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
Gather these items before starting the experiment. Check off items as you prepare.
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
Eppendorf • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
Allen Institute • not mentioned
3 items available from ConductScience
Estimated: $2,554.60
As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
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”
NET-cre mice required 10 days habituation; wild-type mice required 5 days habituation