"text": "To understand the coordination of Ca 2+ and cAMP signaling, we next imaged GCaMP and Pink Flamindo simultaneously. Cortical astrocytes were imaged with 1040-1060 nm wavelength laser which while optimized towards Pink Flamindo permits the observation of GCaMP responses with a comparable dynamic range. We employed two patterns of PS, which provided equal stimulation duration but with distinct temporal patterns. In the \"repetitive\" stimulation regime, 3-s PS, which induced Ca 2+ elevations but not cAMP elevations in astrocytes, was repeated 10 × with 7 s inter-stimulus intervals, whereas in the \"single\" continuous stimulation regime, 30 s of continuous PS was applied. The repetitive stimulation induced significant Ca 2+ elevations in astrocytes (Δ F / F 120.0 ± 6.6%) as well as a small elevation of cAMP (104.4̴..."
},
{
"@type": "HowToStep",
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"name": "Temporally distinct astrocytic Ca 2+ and cAMP surges by NA",
"text": "To gain more insight into the differential time course of Ca 2+ and cAMP dynamics, we examined Ca 2+ and cAMP responses during longer PS (120 s), mimicking a saturated extracellular NA environment. To accommodate two-photon image acquisition we introduced brief photostimulus-free periods during the PS (Fig., see Methods). Under these conditions, we obtained distinct time courses for Ca 2+ and cAMP signals during activation of NAergic fibers. Ca 2+ activity showed an immediate increase after the start of PS (time to peak: 11.3 ± 1.3 s) and gradually decreased to basal levels after 70 s despite the presence of additional PS. Ca 2+ increase was not observed thereafter (Fig. ). On the other hand, cAMP signals took 30-40 s (32.9 ± 4.2 s) to reach the peak and did not return to basal level during the PS (Fig...."
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"name": "Bursting NAergic activity induces astrocytic Ca 2+ surges",
"text": "To understand the mechanism behind the temporally distinct astrocytic second messenger dynamics, we imaged cortical NAergic axonal activities with a faster responsive Ca 2+ probe GCaMP6.f (Fig., Supplementary Fig. ). In awake conditions, cortical NAergic axons exhibited continual Ca 2+ activities in awake mice in a range of approximately 0.5-0.6 Hz (Fig. ) (0.56 ± 0.04 Hz), whereas such activities were not observable under deep isoflurane anesthesia (Supplementary Fig. ). To reveal how this NAergic activity influences astrocytes, we simultaneously imaged astrocytic and NAergic axonal Ca 2+ activities with R-CaMP1.07 and GCaMP6.f, respectively. We observed two types of NAergic Ca 2+ activity patterns: single peak (SP) signals whereby Δ F / F returns to the basal level before next firing initiation and multipeak (MP) signa..."
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"name": "Prolonged vigilance induces astrocytic cAMP increases",
"text": "To place our observations into a behavioral context, we imaged astrocytic Ca 2+ and cAMP dynamics in the auditory cortex of unanesthetized mice. Previous studies have shown that startle causes large Ca 2+ response in cortical astrocytes. Therefore, we first tested whether a startle response drives not only Ca 2+ but also cAMP increases. In this experiment, mice received unpredictable air puffs onto the right side of the face for 10 s while the auditory cortex was imaged (Fig. ). In stark contrast to the expected astrocytic Ca 2+ elevations ( F / F 0 162.2 ± 15.6%) (Fig. ), we observed that air puffs did not lead to significant cAMP increases ( F / F 0 104.0 ± 1.7%) (Fig. ). These responses were qualitatively similar to the short stimulation of NA axons demonstrated in Fig. (e.g. 3- or 5-s PS), suggesting that transient a..."
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"name": "High vigilance induced prolonged NAergic activity",
"text": "Finally, to investigate functional importance related to astrocytic cAMP signaling, we induced activated Gs signaling using a Gs-type DREADD (designer receptors exclusively activated by designer drugs) expressed in astrocytes via AAV8-GFAP-HA-rM3D-mCitrine (Fig. ). A clear sustained cAMP elevation in astrocytes was observed by Pink Flamindo after CNO (Clozapine-N-Oxide) injection, lasting more than 2 h (Fig. ) (0 min: 99.2 ± 0.6%, 60 min: 180.6 ± 21.4%, 160 min: 174.9 ± 16.6%). Considering that one of the downstream functions of cAMP signaling in astrocytes is glycogenolysis, we visualized glycogen after CNO injection by immunohistochemistry. We confirmed that mice with rM3D-mCitrine expression in cortical astrocytes had distinctively lower levels of glycogen compared to control animals (Fig...."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Methods",
"text": "Adult mice (postnatal 2-4 months) were anesthetized with ketamine and xylazine (70 and 10 mg/kg, respectively, i.p.) for introduction and kept under stable anesthesia with isoflurane (0.5-1.0%) until the end of surgery. Surgery was performed using a stereotaxic apparatus. After a small cranial hole was made above the cerebellum at the stereotaxic coordinate of AP -5.5 mm, ML +0.9 mm by a dental drill. Intracranial microinjection of AAV (AAV-DJ/8-EF1a-DIO-ChR2-EYFP, AAV-DJ/8-EF1a-DIO-GCaMP6.f, or AAV-DJ/8-EF1a-DIO-EYFP (1-3 × 10 12 vg/mL) was conducted using a glass micropipette connected to a Femtojet microinjector (Eppendorf) at three depths (2.5, 3.0, and 3.5 mm from the surface, 300 nL at each location). Microinjection of AAV to the cerebral cortex was performed after attachment of a stainless headplate t..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Methods",
"text": "For head-fixed fear conditioning experiments, additional surgery for electromyography (EMG) electrode implantation was performed. One week after the preparation of cranial window, tungsten wires (50 µm in diameter) were inserted in neck muscles and fixed with dental cement. After surgery, mice were kept on a heat pad for recovery (37 °C, 2 days)."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Temporally distinct astrocytic Ca 2+ and cAMP surges by NA"
},
{
"@type": "HowToTool",
"name": "Temporally distinct astrocytic Ca 2+ and cAMP surges by NA"
},
{
"@type": "HowToTool",
"name": "Prolonged vigilance induces astrocytic cAMP increases"
},
{
"@type": "HowToTool",
"name": "Prolonged vigilance induces astrocytic cAMP increases"
},
{
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"name": "Methods"
},
{
"@type": "HowToTool",
"name": "Methods"
},
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"name": "Histology"
},
{
"@type": "HowToTool",
"name": "Imaging and head-fixed fear conditioning"
}
],
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"name": "Histology"
},
{
"@type": "HowToSupply",
"name": "Imaging and head-fixed fear conditioning"
},
{
"@type": "HowToSupply",
"name": "Histology"
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"headline": "Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance",
"datePublished": "2020",
"author": [
{
"@type": "Person",
"name": "Yuki Oe"
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{
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"name": "Xiaowen Wang"
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"name": "Tommaso Patriarchi"
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{
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"name": "Ayumu Konno"
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{
"@type": "Person",
"name": "Katsuya Ozawa"
},
{
"@type": "Person",
"name": "Kazuko Yahagi"
},
{
"@type": "Person",
"name": "Hirokazu Hirai"
},
{
"@type": "Person",
"name": "Lin Tian"
},
{
"@type": "Person",
"name": "Thomas J. McHugh"
},
{
"@type": "Person",
"name": "Hajime Hirase"
}
],
"identifier": "10.1038/s41467-020-14378-x"
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