Motor Cortex Fiber Tracing in Grafted Rats

Three weeks before sacrifice, descending CST fibers of grafted rats were labeled with BDA (10% BDA in 0.9% saline, molecular probes) by injecting into five spots of the right motor cortex. The skulls of anesthetized rats (ketamine (80 mg kg⁻¹) and xylazine (10 mg kg⁻¹) were tightly fixed to a stereotaxic apparatus (RWD). A vertical midline incision was made from between the eyes to the posterior skull. The injection area on the right hemisphere defined in a rectangle measuring 2 mm (from 1.0 mm anterior to −1.0 mm posterior to the bregma) by 1.5 mm (lateral to the bregma). A drill was used to create the injection sites on the skull. Injections were performed using a 33G syringe (Hamilton) attached to a micropump (RWD). Each injection delivered 100 nL of the BDA solution into the motor cortex at a rate of 100 nL min⁻¹. The injector tip was left in place for an additional 5 min before and after the injections. For visualization, BDA staining was performed by incubating the sections in 3% H₂O₂ for 30 min to reduce endogenous peroxidase, followed by 2 h incubation with streptavidin–horseradish peroxidase (Vectastain R.T.U. Elite ABC Reagent, Cat. No: PK‐7100) at room temperature. PerkinElmer Biotinyltyramide (1:100 in amplification diluent) was then added to the sections for another 1 h. Detection was accomplished by incubation with Extra‐Avidin@ TRITC (1:200, Sigma) in 0.1% PBS‐T for 2 h.