Name: direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation
Uploaded: 2016-06-07T14:15:00
Description: Watch this Scientific Journal Video about Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation at JoVE.com

We are grateful for the technical support from the Neural Circuit Electrophysiology Core at Academia Sinica. This work was supported by the National Science Council (102-2320-B-001-026-MY3 and 100-2311-B-001-003-MY3) and Neuroscience Program of Academia Sinica.

Процедуры, которые включают предметы животных были одобрены Уходу за животными и Комитетом по утилизации, Академии Синика, Тайбэй, Тайвань. 1. Подготовка экспериментального решения и оборудование для многоэлектродная регистрирующей <ol><li> Готовят искусственную спинно?...

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В настоящем исследовании, были протестированы эффекты продолжительности и ориентации DCS на АСС припадков типа деятельности. Для получения стабильных данных в срезах мозга мышей, как сохранить целостность пути МТ-ACC и во избежание повреждений он является ключевым, особенно шаги, в кото?...

Epilepsy is a common neurological disorder. Thirty percent of patients with epilepsy suffer from drug-resistant seizures1. Transcranial direct-current stimulation (tDCS) provides a noninvasive approach to control or alter network activities across large brain areas, such as seizures. Clinical studies have shown that tDCS effectively treats intractable seizures2 and can produce both short- and long-term suppressive effects on seizures3-5. However, the therapeutic mechanism of tDCS actions is still unclear. The brain slice model presented is an in vitro method to investigate how the therapeutic mechanism of tDCS actions alters the symptoms of seizure-like brain activities. Accordingly, to achieve its optimal effects, specific stimulation parameters including orientation, field strength, and stimulation duration need to be tested in an experimental model. Previous studies have shown that the orientation of the electric field is important to obtain therapeutic effects6. Thus, testing and arranging the orientation of electrodes relative to the position of the tested brain slice are feasible.
Frontal lobe epilepsy and anterior cingulate cortex (ACC) seizures are often drug-resistant7,8. Some studies have reported the application of tDCS in the cingulate cortex9-11. tDCS is shown to affect vigilance, decision making and emotion through alteration of ACC activities, and can modulate neuronal excitability and seizure activity in this brain region12. Therefore, suppressive effects of tDCS on ACC seizures might be helpful for clinical treatment and the evaluation of alternative treatments.
The present protocol describes the preparation of an electrode in the recording chamber for DCS of a brain slice and its effect on seizure-like activity recording with a multielectrode array (MEA).

<table border="0" cellpadding="0" cellspacing="0" width="1018">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1017px;">Anesthetic:</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>Halocarbon Products Corporation&#160;</td>
			<td>NDC 12164-002-25</td>
			<td style="width:187px;">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">aCSF (total:1L):</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-Glucose</td>
			<td>MERCK</td>
			<td>1.08337.1000</td>
			<td style="width:187px;">10 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydrogen carbonate</td>
			<td>MERCK</td>
			<td>1.06329.0500</td>
			<td style="width:187px;">25 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>MERCK</td>
			<td>1.06404.1000</td>
			<td style="width:187px;">124 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">(+)-Sodium L-ascorbate, &#62;=98%</td>
			<td>SIGMA</td>
			<td>A4034-100G</td>
			<td style="width:187px;">0.15 g / 2 c.c</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium sulfate, anhydrous,ReagentPlus</td>
			<td>SIGMA</td>
			<td>M7506-500G</td>
			<td style="width:187px;">2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>MERCK</td>
			<td>1.02382.1000</td>
			<td style="width:187px;">2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dihydrogen phosphate monohydrate</td>
			<td>MERCK</td>
			<td>1.06346.1000</td>
			<td style="width:187px;">1 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>May &#38; Baker LTD Dagenham England</td>
			<td>MS 7616</td>
			<td style="width:187px;">4.4 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Drugs:</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Bicuculline</td>
			<td>TOCRIS</td>
			<td>0130</td>
			<td style="width:187px;">5 &#181;M in aCSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Aminopyridine</td>
			<td>TOCRIS</td>
			<td>0940</td>
			<td style="width:187px;">250 &#181;M in aCSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Brain slice Preparation:</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Vibratome</td>
			<td>Vibratome</td>
			<td>Series 1000</td>
			<td style="width:187px;">Block slicing into 500 &#181;m thick slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">MEA system:</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:355px;">Multielectrode array (MEA) probes: 6 x 10 planar MEA</td>
			<td>Multi Channel Systems</td>
			<td style="width:184px;">60MEA500/30iR-Ti-pr MEAS 6x10</td>
			<td style="width:187px;">electrode diameter, 30 &#181;m; electrode spacing, 500 &#181;m; impedance, 50 k&#937; at 200 Hz</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:355px;">Multielectrode array (MEA) probes: 8 x 8 MEA&#160;</td>
			<td>Ayanda Biosystems</td>
			<td style="width:184px;">60MEA200/10iR-Ti-pr MEAS 8x8</td>
			<td style="width:187px;">pyramidal-shaped electrode; diameter, 40 &#181;m; tip height, 50 &#181;m; electrode spacing, 200 &#181;m; impedance, 1000 k&#937; at 200 Hz</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:355px;">A 60-channel amplifier was used with a band-pass filter set between 0.1 Hz and 3 KHz at 1200X amplification</td>
			<td>Multi-Channel Systems</td>
			<td>MEA-1060-BC</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MC Rack software at a 10 KHz sampling rate</td>
			<td>Multi-Channel Systems</td>
			<td></td>
			<td style="width:187px;">Software for data collect and recordings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">control of a pulse generator</td>
			<td>Multi-Channel Systems</td>
			<td>STG 1002</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">slice anchor kits and hold-downs</td>
			<td>Warner Instruments</td>
			<td style="width:184px;">SHD-26H/10; WI64-0250</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump-minipuls3</td>
			<td>Gilsom</td>
			<td style="width:184px;">MINIPULS3</td>
			<td>perfusion rate : 8 ml/min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Name</td>
			<td style="width:292px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Stimulation system:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isolated stimulator</td>
			<td>A-M Systems</td>
			<td>Model 2100</td>
			<td style="width:187px;">intensity of &#177;350 &#956;A , duration of 200 &#956;s</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tungsten electrode</td>
			<td>A-M Systems</td>
			<td>575300</td>
			<td style="width:187px;">placed in thalamus</td>
		</tr>
	</tbody>
</table>

direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation

Катодная микрополяризация (ТОК) индуцирует подавляющие действие на лекарственно-устойчивых судорог. Для выполнения эффективных действий, параметры стимуляции (например, ориентация, напряженность поля, и длительность стимуляции) должны быть изучены в препаратах срезов головного мозга мышей. Тестирование и упорядочивание ориентацию электрода по отношению к положению среза мыши мозга осуществимы. Данный способ сохраняет thalamocingulate путь для оценки влияния DCS на передней части поясной извилины припадок, как деятельность. Результаты записей массива многоканальных показали, что катодный DCS значительно уменьшил амплитуду стимуляции вызвали ответы и продолжительность 4-аминопиридина и бикукуллин-индуцированных припадков подобной активностью. Это исследование также показало, что катодная приложения DCS в 15 мин вызвала длительная депрессия в thalamocingulate пути. Настоящее исследование посвящено изучению влияния DCS на thalamocingulatе синаптической пластичности и острых приступов, как деятельность. Действующая процедура может проверить оптимальные параметры стимуляции , в том числе ориентации, напряженности поля и продолжительности стимуляции в качестве экстракорпорального модели мыши в. Кроме того, метод может оценить влияние DCS на корковых припадок, как деятельность на обоих клеточном и сетевых уровнях.

Подготовка установки Thalamocingulate Slice и MEA системы записи MT-ACC ломтика от мышей является специальной подготовки срез , который позволяет изучение электрофизиологических свойств thalamocingulate пути. Рисунок 1А показывает, каким образом ...

Watch this Scientific Journal Video about Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation at JoVE.com

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Прямая тока Стимуляция и Многоэлектродные массив Запись припадков, как активность мозга у мышей Кусочек Подготовка

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

The overall goal of this experimental procedure, is to access the arrangement of electrodes in the recording chamber for direct current stimulation of a brain slice and it's effect on seizure-like activity recorded with a multi-electrode array. This better can help answer key questions in direct current stimulation, and a multi-electrode array recorded seizure-like activity in mouse brain slice. An advantage of this technique is the effects of direct current stimulation can be evaluated in the specific pathway of the mouse brain slice.Demonstrating the procedure will be Hsiang Chin, and Wei-Jen Chang, technicians from my laboratory. In this procedure, sterilize all of the surgical instruments with 75%ethanol solution before the experiment. Then, expose the skull of the animal and trim off the remaining muscle.Next, peel away the dorsal surface of the skull using rongeurs, and trim away the sides. Subsequently, using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain before removing it. Quickly transfer the brain to a beaker filled with ice cold, oxygenated, ACSF.To prepare slices containing the MT-ACC pathway, make a sagittal cut, 2 mm lateral to the midline in each hemisphere, to display the subcortical anatomy. Then, make an angled cross cut parallel to the visible fiber tract in the striatum. Make the second angled cross cut from the connection between the cerebellum and visual cortex, to the midpoint between the anterior commissure and the optic tract, that are ventral and parallel to the thalamus cingulate pathway.After that, attach the brain block to an angular plate with a cyanoacrylate adhesive. Make a cut just above the turning point of the pathway. After that, unfold the plate, flatten it, and glue it on to the chamber stage of a vibratome.Subsequently section the brain slices at 500 micrometers thickness, and keep them in ice cold oxygenated ACSF. Transfer a slice to the recording chamber at 32 degrees Celsius with continuous profusion of oxygenated ACSF for one hour. Confirm the placement of a MEA probe on the multichannel system.Use one tube to guide the ACSF into the MEA chamber and the other tube to guide the ACSF out of the chamber. Continuously perfuse the preparation with warm, oxygenated ACSF at 30 degrees Celsius. Using a wet cotton swab, transfer a brain slice to the MEA.Carefully move the brain slice to orient the ACC above the electrodes, then, stabilize the brain slice with a slice anchor to ensure a good electrical connection between the slice and the electrodes. Afterward, place the anode electrode proximal to the ACC and the cathode electrode distal to the ACC. Record the field strength by the two field orientations of MEA.Then deliver the electric currents using a stimulator. Adjust the distance between the two silver chloride electrodes to about 1.5 to 2 cm and adjust the stimulators current strength to make the DCS between 0.5 and 2 milliamps. In this procedure, place a tungsten electrode in MT, and deliver pulses from the stimulator to the thalamic region of the slice.Next, use various current intensities, to determine the threshold that elicits an ACC response. Move the tungsten electrode along the thalamus cingulate pathway to obtain the optimal response profile. Record 1-20 ACC responses and use the software to automatically average all of the ACC responses evoked my MT stimulation.To induce seizure-like activity, add 250 micromolar 48P and 5 micromolar bicuculline to the perfusion solution, and continue to profuse the slice for two to three hours. Maintain the pump at a relatively fast perfusion rate, which can help prevent the build up of a pH gradient. Next, place the tungsten electrode at MT and deliver electrical stimulation to obtain an ACC response profile.Record 10-20 sweeps and average the responses. Afterward, replace the prefusion solution with fresh ACSF to wash out the drugs. 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 10-20 responses and then 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 48P, and 5 micromolar bicuculline to the perfusion solution, and wait 2-3 hours.If the drugs effect the brain slice, the slice should produce cortical seizure responses. Subsequently, collect 10-20 anterior cingulate cortical responses, and measure the amplitude and duration of the electrically evoked cortical seizure responses. Nest, 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. This figure shows different evoked responses, which include the thalamic stimulation evoked responses in ACC, drug-induced, seizure-like activity, and both the thalamic stimulation and drug induced seizure-like activity.This figure shows the affect of different orientations in DCS, such as different orientations in the electric field, thalamic stimulation evoked responses with DCS, and the effect of cathodal DCS on seizure-like activity. And in this figure, 15 minutes of cathodal DCS is shown the have effectively induced long term depression and depressed evoked responses. The seizure duration was significantly decresed after 15 minutes of cathodal DCS compared with no DCS application.faster, this technically can be done in four hours if it is performed properly. Following this procedure, other matters like transcranial magnetic stimulation can be performed, in order to answer additional questions like providing noninvasive approaches for controlling resistance seizures. After this development, this technique paved the way for researchers in the field of noninvasive therapy to explore seizure treatments in of animal model.After watching this video, you should have a good understanding of how to use the current stimulation to assess seizure-like activity in brain slices.

Research

JoVE Journal

Neuroscience

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

Подготовка фрагментов, органотипической культивирования тканей и люциферазы Запись часы активность генов в супрахиазматическое ядро

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Позиционирование электродов и монтаж в Транскраниальная постоянного тока стимуляции

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Multi-электрод массив записей из нейронов Лавины в органотипической культур

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

A Computer-assisted Multi-electrode Patch-clamp System

Компьютерный многоэлектродной патч-зажим система

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Недорогой метод для анализа захват как активность и движение в<em&gt; Дрозофилы</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

Проектирование и изготовление Ultralight Вес, Регулируемые многоэлектродной Зонды для Электрофизиологические записи в мышах

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Remotely Supervised Transcranial Direct Current Stimulation: An Update on Safety and Tolerability

Удаленно контролируемых транскраниальной прямые текущие стимуляции: Обновленная информация о безопасности и переносимости

The remotely supervised tDCS (RS-tDCS) protocol enables participation from home through guided and monitored self-administration of tDCS treatment while ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial Direct Current Stimulation tDCS in Mice

Постоянного тока транскраниальной стимуляции (ЦТД) у мышей

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Stimulation Location Determination using a 3D Digitizer with High-Definition Transcranial Direct Current Stimulation

Определение местоположения стимуляции с помощью 3D Digitizer с высокой четкостью Transcranial Прямого тока стимуляции

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, ...