Mo 10

This method has been reported in our previous study [18 (link)]. Briefly, after exposing the lumbar enlargement (L3–L5 spinal segments) by laminectomy, rats were mounted in a stereotaxic head holder (SR-5R, Narishige, Japan) and stabilized with clamps attached to L2 and T12 vertebral processes. The dura was carefully removed, and the spinal cord was covered with warm mineral oil (37°C). Extracellular recordings of the activity of single WDR neurons in SDH with receptive fields (RFs) located on ST36 were obtained with a glass microelectrode (impedance: 10~15 MΩ; the tip of the microelectrode: 1-2 μm (A-M Systems Co.)) filled with 2% Pontamine Sky Blue in 0.5 mol/L sodium acetate. Recording electrodes were lowered into the spinal cord using an electronic micromanipulator (MO-10, Narishige, Japan) in 1 μm steps: 0.5–1.2 mm lateral to the midline in the right side of spinal cord and 0.3–1.3 mm from the dorsal surface [23 (link)]. Recordings were made only from single neurons whose amplitude could be easily discriminated. Electrophysiological activity was amplified (DAM50, WPI, USA), filtered (bandwidth: 300–5000), audiomonitored, and recorded with data acquisition systems (MP150, Biopack, CA, USA). The recording site and ST36 were all in the right side of the rats.

To label a specific group of orofacial motor neurons, AAV2-retro-CAG-Cre (1000 nl) was injected into either the mystacial pad, genioglossus, or masseter muscles at postnatal day 17 using a volumetric injection system (based on a single-axis oil hydraulic micromanipulator MO-10, Narishige International USA, Inc, East Meadow, NY, USA) (Petreanu et al., 2009 (link)) equipped with a pulled and beveled glass pipette (Drummond, 5-000-2005). Before injection, mice were anesthetized by a cocktail of ketamine and xylazine (100 mg/kg and 10 mg/kg, i.p.). For the mystacial pad, the virus was injected subcutaneously into the areas around C2 and B2 vibrissae (500 nl each). For the genioglossus, the virus was injected directly into the muscle after exposing it by ventral neck dissection. Briefly, the genioglossus muscle was exposed by making a small incision in the mylohyoid muscle after the anterior digastric muscle was split open in the midline. For the masseter, the virus was injected into the area between the buccal and marginal nerves after making a small incision on a skin.

Intracerebral administration of cryopreserved GFP⁺ GABAergic progenitors was performed with beveled glass micropipettes (50–70μm diameter, Drummond Scientific, USA) coupled to a stereotaxic apparatus and controlled with the help of a microinjector (MO-10, Narishige, Tokyo, Japan), as previously described [12 (link),13 (link)]. Cells were pelleted by centrifugation (5 min, 300xg) and resuspended in DMEM/F12 at 0.1–0.5x10⁶ cells/μl. MGE cell suspension were loaded into glass micropipettes prefilled with pure mineral oil and then with DMEM/F12.
Transplants were performed in neonatal mice (P3-P5) as previously described [12 (link)]. Mice were anesthetized by hypothermia until pedal reflex was abolished (approximately 1–2 minutes). Anesthesia was maintained by performing surgery on a cold aluminum plate. We used the following stereotaxic coordinates from Bregma (according to the Atlas of Developing Mouse Brains E17, 5, P0 and P6. Paxino, George / Halliday) for cortex (2.2mm A, 3.5 mm L, 1.2 mm D), and hippocampus (-1.2 mm A, 1.7 mm L, 2.0 mm D). After surgical procedure, grafted mice were returned to their cages and analyzed one month later.