Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Summary

Potassium ions contribute to the resting membrane potential of cells and extracellular K⁺ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K⁺-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K⁺ concentration dynamics in adult hippocampal slices.

Abstract

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K⁺ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K⁺ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K⁺ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K⁺-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K⁺ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K⁺ concentrations in the nervous system.

Introduction

Potassium ion concentrations are tightly regulated in the brain, and their fluctuations exert a powerful influence on the resting membrane potential of all cells. In light of these critical contributions, an important goal of biology is to determine the cellular and biophysical mechanisms that are used to tightly regulate the concentration of K⁺ in the extracellular space in different organs of the body^1,2. An important requirement in these studies is the ability to measure K⁺ concentrations accurately. Although many components which contribute to potassium homeostasis in the brain in hea....

Protocol

All animal experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Chancellor's Animal Research Committee at the University of California, Los Angeles. All mice were housed with food and water available ad libitum in a 12 h light-dark environment. All animals were healthy with no obvious behavioral changes, were not involved in previous studies, and were sacrificed during the light cycle. Data for experiments were collected from adult mice (6-8 weeks old for all experiments).

1. Preparation of K⁺ selective microelectrodes

Results

For selective measurement of extracellular K⁺, we prepared ion-selective microelectrodes coated with a hydrophobic layer through silanization of clean borosilicate glass pipettes (Figure 1A). This coating enables the K⁺ ionophore containing valinomycin to rest at the tip of the electrode and permit only K⁺ flux through a narrow opening at the electrode tip (Figure 1B). After priming the electrodes.......

Discussion

The method that we describe here has allowed us to assess K⁺ dynamics in response to electrical stimulation of Schaffer collaterals in acute hippocampal slices from adult mice. Our method of preparing K⁺ ion selective microelectrodes is similar to earlier described procedures^12,13,14,15. However, this method has advantages over alternative electrode configurations in that .......

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
Electrode puller	Sutter	P-97
Ag/AgCl ground pellet	WPI	EP2
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