Measurement of Evoked Potassium Ion Concentration Dynamics in Coronal Hippocampal Slices
Transcrição
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.
