Anesthesia induction

Anesthetize adult mice with ketamine and xylazine via intraperitoneal injection

not specifiednot specified

Note: Dosages: ketamine 70 mg/kg, xylazine 10 mg/kg

View evidence from paper

“Adult mice (postnatal 2–4 months) were anesthetized with ketamine and xylazine (70 and 10 mg/kg, respectively, i.p.)”

2

Maintain surgical anesthesia

Maintain stable anesthesia with isoflurane throughout surgical procedure

until end of surgerynot specified

Note: Isoflurane concentration: 0.5-1.0%

View evidence from paper

“kept under stable anesthesia with isoflurane (0.5–1.0%) until the end of surgery”

3

Position animal in stereotaxic apparatus

Secure anesthetized mouse in stereotaxic apparatus for precise surgical positioning

not specifiednot specified

Conscious Mouse Stereotaxic Precision Locator

Matches: Stereotaxic apparatus

0View

Note: Required for accurate intracranial injections

View evidence from paper

“Surgery was performed using a stereotaxic apparatus”

4

Create cranial hole for cerebellar injection

Using dental drill, create small cranial hole above cerebellum at specified stereotaxic coordinates

not specifiednot specified

Note: Coordinates: AP −5.5 mm, ML +0.9 mm

View evidence from paper

“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”

5

Intracranial AAV microinjection to cerebellum

Inject AAV vectors into cerebellum using glass micropipette connected to Femtojet microinjector at three depths

not specifiednot specified

Single Channel Micropipettes

Matches: Glass micropipette

$54.6View

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

View evidence from paper

“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)”

6

Attach stainless steel headplate

Secure stainless steel headplate to skull using dental cement for head-fixation during future experiments

not specifiednot specified

Note: Performed after cerebellar injection

View evidence from paper

“Microinjection of AAV to the cerebral cortex was performed after attachment of a stainless headplate to the skull with dental cement”

7

Intracranial AAV microinjection to auditory cortex

Inject AAV vectors into auditory cortex using glass micropipette at specified coordinates

not specifiednot specified

Single Channel Micropipettes

Matches: Glass micropipette

$54.6View

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)

View evidence from paper

“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)”

8

Intracranial AAV microinjection to parietal cortex

Inject AAV vectors into parietal cortex using glass micropipette at specified coordinates

not specifiednot specified

Single Channel Micropipettes

Matches: Glass micropipette

$54.6View

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

View evidence from paper

“or the parietal cortex (AP −1.5 to −3.0 mm, ML 1.5 to 3.5 mm, DV −0.3 mm)”

9

Install cranial window

Cover virus-injected cortical area with sterilized round cover glass to serve as cranial window for two-photon imaging

not specifiednot specified

Automatic Staining and Coverslipping Machine

Matches: Round cover glass

$2,500View

Note: Cover glass diameter: 3 or 4 mm

View evidence from paper

“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”

10

Post-operative recovery after cranial window surgery

Place mice on heated pad for recovery following cranial window implantation

2 days37°C

Note: Maintain constant temperature during recovery period

View evidence from paper

“After surgery, mice were kept on a heat pad for recovery (37°C, 2 days)”

11

EMG electrode implantation surgery

Perform additional surgery for electromyography electrode implantation in neck muscles

not specifiednot specified

Note: Performed one week after cranial window preparation

View evidence from paper

“One week after the preparation of cranial window, tungsten wires (50 μm in diameter) were inserted in neck muscles and fixed with dental cement”

12

Insert tungsten EMG electrodes

Insert tungsten wires into neck muscles and secure with dental cement for electromyography recording

not specifiednot specified

Note: Electrode diameter: 50 μm

View evidence from paper

“tungsten wires (50 μm in diameter) were inserted in neck muscles and fixed with dental cement”

13

Post-operative recovery after EMG electrode implantation

Place mice on heated pad for recovery following EMG electrode implantation

2 days37°C

Note: Maintain constant temperature during recovery period

View evidence from paper

“After surgery, mice were kept on a heat pad for recovery (37°C, 2 days)”

14

Wait for viral expression

Allow time for AAV-expressed fluorescent indicators to become observable

2 weeks minimumnot specified

Note: For ChR2-EYFP, wait at least 3 weeks for sufficient protein expression

View evidence from paper

“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”

15

Habituation and handling for wild-type mice

Conduct 5 days of handling and habituation for wild-type mice before imaging experiments

20-40 minutes per day per mousenot specified

Note: Individual mouse handling

View evidence from paper

“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”

16

Habituation and handling for NET-cre mice

Conduct 10 days of handling and habituation for NET-cre mice before imaging experiments

20-40 minutes per day per mousenot specified

Note: Individual mouse handling; longer habituation period than wild-type mice

View evidence from paper

“and 10 days for NET-cre mice before imaging”

Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance

Head-Fixed Fear Conditioning

Equipment5

Materials15

Software1

Protocol Steps

Anesthesia induction

Maintain surgical anesthesia

Position animal in stereotaxic apparatus

Create cranial hole for cerebellar injection

Intracranial AAV microinjection to cerebellum

Attach stainless steel headplate

Intracranial AAV microinjection to auditory cortex

Intracranial AAV microinjection to parietal cortex

Install cranial window

Post-operative recovery after cranial window surgery

EMG electrode implantation surgery

Insert tungsten EMG electrodes

Post-operative recovery after EMG electrode implantation

Wait for viral expression

Habituation and handling for wild-type mice

Habituation and handling for NET-cre mice