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>

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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 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 ...

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

Research

JoVE Journal

Neuroscience

11.9K 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

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.