This method allows the functional isolation of single motor units in handling masses of experimental animals. The main advantage of this technique is recording of force and action potentials of individual motor units of identified physiological type. Helping to demonstrate the procedure will be Professor Jan Celichowski, head of the department of neurobiology.
Begin by cutting the skin along the spinal column, from the sacrum up to the thoracic vertebrae, with sharp-blunt scissors. Identify the S1 vertebra as the lowest segment. Then cut and remove the longissimus muscles, and the spinous processes from L6 to L2 vertebrae.
Use fine rongeurs to remove the transverse processes from L6 to L2, and perform a laminectomy over L6 to L2 segments to expose the lumbar segments of the spinal cord covered by the dura mater. Using sharp scissors, cut the spinal cord and the dorsal and ventral roots at L2 vertebrae segment, level at the upper border of the laminectomy. Place small pieces of dry gel foam to stop the bleeding.
Next, use sharp-blunt scissors to make a longitudinal cuddle on the posterior side of the left hind limb, from the Achilles tendon to the hip. Locate the popliteal fossa at the back of the knee joint, which is covered by the biceps femoris muscle, and make a cup between the anterior and posterior part of this muscle. Moving upwards, cut the two heads of the biceps femoris all the way to the hip to expose the sciatic nerve.
Using blunt forceps and scissors, separate the lateral from the medial head of the gastrocnemius muscle, and cut the distal insertion of the medial gastrocnemius muscle. Identify the medial gastrocnemius or MG nerve, then use forceps and scissors to cut all remaining collaterals of the sciatic nerve including collaterals to posterior biceps and semitendinosus. Bred a non elastic ligature through the Achilles tendon and make three knots.
Then make a two centimeter incision in the skin and underlying connective tissue along the anterior side of the left hind limb for a mobilization with a middle clan. Fix the left hind limb by putting a steel clamp on the tibia. Place the rat in a custom made adjustable frame.
Pull the skin flaps around the laminectomy with four ligatures. And suture them to the frame in order to form a pool for paraffin oil over the exposed spinal cord. Use a Dumont 55 forceps to lift the dura mater at the intersection of the spinal cord.
Then cut it caudally up to the sacral bone and retract it. Separate the left and right dorsal and ventral roots at successive levels with a blunt glass rod. Fill the pool over the spinal cord with warm paraffin oil, covering the exposed ventral and dorsal roots.
Place the rat on a custom made aluminum plate with a pool for its hind limbs, connected to the closed loop heating system. Fix the clamp on the left hind limb with the metal bar to immobilize it. Fix the vertebral column by putting steel clamps at the sacral bone and the L1 vertebra.
Then connect the left medial gastrocnemius muscle with the non elastic ligature to the force transducer via the Achilles tendon. Insert a bipolar silver wire or electrode through the middle part of the muscle, perpendicular to its long axis, and fill the chamber for hind limbs with warm paraffin oil to cover the medial gastrocnemius muscle. Stretch the operated muscle to a passive tension of 100 millinewton, controlled by the force transducer.
Then use sharp-blunt scissors to make a two centimeter incision in the skin of the right hind limb, and insert a silver wire electrode to be used as a reference electrode. Place and fix a custom made insulated metal plate above the exposed spinal roots. Put left pairs of ventral and dorsal roots on the plate.
And add saline to the pool formed by the skin around the laminectomy. Place a silver wire stimulating electrode over the exposed spinal roots, then place a positive pole three millimeters above the plate in oil, and the negative pole in the saline. Stimulate the ventral roots with electrical rectangular pulses, evoking contraction of muscles.
Use a pair of Dumont 55 forceps and magnifying glasses to split L5 or L4 ventral roots into very fine bundles of axons. Place one of these bundles on a silver wire electrode and stimulate it to observe activity of a single motor unit. By progressively increasing the intensity of the stimulus, identify a single motor unit on the basis of the evoked all-or-none character of the twitch contraction and action potential stimulus.
Parameters of motor unit contractions and action potentials can be calculated using recordings when stable conditions of recordings are insured. A representative recording of the single twitch of a fast motor unit is shown here. The upper trays shows the motor unit action potential.
The delay between stimulus delivery and onset of the motor unit action potential is due to conduction time from the ventral root to the muscle. A representative recording of the unfused tetanus force of a fast motor unit and a train of motor unit action potentials are shown here along with the time positions of the applied stimuli. Before attempting this procedure, keep in mind that the splitting of the ventral root into thin filaments needs some training and experience.
