impact of direct current stimulation on evoked epileptic seizures in mouse brain slices

Impact of Direct Current Stimulation on Evoked Epileptic Seizures in Mouse Brain Slices

In this procedure, ensure the generation of uniform electric fields by passing currents between two parallel silver chloride-coated silver wires that are placed inside the MEA chamber. If there are no issues, the DCS should stay between 0.5 and 2 milliamps.
Next, turn off the DCS and stimulate the thalamus with a tungsten electrode. To obtain maximal synaptic responses in ACC, record 10 to 20 responses and average them. Then, turn on the DCS and stimulate the thalamus simultaneously. Evaluate the amplitude changes of the thalamic stimulation-evoked ACC response during DCS.
Now, turn off the DCS. Add 250 micromolar 4-AP and 5 micromolar bicuculline to the perfusion solution, and wait two to three hours. If the drugs affect the brain slice, the slice should produce cortical seizure responses. Subsequently, collect 10 to 20 anterior cingulate cortical responses and measure the amplitude and duration of the electrically-evoked evoked cortical seizure responses.
Next, turn on the DCS and stimulate the thalamus simultaneously. Evaluate the changes in the amplitude and duration of the evoked cortical seizure responses during DCS application. After that, replace the perfusion solution with fresh aCSF to wash out the drugs.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparing Experimental Solution and Equipment for Multielectrode Array Recording
<ol>
	<li>Prepare artificial cerebral spinal fluid (artificial cerebrospinal fluid [aCSF]; 124 mM NaCl, 4.4 mM KCl, 1 mM NaH2PO3, 2 mM MgSO4, 2 mM CaCl2, 25 mM NaHCO3, and 10 mM glucose, bubbled with 95% O2&#160;and 5% CO2).</li>
	<li>Use two types of MEA probes: 6 x 10 planar MEA and 8 x 8 MEA. The former probe covers the region that comprises the cortex, striatum, and thalamus. The latter probe covers only the cortical region.</li>
	<li>Use a 60-channel amplifier with a band-pass filter set between 0.1 Hz and 3 kHz at 1,200 amplification. Acquire data at a 10 kHz sampling rate.</li>
	<li>Place two AgCl-coated silver wires inside the MEA chamber for DCS. Use the AgCl-coated silver wires to produce electric fields that are generated by an isolated stimulator.</li>
	<li>Place a tungsten electrode (diameter, 127 &#181;m; length, 7.62 cm; 8&#176; AC tapered tip; resistance, 5 M&#937;) for thalamic stimulation, and place the reference electrode in the MEA chamber. Deliver the tungsten electrode&#39;s currents using an isolated stimulator that is controlled by a pulse generator.</li>
</ol>
2. Brain Slice Preparation
<ol>
	<li>Use male C57BL/6J mice, 4-8 weeks old. House the animals in an air-conditioned room (21-23 &#176;C; 50% humidity; 12 hr/12 hr light /dark cycle, lights on at 8:00 AM) with free access to food and water.</li>
	<li>Take a 250 ml&#160;aliquot of the aCSF that was prepared in Step 1.1 and place it in a beaker that contains ice. At the same time, supply continuous gas that is composed of 95% O2&#160;and 5% CO2.</li>
	<li>Surgery
	<ol>
		<li>Anesthetize the animal with 4% isoflurane in a glass box for approximately 3 min. Once the animal reaches a surgical depth of anesthesia (indicated by the lack of a response to toe pinch), place it on a shallow tray that is filled with crushed ice, and remove the head using scissors.</li>
		<li>Expose the skull, and trim off the remaining muscle. Next, using rongeurs, peel away the dorsal surface of the skull from the brain. Trim away the sides of the skull using rongeurs. Sterilize all of the surgical instruments with a 75% ethanol solution.</li>
		<li>Using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain, and remove the brain. After decapitation, quickly transfer the brain to a beaker filled with ice-cold oxygenated aCSF.</li>
	</ol>
	</li>
	<li>Preparation of MT-ACC Brain Slice 
	Note: Prepare slices that contain the pathway from the MT to ACC.
	<ol>
		<li>Hand-cut the brain block with two sagittal cuts 2.0 mm lateral to the midline in each hemisphere to display the subcortical anatomy. Then make two angled cuts. Make the first cross-cut parallel to the visible fiber tract in the striatum.</li>
		<li>Make the second crosscut from the connection between the cerebellum and visual cortex to the midpoint between the anterior commissure and optic tract that are ventral and parallel to the thalamocingulate pathway.</li>
		<li>Attach the brain block to an angular plate (~120&#176;) with cyanoacrylate adhesive and make a cut just above the turning point of the pathway. Unfold the plate, flatten it, and glue it onto the chamber stage of a vibratome.</li>
		<li>Make medial thalamus-ACC brain slices (500 &#181;m thick) and then immerse them in ice-cold oxygenated aCSF. Transfer slices to the recording chamber, and keep at 32 &#176;C under continuous perfusion (12 ml/min) with oxygenated aCSF for 1 hr.</li>
	</ol>
	</li>
</ol>
3. Preparation of Perfusion Chamber for Multielectrode Array Recording
<ol>
	<li>Preparation of Perfusion Chamber
	<ol>
		<li>Place a MEA probe on a multi-channel system and use two separate polyethylene tubes to connect the probe to a peristaltic pump. Use one tube to guide the aCSF into the MEA chamber and the other tube to guide the aCSF out of the chamber. Finally, continuously perfuse the preparation with warm (29-30 &#176;C) oxygenated aCSF (8 ml/min).</li>
	</ol>
	</li>
	<li>Transfer brain slice to MEA. Hold down the brain slice on the MEA using a wet cotton swab. Carefully move the brain slice to ensure the ACC is oriented above the electrodes.</li>
	<li>Use slice anchor kits and hold-downs to press the brain slice. This step ensures a good electrical connection between the slice and electrodes.</li>
</ol>
4. Generation of Electric Fields by DCS
Note: The definition of the electric field orientation was based on the direction of the axodendritic axis in the ACC. The orientations of dendrite and soma compartments were confirmed using Golgi staining.
<ol>
	<li>Place the AgCl electrode (defined as the anode) proximal to the ACC and place the other electrode (defined as the cathode) distal to the ACC. Record the field strength that is generated by the two field orientations (parallel and perpendicular to the ACC axodendritic fibers) by the MEA, and deliver the currents of the electric fields using a stimulator.</li>
	<li>Fix the distance of the AgCl electrodes (about 1.5-2 cm) and adjust the stimulator&#39;s current strength to make the DCS between 0.5 and 2 mA.</li>
</ol>
5. Electrically induced Cortical Synaptic Responses
Note: Induce synaptic responses in the ACC by electrical stimulation in the MT, in which a programmable electrical stimulus generator produces rectangular biphasic current pulses.
<ol>
	<li>Repeat Section 3 above.</li>
	<li>Place a tungsten electrode in the MT and deliver pulses from the stimulator to the thalamic region of the slices via bipolar tungsten electrodes.</li>
	<li>Use various current intensities to determine the threshold that elicits an ACC response. Here, use an intensity of &#177;150 &#181;A and duration of 200 &#181;sec, which elicited an 80% maximal response in the ACC in most slices.</li>
	<li>Move the tungsten electrode along the thalamocingulate pathway (from MT to corpus callosum) in the MT-ACC slice to obtain the optimal response profiles.</li>
	<li>Make 10-20 sweeps of ACC responses and use the software to automatically average all of the ACC evoked by MT stimulation. The result is the synaptic responses in ACC induced from MT stimulation by MT-ACC pathway.</li>
</ol>
6. Electrically induced Seizure-like Activity
Note: Seizure-like activity was induced by the application of 4-aminopyridine (4-AP; 250 &#181;M) and bicuculline (5 &#181;M). Previous time-control studies showed that maximal and stable responses appeared 2-3 hr after drug application.
<ol>
	<li>Repeat Section 5 above.</li>
	<li>Add drugs to the perfusion solution. Use 4-AP (250 &#181;M) and bicuculline (5 &#181;M). Mix the drugs uniformly, and continue perfusion for 2-3 hr.</li>
	<li>To facilitate seizure-like activity, maintain the perfusion pump at a relatively fast perfusion rate (8 ml/min), which can also help prevent the build-up of a pH gradient.</li>
	<li>Place a tungsten electrode in the MT, and deliver electrical stimulation (150 &#181;A, 200 &#181;sec duration) to obtain ACC response profiles.</li>
	<li>Make 10-20 sweeps and average the responses.</li>
	<li>Replace the perfusion solution with fresh aCSF to wash out the drugs. Repeat Step 6.5.</li>
</ol>
7. Testing Effect of DCS on Evoked Cortical Responses
<ol>
	<li>Repeat Sections 3 and 4. Ensure that uniform electric fields are generated by passing currents between two parallel AgCl-coated silver wires that are placed inside the MEA chamber. If there are no issues, the DCS stays between 0.5 and 2 mA.</li>
	<li>Turn off the DCS and place a tungsten electrode to stimulate the thalamus (&#177;150 &#181;A, 200 &#181;sec duration). To obtain maximal synaptic responses in the ACC, make 10-20 sweeps and average the responses.</li>
	<li>Simultaneously turn on the DCS (2 mV/mm DCS strength) and thalamic stimulation (350 &#181;A, 200 &#181;sec duration). Evaluate the changes of amplitude of the thalamic stimulation-evoked ACC response during DCS.</li>
	<li>Turn off the DCS and add 4-AP (250 &#181;M) and bicuculline (5 &#181;M) to the perfusion solution. Then wait 2-3 hr. When the drugs affect the brain slice, the slice produces cortical seizure responses.</li>
	<li>Make 10-20 sweeps of ACC responses, and then measure the amplitude and duration of electrical evoked cortical seizure responses.</li>
	<li>After step 7.5, simultaneously turn on the DCS (2 mV/mm DCS strength) and thalamic stimulation (150 &#181;A, 200 duration &#181;sec). Evaluate changes in the amplitude and duration of evoked cortical seizure responses during DCS application.</li>
	<li>Replace the perfusion solution with fresh aCSF to wash out the drugs, and repeat steps 7.2 and 7.3.</li>
	<li>Collect all of the recording data and group the data into the different experimental conditions. Evaluate the amplitude and duration of cortical seizure responses under different experimental conditions.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthetic:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Halocarbon Products Corporation&#160;</td>
			<td>NDC 12164-002-25</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>aCSF (total:1 L):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>MERCK</td>
			<td>1.08337.1000</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>MERCK</td>
			<td>1.06329.0500</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>MERCK</td>
			<td>1.06404.1000</td>
			<td>124 mM</td>
		</tr>
		<tr>
			<td>(+)-Sodium L-ascorbate,&#62;=98%</td>
			<td>SIGMA</td>
			<td>A4034-100G</td>
			<td>0.15 g/2 c.c</td>
		</tr>
		<tr>
			<td>Magnesium sulfate, anhydrous, ReagentPlus</td>
			<td>SIGMA</td>
			<td>M7506-500G</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>MERCK</td>
			<td>1.02382.1000</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>MERCK</td>
			<td>1.06346.1000</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>May &#38; Baker LTD Dagenham England</td>
			<td>MS 7616</td>
			<td>4.4 mM</td>
		</tr>
		<tr>
			<td>Drugs:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(+)-Bicuculline</td>
			<td>TOCRIS</td>
			<td>130</td>
			<td>5 &#181;M in aCSF</td>
		</tr>
		<tr>
			<td>4-Aminopyridine</td>
			<td>TOCRIS</td>
			<td>940</td>
			<td>250 &#181;M in aCSF</td>
		</tr>
		<tr>
			<td>Brain slice Preparation:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Vibratome</td>
			<td>Series 1000</td>
			<td>Block slicing into 500 &#181;m thick slices</td>
		</tr>
		<tr>
			<td>MEA system:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multielectrode array (MEA) probes: 6 x 10 planar MEA</td>
			<td>Multi Channel Systems</td>
			<td>60MEA500/30iR-Ti-pr MEAS 6x10</td>
			<td>electrode diameter, 30 &#181;m; electrode spacing, 500 &#181;m; impedance, 50 k&#937; at 200 Hz</td>
		</tr>
		<tr>
			<td>Multielectrode array (MEA) probes: 8 x 8 MEA</td>
			<td>Ayanda Biosystems</td>
			<td>60MEA200/10iR-Ti-pr MEAS 8x8</td>
			<td>pyramidal-shaped electrode; diameter, 40 &#181;m; tip height, 50 &#181;m; electrode spacing, 200 &#181;m; impedance, 1,000 k&#937; at 200 Hz</td>
		</tr>
		<tr>
			<td>A 60-channel amplifier was used with a band-pass filter set between 0.1 Hz and 3 KHz at 1,200X amplification</td>
			<td>Multi Channel Systems</td>
			<td>MEA-1060-BC</td>
			<td></td>
		</tr>
		<tr>
			<td>MC Rack software at a 10 KHz sampling rate</td>
			<td>Multi-Channel Systems</td>
			<td></td>
			<td>Software for data collect and recordings</td>
		</tr>
		<tr>
			<td>control of a pulse generator</td>
			<td>Multi-Channel Systems</td>
			<td>STG 1002</td>
			<td></td>
		</tr>
		<tr>
			<td>slice anchor kits and hold-downs</td>
			<td>Warner Instruments</td>
			<td>SHD-26H/10; WI64-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump-minipuls3</td>
			<td>Gilsom</td>
			<td>MINIPULS3</td>
			<td>perfusion rate : 8 ml/min</td>
		</tr>
		<tr>
			<td>Stimulation system:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated stimulator</td>
			<td>A-M Systems</td>
			<td>Model 2100</td>
			<td>intensity of &#177;350 &#956;A , duration of 200 &#956;sec</td>
		</tr>
		<tr>
			<td>Tungsten electrode</td>
			<td>A-M Systems</td>
			<td>575300</td>
			<td>placed in thalamus</td>
		</tr>
	</tbody>
</table>

Source: Lu, H., et al. <a href="https://app.jove.com/v/53709">Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation.</a> J. Vis. Exp. (2016)
The video demonstrates a study for assessing the impact of direct current stimulation (DCS) on epileptic seizures in mouse brain slices. The brain slices containing the connected medial thalamus (MT) and anterior cingulate cortex (ACC) regions are placed in a multielectrode array (MEA). Electrical stimuli to the MT and seizure-inducing drugs are applied to induce epileptic seizures in the ACC. DCS is then applied to assess its impact on evoked seizures.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparing Experimental Solution ...

Take a mouse brain slice containing the medial thalamus, or MT, projecting to the anterior cingulate cortex, or ACC.
Secure the slice in a multielectrode array chamber perfused with oxygenated aCSF to record electrical signals.
Place positive and negative electrodes and apply direct current stimulation, or DCS, generating an electric field across the slice.
Disable DCS and place an electrode in the MT. Deliver a stimulus to evoke action potentials in the thalamic neurons, triggering neurotransmitter release.
The neurotransmitters trigger ion influx in ACC neurons and generate electrical signals, termed stimulation-evoked synaptic responses.
Reactivate DCS to monitor its effects on ACC synaptic responses.
Next, deactivate DCS and introduce seizure-inducing drugs.
The drugs increase neuronal excitability, causing the thalamic stimulus to produce continuous bursts of signals in the ACC, termed evoked epileptic seizures.
Finally, reactivate DCS to evaluate its effect on seizures by measuring the reduction in seizure amplitude and duration.

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Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the cases, serious side effects affect the quality of life of patients. Moreover, a high percentage of epileptic patients are drug resistant; in their case, neurosurgery or neurostimulation are necessary. Therefore, the major goal of epilepsy research is to discover new therapies which are either capable of curing epilepsy without side effects or preventing recurrent seizures in drug-resistant patients. Neuroengineering provides new approaches by using novel strategies and technologies to find better solutions to cure epileptic patients at risk.
As a demonstration of a novel experimental protocol in an acute mouse model of epilepsy, a direct in situ electrophoretic drug delivery system is used. Namely, a neural probe incorporating a microfluidic ion pump (&#181;FIP) for on-demand drug delivery and simultaneous recording of local neural activity is implanted and demonstrated to be capable of controlling 4-aminopyridine-induced (4AP-induced) seizure-like event (SLE) activity. The &#947;-aminobutyric acid (GABA) concentration is kept in the physiological range by the precise control of GABA delivery to reach an antiepileptic effect in the seizure focus but not to cause overinhibition-induced rebound bursts. The method allows both the detection of pathological activity and intervention to stop seizures by delivering inhibitory neurotransmitters directly to the epileptic focus with precise spatiotemporal control.
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Electrophoretic Delivery of &#947;-aminobutyric Acid GABA into Epileptic Focus Prevents Seizures in Mice

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Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

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Impact of Direct Current Stimulation on Evoked Epileptic Seizures in Mouse Brain Slices

Transcripción

Impact of Direct Current Stimulation on Evoked Epileptic Seizures in Mouse Brain Slices