Measurement of Evoked Potassium Ion Concentration Dynamics in Coronal Hippocampal Slices

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Preparation of Acute Hippocampal Brain Slices

1. Preparation of slice solutions
   1. Prepare 500 mL sucrose cutting solution composed of: 194 mM sucrose, 30 mM NaCl, 4.5 mM KCl, 10 mM D-glucose, 1 mM MgCl₂, 1.2 mM NaH₂PO₄, and 26 mM NaHCO₃, 290-300 mOsm, saturated with 95% O₂ and 5% CO₂.
   2. Prepare 1-2 liters of recording solution (artificial cerebrospinal fluid, ACSF) composed of: 124 mM NaCl, 4.5 mM KCl, 1 mM MgCl₂, 10 mM D-glucose, 2 mM CaCl₂, 1.2 mM NaH₂PO₄, and 26 mM NaHCO₃; pH 7.3 – 7.4 (after bubbling), 290 – 300 mOsm, saturated with 95% O₂ and 5% CO₂. Fill a beaker containing a brain slice holder chamber with recording solution and keep it at 32-34 °C. Fill the vibratome chamber with ice-water slush.
2. Acute slice preparation
   1. Deeply anesthetize a mouse by placing it in a bell jar pre-charged with 2-3 mL isoflurane. Check for toe pinch reflex, and if non-responsive, rapidly decapitate it using a pair of sharp shears or guillotine as your animal protocol requires.
   2. Make a 2-3 cm incision using shears from the caudal portion of the skull to cut the scalp along the midline. While manually retracting the scalp, make two 1 cm horizontal incisions from the foramen magnum along the sides of the skull. Then, using fine shears, cut an incision the length of the skull, along the midline from the back of the skull to the nose.
   3. Using fine forceps inserted near the midline, retract the incised skull in two portions. Extract the mouse brain from the skull and use a blade to remove the cerebellum and olfactory bulbs, which are respectively located at the caudal and rostral portions of the brain. These can be identified by the large fissures, which separate them from the cortex.
   4. Mount the brain block onto the vibratome tray using super glue. Fill the vibratome tray with ice cold cutting solution.
   5. Cut tissue sections on the coronal plane at 300 µm thickness. Usually, 6 coronal hippocampal slices can be collected.
   6. After each section is cut, immediately transfer the slices to the slice holding beaker warmed to 32-34°C. Keep the sections at this temperature for 20 min before removing the beaker containing the sections and place this at room temperature for at least 20-30 minutes prior to recording.

2. Measurement of Electrically Evoked K⁺ Dynamics

1. Setting up the slice preparation
   1. 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.
   2. Ensure the tips of the bipolar stimulating electrode are parallel to one another and are level with the plane of the slice. Slowly, over the course of 6-7 seconds, insert the electrodes into CA3 stratum radiatum approximately 40-50 µm deep to stimulate Schaffer collaterals. In coronal sections, CA3 can be approximately identified as the portion of the hippocampus proper lateral to the granule cell layer at the hippocampal genu, with the stratum radiatum falling medial and ventral to the pyramidal cell layer.
   3. Carefully insert the K⁺-selective electrode into CA1 stratum radiatum approximately 50 µm deep, by slowly lowering the electrode over approximately 3-4 seconds. Allow the potential to stabilize across the electrode before applying stimulations to the slice: this usually takes 5 to 10 minutes. If the slice exhibits spontaneous changes in extracellular K⁺ then discard and repeat this process with a new slice.
2. Measure evoked K⁺ release
   1. Apply trains of electrical stimulation (8 pulses) by manually depressing the trigger on the stimulator while digitally recording responses. Apply stimulation at 10 Hz and 1 ms pulse width, starting at 10 µA stimulus amplitude.
   2. Apply increasing stimulation amplitudes by a factor of 2 until a maximum K⁺ response amplitude is detected. If you do not see any response, move the position of the K⁺ electrode closer to the stimulation site in 100 µm in increments
   3. Determine the stimulus amplitude that produces the half maximal response. In our experience, this is between 40-160 µA, depending upon the preparation quality, age of the animal, and the distance between stimulation electrodes and K⁺ selective electrode.
   4. In the same slice, using a stimulus amplitude at one step lower than the amplitude that produces the half maximal response (e.g. if 80 µA produces a half-maximal response, use 40 µA) apply stimulus trains of increasing number of pulses. Initially, we have used trains of 1, 2, 4, 8, 16, 32, 64 and 128 pulses.
   5. To confirm that K⁺ signals are mediated by action potential firing of the electrically stimulated Schaffer collaterals bath, apply 0.5 µM tetrodotoxin (TTX) in ACSF for 10 minutes and repeat the stimulation protocol. No evoked responses should be observed.
   6. After finishing the slice experiment, confirm the electrode has maintained its responsivity by re-calibrating the electrode in the calibration solutions and ensuring the response has not deviated by more than 10% from the initial calibration