Head-Fixed Fear Conditioning

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

“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 × 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)”

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

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

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

“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)”

“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”

“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”

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