This article details a method for recording rhythmic electrical activity from a brainstem-spinal cord preparation in newborn rodents. The procedure involves manipulating the neuronal network responsible for breathing control and assessing the effects of oxygen levels on action potentials.
Secure a brainstem-spinal cord preparation from a newborn rodent in a recording chamber with a flow of oxygenated aCSF. Remove the thin tissue covering the preparation.
The brainstem's neuronal network generates a rhythmic electrical activity, termed inspiratory burst, transmitted via motor neurons of the spinal cord's fourth ventral, or C4, nerve root to control breathing.
Position a recording pipette near the nerve root and use suction to draw the root inside, forming a seal.
Intrinsic properties of the brainstem neurons cause sodium influx, depolarizing the membrane and generating an action potential, followed by potassium outflow for repolarization.
Ensuing hyperpolarization prevents new action potentials until the sodium-potassium ATPase utilizes ATP to restore the membrane potential.
The action potentials propagate to the nerve root as rhythmic bursts.
Flow oxygen-deprived aCSF to disrupt ATP production and impair ATPase function, delaying action potentials and decreasing rhythmic bursts.
Switching back to oxygenated aCSF recovers the number of rhythmic bursts.
In this procedure, place the nervous tissue in the recording chamber with its ventral side facing up. Fix it with pins in the lowest part of the spinal cord, and the rostral-most part of the brain stem. After that, remove the arachnoid membrane, which is a thin tissue covering the surface of the nervous tissue. Be careful not to remove the pia mater and blood vessels of the nervous tissue. Next, fill the electrode with ACSF.
Using a micromanipulator, carefully place the electrode close to the fourth ventral root. Gently aspirate the nerve rootlet using the syringe. Then, place the electrode with the nerve rootlet against the spinal cord. Subsequently, record the electrophysiological activities generated by the nervous tissue under normoxic conditions for at least 20 minutes to determine the baseline parameters of the preparation.
Switch the perfusion solution from carbogen-bubbled ACSF to hypoxia stimulus ACSF, and record for 15 minutes. After that, switch the perfusion solution back to standard carbogen-bubbled ACSF, and record for at least another 15 minutes before ending the recording.
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