Name: making, testing, and using potassium ion selective microelectrodes in tissue slices of adult brain
Uploaded: 2018-05-07T14:45:00.000+00:00
Duration: 11 min 20 s
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 were collected from adult mice (6-8 weeks old for all experiments).
1. Preparation of K+ selective microelectrodes
<ol>
	<li>Silanization of borosilicate glass
	<ol>
		<li>Remove sufficient glass capillaries from packaging and place into a 50 mL conical tube. Fill conical tube to top with 1 M HCl. Wash electrodes with gentle agitation in HCl overnight or for a minimum of 6 hours.</li>
		<li>Briefly rinse capillaries with 70% ethanol and then dry completely at 100-120 &#176;C for 6-8 hours. Store washed capillaries in containers with anhydrous calcium sulfate desiccant for up to 4 weeks before further use.</li>
		<li>Prior to silanization, pull capillaries to a fine tip using a microelectrode puller. The microelectrodes that we use are approximately 2-5 microns in diameter. Always handle washed capillaries with gloves, as oils from skin can interfere with silanization.</li>
		<li>Place microelectrodes into a glass container so that the electrodes are elevated from the bottom to prevent tip breakage. Fix the microelectrodes to the container using autoclavable tape or similar adhesive tape.</li>
		<li>Remove approximately 0.5 mL of 5% dichlorodimethylsilane (DDS) silanization solution from its container using the nitrogen replacement method (see Figure 2). Fill a balloon with nitrogen gas and attach a syringe or tube and needle to the balloon. Insert the needle into the DDS container while drawing up DDS into a separate syringe via a longer needle.</li>
		<li>Apply the silanization solution dropwise to the tips of the pipettes and immediately cover. Place the container holding the microelectrodes with silanization solution into a pre-warmed (170-180 &#176;C) laboratory oven for 10-12 hours or at 200-220 &#176;C for 30 minutes</li>
		<li>After the incubation, turn off the oven and then remove the plate from the oven. Be careful when removing the plate from the incubator, as it is extremely hot. Place the plate on a bench at room temperature for 10-15 minutes to allow the glassware to cool.</li>
		<li>Remove the microelectrodes from the plate (using a razor blade or scalpel blade to cut the tape) and place them into a desiccant filled airtight container. Silanized microelectrodes kept free of moisture can be used for up to 1 week following silanization.</li>
	</ol>
	</li>
	<li>Priming the electrodes
	<ol>
		<li>Prepare a stock solution of K+ ionophore cocktail: 5% w/v valinomycin, 93% v/v 1,2-dimethyl-3-nitrobenzene, 2% w/v potassium tetrakis(4-chlorophenyl)borate11. This solution is a faint yellow color. Keep in an airtight, opaque container at room temperature. If properly stored this solution can last many months.</li>
		<li>Prepare a stock solution of 10 mM HEPES buffered 300 mM NaCl at pH 7.4. Fix the electrode into a clamp and backfill with the buffered NaCl using a 28G microfil tip connected to a syringe. Observe that the saline solution has reached the end of the microelectrode tip. Confirm that the microelectrode is free of large bubbles that could interfere with the flow of current.</li>
		<li>Break the tip of the electrode to approximately 10-20 &#181;m wide, using the blunt side of a scalpel or razor blade.</li>
		<li>Using a micropipette, apply a small droplet (~0.1 &#181;l) of the K+ ionophore near the tip of the microelectrode. If the electrode has been properly silanized the droplet will be absorbed into the broken tip. Fill the electrode to 1-2 mm with the K+ ionophore, and remove excess using tissue paper.</li>
	</ol>
	</li>
</ol>
2. Calibration of K+ Selective Microelectrodes
<ol>
	<li>Preparation of calibration solutions
	<ol>
		<li>Prepare solutions of various concentrations of KCl in equal osmolarity artificial cerebrospinal fluid (ACSF) by replacing NaCl with KCl. We used 0.1, 1, 4.5, 10 and 100 mM K+ ACSF, for calibration of the electrodes. The recipes for these calibration solutions are listed in Table 1.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Chemical</td>
			<td>MW&#160;</td>
			<td rowspan="2">final mM</td>
			<td rowspan="2">0.1 mM [K+]</td>
			<td rowspan="2">1 mM [K+]</td>
			<td rowspan="2">4.5 mM [K+]</td>
			<td rowspan="2">10 mM [K+]</td>
			<td rowspan="2">100 mM [K+]</td>
		</tr>
		<tr>
			<td>(g / mol)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>58.44</td>
			<td>varies</td>
			<td>1.51 g</td>
			<td>1.50 g</td>
			<td>1.44 g</td>
			<td>1.4 g</td>
			<td>0.345 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>1 M stock</td>
			<td>varies</td>
			<td>20 &#181;l</td>
			<td>200 &#181;l</td>
			<td>900 &#181;l</td>
			<td>2 ml</td>
			<td>20 ml</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 M stock</td>
			<td>2</td>
			<td colspan="5">400 &#181;l</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 M stock</td>
			<td>1</td>
			<td colspan="5">200 &#181;l&#160;&#160;</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>119.98</td>
			<td>1.2</td>
			<td colspan="5">0.29 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>84.01</td>
			<td>26</td>
			<td colspan="5">0.437 g</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>180.16</td>
			<td>10</td>
			<td colspan="5">0.360 g</td>
		</tr>
		<tr>
			<td>Water</td>
			<td colspan="7">q.s. 200 ml</td>
		</tr>
	</tbody>
</table>
Table 1. Potassium calibration solutions
<ol start="2">
	<li>Microelectrode calibration
	<ol>
		<li>Bubble all solutions with 95% O2/5% CO2 for at least 20 minutes before beginning the experiment. Begin perfusing the bath with 4.5 mM [K+] ACSF at a rate of 3 mL per minute. Place the K+ selective electrode into electrode holder attached to the electrode headstage on the manipulator. This headstage is connected to an appropriate amplifier. Insert the tip of the electrode into the bath perfusate.</li>
		<li>Ensure the Ag/AgCl ground electrode is bathed in the same solution and that the flow is steady. Apply calibration solutions in a stepwise fashion and record the potential changes in mV across the electrode tip. Wait for the potential at the electrode tip to reach a stable value before switching to the next solution</li>
		<li>Measure the steady state voltage change in response to the application of the calibration solutions to the electrode tip. Confirm that the slope of the electrode response is at least 52 and no greater than 58 mV per log change in [K+].</li>
	</ol>
	</li>
</ol>
3. Preparation of Acute Hippocampal Brain Slices
<ol>
	<li>Preparation of slice solutions
	<ol>
		<li>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.</li>
		<li>Prepare 1-2 liters of recording solution (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 &#176;C. Fill the vibratome chamber with ice-water slush.</li>
	</ol>
	</li>
	<li>Acute slice preparation
	<ol>
		<li>Deeply anesthetize a mouse by placing it in a bell jar precharged 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Mount the brain block onto the vibratome tray using super glue. Fill the vibratome tray with ice cold cutting solution.</li>
		<li>Cut tissue sections on the coronal plane at 300 &#181;m thickness. Usually 6 coronal hippocampal slices can be collected.</li>
		<li>After each section is cut, immediately transfer the slices to the slice holding beaker warmed to 32-34&#176;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.</li>
	</ol>
	</li>
</ol>
4. Measurement of Electrically Evoked K+ Dynamics
<ol>
	<li>Setting up the slice preparation
	<ol>
		<li>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.</li>
		<li>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 &#181;m 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.</li>
		<li>Carefully insert the K+-selective electrode into CA1 stratum radiatum approximately 50 &#181;m 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.</li>
	</ol>
	</li>
	<li>Measure evoked K+ release
	<ol>
		<li>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 &#181;A stimulus amplitude.</li>
		<li>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 &#181;m in increments</li>
		<li>Determine the stimulus amplitude that produces the half maximal response. In our experience, this is between 40-160 &#181;A, depending upon the preparation quality, age of the animal, and the distance between stimulation electrodes and K+ selective electrode.</li>
		<li>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 &#181;A produces a half-maximal response, use 40 &#181;A) apply stimulus trains of increasing number of pulses. Initially, we have used trains of 1, 2, 4, 8, 16, 32, 64 and 128&#160;pulses.</li>
		<li>To confirm that K+ signals are mediated by action potential firing of the electrically stimulated Schaffer collaterals bath, apply 0.5 &#181;M TTX in ACSF for 10 minutes and repeat the stimulation protocol. No evoked responses should be observed.</li>
		<li>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</li>
	</ol>
	</li>
</ol>

<ol>
	<li>McDonough, A. A., Youn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+homeostasis:+The+knowns,+the+unknowns,+and+the+health+benefits.">Potassium homeostasis: The knowns, the unknowns, and the health benefits.</a> Physiol Bethesda Md. 32 (2), 100-111 (2017).</li><li>Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion channels of excitable membranes. , 507 (2001).</li><li>Kofuji, P., Ceelen, P., Zahs, K. R., Surbeck, L. W., Lester, H. A., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+inactivation+of+an+inwardly+rectifying+potassium+channel+(Kir4.1+subunit)+in+mice:+Phenotypic+impact+in+retina.">Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: Phenotypic impact in retina.</a> J Neurosci. 20 (15), 5733-5740 (2000).</li><li>Sibille, J., Dao Duc, K., Holcman, D., Rouach, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroglial+potassium+cycle+during+neurotransmission:+role+of+Kir4.1+channels.">The neuroglial potassium cycle during neurotransmission: role of Kir4.1 channels.</a> PLoS Comput Biol. 11 (3), e1004137 (2015).</li><li>Tong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+Kir4.1+ion+channel+deficits+contribute+to+neuronal+dysfunction+in+Huntington's+disease+model+mice.">Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice.</a> Nat Neurosci. 17 (5), 694-703 (2014).</li><li>Datta, D., Sarkar, K., Mukherjee, S., Meshik, X., Stroscio, M. A., Dutta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphene+oxide+and+DNA+aptamer+based+sub-nanomolar+potassium+detecting+optical+nanosensor.">Graphene oxide and DNA aptamer based sub-nanomolar potassium detecting optical nanosensor.</a> Nanotechnology. 28 (32), 325502 (2017).</li><li>Bandara, H. M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palladium-Mediated+Synthesis+of+a+Near-Infrared+Fluorescent+K++Sensor.">Palladium-Mediated Synthesis of a Near-Infrared Fluorescent K+ Sensor.</a> J Org Chem. 82 (15), 8199-8205 (2017).</li><li>Depauw, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+selective+potassium+sensor+for+the+detection+of+potassium+in+living+tissues.">A highly selective potassium sensor for the detection of potassium in living tissues.</a> Chem Weinh Bergstr Ger. 22 (42), 14902-14911 (2016).</li><li>Machado, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofouling-Resistant+Impedimetric+Sensor+for+Array+High-Resolution+Extracellular+Potassium+Monitoring+in+the+Brain.">Biofouling-Resistant Impedimetric Sensor for Array High-Resolution Extracellular Potassium Monitoring in the Brain.</a> Biosensors. 6 (4), (2016).</li><li>Rose, M. C., Henkens, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+sodium+and+potassium+complexes+of+valinomycin.">Stability of sodium and potassium complexes of valinomycin.</a> Biochim Biophys Acta BBA - Gen Subj. 372 (2), 426-435 (1974).</li><li>Ammann, D., Chao, P., Simon, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valinomycin-based+K++selective+microelectrodes+with+low+electrical+membrane+resistance.">Valinomycin-based K+ selective microelectrodes with low electrical membrane resistance.</a> Neurosci Lett. 74 (2), 221-226 (1987).</li><li>Amzica, F., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+glial+membrane+potentials+during+sleep+and+paroxysmal+oscillations+in+the+neocortex.">Neuronal and glial membrane potentials during sleep and paroxysmal oscillations in the neocortex.</a> J Neurosci. 20 (17), 6648-6665 (2000).</li><li>Amzica, F., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+significance+of+K-complexes.">The functional significance of K-complexes.</a> Sleep Med Rev. 6 (2), 139-149 (2002).</li><li>MacVicar, B. A., Feighan, D., Brown, A., Ransom, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+optical+signals+in+the+rat+optic+nerve:+role+for+K(+)+uptake+via+NKCC1+and+swelling+of+astrocytes.">Intrinsic optical signals in the rat optic nerve: role for K(+) uptake via NKCC1 and swelling of astrocytes.</a> Glia. 37 (2), 114-123 (2002).</li><li>Chever, O., Djukic, B., McCarthy, K. D., Amzica, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implication+of+Kir4.1+channel+in+excess+potassium+clearance:+an+in+vivo+study+on+anesthetized+glial-conditional+Kir4.1+knock-out+mice.">Implication of Kir4.1 channel in excess potassium clearance: an in vivo study on anesthetized glial-conditional Kir4.1 knock-out mice.</a> J Neurosci. 30 (47), 15769-15777 (2010).</li><li>Hall, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-selective+membrane+electrodes:+A+general+limiting+treatment+of+interference+effects.">Ion-selective membrane electrodes: A general limiting treatment of interference effects.</a> J Phys Chem. 100 (17), 7230-7236 (1996).</li><li>Haack, N., Durry, S., Kafitz, K. W., Chesler, M., Rose, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-barreled+and+Concentric+Microelectrodes+for+Measurement+of+Extracellular+Ion+Signals+in+Brain+Tissue.">Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.</a> J Vis Exp. (103), e53058 (2015).</li><li>Larsen, B. R., MacAulay, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kir4.1-mediated+spatial+buffering+of+K(+):+Experimental+challenges+in+determination+of+its+temporal+and+quantitative+contribution+to+K(+)+clearance+in+the+brain.">Kir4.1-mediated spatial buffering of K(+): Experimental challenges in determination of its temporal and quantitative contribution to K(+) clearance in the brain.</a> Channels Austin Tex. 8 (6), 544-550 (2014).</li><li>Mei, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+recording+of+circadian+rhythms+in+brains+of+freely+moving+mice.">Long-term in vivo recording of circadian rhythms in brains of freely moving mice.</a> Proceedings of the National Academy of Sciences. 115, 4276-4281 (2018).</li></ol>

The authors have nothing to disclose.

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

making-testing-using-potassium-ion-selective-microelectrodes-tissue

Research

JoVE Journal

Neuroscience

12.0K Views.  University of California Los Angeles. 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.

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

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Medicine

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Characterizing Extracellular Space in a Brain Slice Using Ion-Selective and Iontophoresis Microelectrodes

Source:&#160; Odackal, J., et al. <a href="https://app.jove.com/v/55755">Real-time iontophoresis with tetramethylammonium to quantify volume fraction and tortuosity of brain extracellular space.</a> J. Vis. Exp. (2017)
This video demonstrates how to characterize the extracellular space in a brain slice by using ion-selective and iontophoresis microelectrodes to measure and compare electrical signals. This process helps determine the space&#39;s volume fraction and degree of twisting.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;&#160;&#160;&#160;
1. ...

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

Summary

Explore More Videos

Chapters in this video

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Horizontal Hippocampal Slices of the Mouse Brain

Characterizing Extracellular Space in a Brain Slice Using Ion-Selective and Iontophoresis Microelectrodes