Name: making, testing, and using potassium ion selective microelectrodes in tissue slices of adult brain
Uploaded: 2018-05-07T14:45:00
Description: Watch this Scientific Journal Video about Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain at JoVE.com

The Khakh lab was supported by NIH MH104069. The Mody lab was supported by NIH NS030549. J.C.O. thanks the NIH T32 Neural Microcircuits Training Grant(NS058280).

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&#39;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 wer...

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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 procedures12,13,14,15. However, this method has advantages over alternative electrode configurations in that ...

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 body1,2. 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 healthy and diseased states have been identified3,4,5, further progress has been slowed due to the specialized nature of preparing ion selective microelectrodes for potassium measurement. Microelectrode sensors represent the gold standard for measuring K+ concentrations in vitro, in tissue slices and in vivo.
Newer approaches for K+ monitoring are under development using optical sensors, however these do not detect a biologically relevant range of K+ concentrations or have not been fully vetted in biological systems, although initial results appear promising6,7,8. Compared to optical sensors, microelectrodes are fundamentally limited to a point source measurement of ions, although electrode arrays could improve the spatial resolution9. This article focuses on the single-barreled microelectrode sensors for monitoring K+ dynamics.
In this work, we report detailed stepwise procedures to make K+ selective microelectrodes, using a valinomycin-based potassium ionophore that permits highly selective (104 fold K+ to Na+ selectivity) K+ movement over membranes10. A naturally occurring polypeptide, valinomycin acts as a K+ permeable pore and facilitates the flow of K+ down it&#39;s electrochemical gradient. We also describe how to calibrate the electrodes, how to store and use them and finally how to deploy them to measure K+ concentration dynamics in acute hippocampal brain slices from adult mice. The use of such electrodes together with genetically modified mice that lack specific ion channels proposed to regulate extracellular K+ dynamics should reveal the cellular mechanisms used by the nervous system to control the ambient concentration of K+ in the extracellular milieu.

<table border="0" cellpadding="0" cellspacing="0" width="997">
	<tbody>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Vibratome</td>
			<td style="width:241px;">DSK</td>
			<td style="width:257px;">Microslicer Zero 1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Mouse: C57BL/6NTac inbred mice</td>
			<td>Taconic</td>
			<td style="width:257px;">Stock#B6</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Electrode puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">Ag/AgCl ground pellet</td>
			<td>WPI</td>
			<td>EP2</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">pCLAMP10.3</td>
			<td style="width:241px;">Molecular Devices</td>
			<td style="width:257px;">n/a</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Custom microfil 28G tip</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">CMF28G</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Tungsten Rod</td>
			<td style="width:241px;">A-M Systems</td>
			<td style="width:257px;">716000</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Bipolar stimulating electrodes</td>
			<td style="width:241px;">FHC</td>
			<td style="width:257px;">MX21XEW(T01)</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Stimulus isolator</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">A365</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Grass S88 Stimulator</td>
			<td style="width:241px;">Grass Instruments Company</td>
			<td style="width:257px;">S88</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Borosilicate glass pipettes</td>
			<td style="width:241px;">World precision instruments</td>
			<td style="width:257px;">1B150-4</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">A to D board</td>
			<td style="width:241px;">Digidata 1322A</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Signal Amplifier</td>
			<td style="width:241px;">Multiclamp 700A or 700B</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Headstage</td>
			<td style="width:241px;">CV-7B Cat 1</td>
			<td style="width:257px;">Axon Instruments</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Patch computer</td>
			<td style="width:241px;">Dell</td>
			<td style="width:257px;">n/a</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S5886</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Potassium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">P3911</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">HEPES</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">H3375</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Bicarbonate</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S5761</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sodium Phosphate Monobasic</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S0751</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">D-glucose</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">G7528</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Calcium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">21108</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Magnesium Chloride</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">M8266</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">valinomycin</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">V0627-10mg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">1,2-dimethyl-3-nitrobenzene</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">40870-25ml</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Potassium tetrakis (4-chlorophenyl)borate</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">60591-100mg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">5% dimethyldichlorosilane in heptane</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">85126-5ml</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">TTX</td>
			<td style="width:241px;">Cayman Chemical Company</td>
			<td style="width:257px;">14964</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Hydrochloric acid</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">H1758-500mL</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Sucrose</td>
			<td style="width:241px;">Sigma</td>
			<td style="width:257px;">S9378-5kg</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Pipette Micromanipulator</td>
			<td style="width:241px;">Sutter</td>
			<td style="width:257px;">MP-285 / ROE-200 / MPC-200</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:332px;">Objective lens</td>
			<td style="width:241px;">Olympus</td>
			<td style="width:257px;">PlanAPO 10xW</td>
			<td></td>
		</tr>
	</tbody>
</table>

making, testing, and using potassium ion selective microelectrodes in tissue slices of adult brain

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.

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...

Watch this Scientific Journal Video about Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain at JoVE.com

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

Potassium ions contribute&#160;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.

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

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