Source Paper
Yuki Oe, Xiaowen Wang, Tommaso Patriarchi, Ayumu Konno, Katsuya Ozawa et al.
Nature Communications • 2020
Objective: To conduct head-fixed fear conditioning experiments in mice with concurrent neural imaging and electromyography recordings to measure neural activity and physiological responses during fear conditioning
This is a Head-Fixed Fear Conditioning protocol using mouse as the model organism. The procedure involves 16 procedural steps, 5 equipment items, 15 materials. Extracted from a 2020 paper published in Nature Communications.
Model and subjects
mouse • wild-type and NET-cre mice • unknown • postnatal 2-4 months • not specified
Study window
~3 week study window | ~1.3 hours hands-on
Core workflow
Anesthesia induction • Maintain surgical anesthesia • Position animal in stereotaxic apparatus
Primary readouts
Key equipment and reagents
Verified items
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Anesthetize adult mice with ketamine and xylazine via intraperitoneal injection
Note: Dosages: ketamine 70 mg/kg, xylazine 10 mg/kg
“Adult mice (postnatal 2–4 months) were anesthetized with ketamine and xylazine (70 and 10 mg/kg, respectively, i.p.)”
Maintain stable anesthesia with isoflurane throughout surgical procedure
Note: Isoflurane concentration: 0.5-1.0%
“kept under stable anesthesia with isoflurane (0.5–1.0%) until the end of surgery”
Secure anesthetized mouse in stereotaxic apparatus for precise surgical positioning
Note: Required for accurate intracranial injections
“Surgery was performed using a stereotaxic apparatus”
Using dental drill, create small cranial hole above cerebellum at specified stereotaxic coordinates
Note: Coordinates: AP −5.5 mm, ML +0.9 mm
“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”
Inject AAV vectors into cerebellum using glass micropipette connected to Femtojet microinjector at three depths
Note: Inject 300 nL at each of three depths: 2.5, 3.0, and 3.5 mm from surface. AAV options: AAV-DJ/8-EF1a-DIO-ChR2-EYFP, AAV-DJ/8-EF1a-DIO-GCaMP6.f, or AAV-DJ/8-EF1a-DIO-EYFP at 1-3 × 10^12 vg/mL
“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)”
Secure stainless steel headplate to skull using dental cement for head-fixation during future experiments
Note: Performed after cerebellar injection
“Microinjection of AAV to the cerebral cortex was performed after attachment of a stainless headplate to the skull with dental cement”
Inject AAV vectors into auditory cortex using glass micropipette at specified coordinates
Note: Coordinates: AP −2.0 to −2.5 mm, ML +3.0 to 4.0 mm, DV −0.3 mm. Volume: 300 nL. AAV options: 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 × 10^13 vg/mL)
“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 × 10^13 vg/mL) was made in the auditory cortex (AP −2.0 to −2.5 mm, ML +3.0 to 4.0 mm, DV −0.3 mm)”
Inject AAV vectors into parietal cortex using glass micropipette at specified coordinates
Note: Coordinates: AP −1.5 to −3.0 mm, ML 1.5 to 3.5 mm, DV −0.3 mm. Volume: 300 nL. Same AAV options as auditory cortex injection
“or the parietal cortex (AP −1.5 to −3.0 mm, ML 1.5 to 3.5 mm, DV −0.3 mm)”
Cover virus-injected cortical area with sterilized round cover glass to serve as cranial window for two-photon imaging
Note: Cover glass diameter: 3 or 4 mm
“The virus-injected area was covered by a sterilized round cover glass (3 or 4 mm in diameter) to serve as a cranial window for two-photon imaging”
Place mice on heated pad for recovery following cranial window implantation
Note: Maintain constant temperature during recovery period
“After surgery, mice were kept on a heat pad for recovery (37°C, 2 days)”
Perform additional surgery for electromyography electrode implantation in neck muscles
Note: Performed one week after cranial window preparation
“One week after the preparation of cranial window, tungsten wires (50 µm in diameter) were inserted in neck muscles and fixed with dental cement”
Insert tungsten wires into neck muscles and secure with dental cement for electromyography recording
Note: Electrode diameter: 50 µm
“tungsten wires (50 µm in diameter) were inserted in neck muscles and fixed with dental cement”
Place mice on heated pad for recovery following EMG electrode implantation
Note: Maintain constant temperature during recovery period
“After surgery, mice were kept on a heat pad for recovery (37°C, 2 days)”
Allow time for AAV-expressed fluorescent indicators to become observable
Note: For ChR2-EYFP, wait at least 3 weeks for sufficient protein expression
“The AAV-expressed fluorescent indicators were observable after 2 weeks. For ChR2-EYFP, we waited at least 3 weeks, so that sufficient amounts of ChR2 protein are expressed in the LC-originated NAergic fibers for cortical PS”
Conduct 5 days of handling and habituation for wild-type mice before imaging experiments
Note: Individual mouse handling
“For head-fixed fear conditioning experiments, we performed 5 days of handling and habituation (20–40 min per day for individual mice) for wild-type mice”
Conduct 10 days of handling and habituation for NET-cre mice before imaging experiments
Note: Individual mouse handling; longer habituation period than wild-type mice
“and 10 days for NET-cre mice before imaging”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
To conduct head-fixed fear conditioning experiments in mice with concurrent neural imaging and electromyography recordings to measure neural activity and physiological responses during fear conditioning
Objective
To conduct head-fixed fear conditioning experiments in mice with concurrent neural imaging and electromyography recordings to measure neural activity and physiological responses during fear conditioning
Subjects
From papermouse • wild-type and NET-cre mice • unknown • postnatal 2-4 months • not specified
Cohort notes
From paperNET-cre mice required 10 days habituation; wild-type mice required 5 days habituation
Anesthesia induction (not specified)
Maintain surgical anesthesia (until end of surgery)
Position animal in stereotaxic apparatus (not specified)
Create cranial hole for cerebellar injection (not specified)
Neural activity recorded via two-photon imaging of fluorescent calcium indicators (GCaMP6.f, GCaMP7.09, RCaMP1.07) and voltage indicators (nLight)
From papernot specified in methods section
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Astrocytic hydrogen peroxide levels via Pink Flamindo imaging
From papernot specified in methods section
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Electromyography recordings from neck muscles during fear conditioning
From papernot specified in methods section
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Channelrhodopsin-2 expression and optogenetic manipulation of neural circuits
From papernot specified in methods section
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Neural activity recorded via two-photon imaging of fluorescent calcium indicators (GCaMP6.f, GCaMP7.09, RCaMP1.07) and voltage indicators (nLight)
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Astrocytic hydrogen peroxide levels via Pink Flamindo imaging
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Electromyography recordings from neck muscles during fear conditioning
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Channelrhodopsin-2 expression and optogenetic manipulation of neural circuits
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
Preprocessing / cleaning
not specified in methods section
Scoring or quantification
Quantify the primary readouts for this experiment: Neural activity recorded via two-photon imaging of fluorescent calcium indicators (GCaMP6.f, GCaMP7.09, RCaMP1.07) and voltage indicators (nLight); Astrocytic hydrogen peroxide levels via Pink Flamindo imaging; Electromyography recordings from neck muscles during fear conditioning; Channelrhodopsin-2 expression and optogenetic manipulation of neural circuits.
Normalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Neural activity recorded via two-photon imaging of fluorescent calcium indicators (GCaMP6.f, GCaMP7.09, RCaMP1.07) and voltage indicators (nLight), Astrocytic hydrogen peroxide levels via Pink Flamindo imaging, Electromyography recordings from neck muscles during fear conditioning, Channelrhodopsin-2 expression and optogenetic manipulation of neural circuits.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Yuki Oe et al. (2020). Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance. Nature Communications
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Current status surfaces were computed from experiment data updated Mar 14, 2026.
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Evidence Quotes
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Canonical Sync
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