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 MgCl....

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