This study investigates the dynamics of potassium ion release in the hippocampus during action potential firing. Using a mouse coronal brain slice, the experiment measures the response of CA1 neurons to stimulation of CA3 neurons.
Secure a mouse coronal brain slice in a recording chamber filled with aCSF.
Insert a bipolar stimulating electrode into the stratum radiatum of the CA3 region in the hippocampus. The CA3 neurons project their axons, termed Schaffer collaterals, to CA1.
Position a potassium ion-selective electrode in the CA1 stratum radiatum.
Electrically stimulate CA3 neurons to open voltage-gated sodium channels. The sodium ion influx turns the membrane potential positive, termed depolarization, and generates an action potential.
After the peak of depolarization, sodium channels deactivate, and voltage-gated potassium channels allow potassium ion outflow, repolarizing the membrane. Subsequently, the membrane potential is restored, and the action potential propagates to CA1.
The potassium ion-selective electrode measures the outflow, shown by a response curve.
Add a blocker to inhibit voltage-gated sodium channel activity. Upon electrical stimulation, a lack of potassium ion response confirms that its dynamics are due to action potentials in the Schaffer collaterals.
To measure the potassium ion dynamics, gently place the brain slice in the bath using a Pasteur pipette and gently hold it in place with a platinum harp with nylon strings. Ensure the tips of the bipolar stimulating electrode are approximately parallel to one another, and are level with the plane of the slice. Over the course of 5 to 7 seconds, slowly insert the electrodes into CA3 stratum radiatum approximately 40 to 50 micrometers deep to stimulate Schaffer collaterals.
Then, carefully insert the potassium ion-selective electrode into CA1 stratum radiatum to approximately 50 micrometers deep by slowly lowering the electrode over approximately 3 to 4 seconds. Allow the potential to stabilize across the electrode before applying stimulations to the slice, which usually takes 5 to 10 minutes. If the slice exhibits excessive spontaneous changes in extracellular potassium ion concentrations, discard and repeat the process with a new slice.
To measure evoked potassium ion release, apply trains of electrical stimulation via the stimulus isolator while digitally recording responses. Apply stimulation at 10 Hertz and 1 millisecond per pulse starting at 10 microamps stimulus amplitude. Apply increasing stimulation amplitudes by a factor of 2 until a maximum potassium response amplitude is detected. If no response is observed, move the position of the potassium ion-selective electrode closer to the stimulation site in 100-micrometer increments.
To confirm that potassium ion concentration changes are mediated by action potential firing of the electrically activated Schaffer collaterals bath, apply 0.5 micromolar TTX in aCSF for 10 minutes and repeat the stimulation protocol. No evoked responses should be observed.
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