Sutter p97 pipette puller

Mice were anesthetized by intraperitoneal injection of 80 mg/kg ketamine and 0.2 mg/kg medetomidine, fixed in a stereotaxic frame (Kopf, Tujunga, CA) and the body temperature was maintained at 37 °C using a heating pad. A surgical procedure to access the cisterna magna was performed [30 (link)]. After a small skin incision over the occipital bone/cervical spinal cord was made, the three covering muscle layers were carefully dissected under a stereomicroscope using fine forceps and scissors. A beveled glass capillary micropipette (Sutter instruments, Novato, CA, USA) with a diameter of < 60 μm was made using a Sutter P97 Pipette puller (Sutter instruments) and was positioned perpendicular to the ear bars and advanced to penetrate the dura until resistance was overcome, indicating entry into the cisterna magna as previously described [2 (link)]. Overall, 5 µL of a Gadospin D solution at 25 mM gadolinium (nanoPET Pharma GmbH) was infused at the speed of 1 µL/min with a NanoJet syringe pump (Chemyx). After the infusion, the micropipette was left in place for 10 min to avoid reflux. Following its withdrawal, the wound was closed.

For whole-cell patch-clamp recording, neurons were infected with a reduced titer of AAV2-scramble-GFP or AAV2-shBrpf1-GFP at DIV3 to achieve sparse labeling, and the recordings were performed at DIV15. Whole-cell recordings were conducted with a conventional patch-clamp technique with a MultiClamp 700B amplifier (Axon Instruments, Burlingame, CA, United States). Patch pipettes were pulled by a Sutter P-97 pipette puller (Sutter Instrument, Novato, CA, United States). The bath solution (pH = 7.3) contained 128 mM NaCl, 5 mM KCl, 25 mM HEPES, 1 mM MgCl₂, 30 mM glucose, and 2 mM CaCl₂. Recording pipettes were filled with an internal solution (pH = 7.2–7.3) containing 125 mM potassium gluconate, 10 mM KCl, 10 mM HEPES, 5 mM EGTA, 10 mM Tris–phosphocreatine, 4 mM MgATP, and 0.5 mM Na₂GTP. All the recordings were performed at room temperature. Data acquisition and analysis were performed with pClamp 10.2 software (Axon Instruments, Burlingame, CA, United States).