Measurement of Evoked Potassium Ion Concentration Dynamics in Coronal Hippocampal Slices

Abstract

Source: Octeau, J. C. et al. Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain. J. Vis. Exp. (2018).

The video demonstrates evoked potassium ion concentration dynamics on electrical stimulation of mouse coronal hippocampal brain slices. A bipolar stimulating electrode is inserted into the stratum radiatum of the CA3 region in the hippocampus, and a potassium-ion selective electrode is placed in the CA1 stratum radiatum. The electrical stimulation causes an action potential, resulting in potassium ion release. Finally, the lack of evoked potassium ion response in the presence of an inhibitory molecule confirms that potassium ion concentration dynamics are due to action potential generation in the Schaffer collaterals.

Protocol

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vibratome	DSK	Microslicer Zero 1
Mouse: C57BL/6NTac inbred mice	Taconic	Stock#B6
Microscope	Olympus	BX51
pCLAMP10.3	Molecular Devices	n/a
Custom microfil 28G tip	World precision instruments	CMF28G
Tungsten Rod	A-M Systems	716000
Bipolar stimulating electrodes	FHC	MX21XEW(T01)
Stimulus isolator	World precision instruments	A365
Grass S88 Stimulator	Grass Instruments Company	S88
Borosilicate glass pipettes	World precision instruments	1B150-4
A to D board	Digidata 1322A	Axon Instruments
Signal Amplifier	Multiclamp 700A or 700B	Axon Instruments
Headstage	CV-7B Cat 1	Axon Instruments
Patch computer	Dell	n/a
Sodium Chloride	Sigma	S5886
Potassium Chloride	Sigma	P3911
HEPES	Sigma	H3375
Sodium Bicarbonate	Sigma	S5761
Sodium Phosphate Monobasic	Sigma	S0751
D-glucose	Sigma	G7528
Calcium Chloride	Sigma	21108
Magnesium Chloride	Sigma	M8266
valinomycin	Sigma	V0627-10mg
1,2-dimethyl-3-nitrobenzene	Sigma	40870-25ml
Potassium tetrakis (4-chlorophenyl)borate	Sigma	60591-100mg
5% dimethyldichlorosilane in heptane	Sigma	85126-5ml
TTX	Cayman Chemical Company	14964
Hydrochloric acid	Sigma	H1758-500mL
Sucrose	Sigma	S9378-5kg
Pipette Micromanipulator	Sutter	MP-285 / ROE-200 / MPC-200
Objective lens	Olympus	PlanAPO 10xW