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.

동물의 과목을 포함하는 절차는 기관 동물 관리 및 활용위원회, 중앙 연구원, 타이페이, 대만에 의해 승인되었다. 1. 다중 전극 배열 녹화에 대한 실험 솔루션 및 장비 준비 <ol><li> 인공 뇌척수액 (ACSF 준비 124 mM의 염화나트륨, 4.4 mM의 KCl을들을 1mM의 NaH를 2 95 % O 버블 PO 3, 2 mM의 황산, 2 mM의 CaCl2를 25 밀리미터의 NaHCO3 및 10 mM의 글루코오스, 2, 5 % CO 2). </li><li> 6 × 10 평면 MEA와 8 × 8 MEA : MEA 프로브의 두 가지 유형을 사용합니다. 전자 프로브는 피질, 선조체 및 시상을 포함하는 영역을 포함한다. 후자의 프로브는 대뇌 피질의 영역을 다룹니다. </li><li> 0.1 Hz에서 1,200 증폭에서 3 kHz의 사이에 밴드 패스 필터를 60 채널 증폭기를 사용한다. 10 kHz 샘플링 레이트로 데이터를 획득. </li><li> DCS의 MEA 챔버 내부에 두 개의 AgCl을 코팅은 와이어를 놓습니다. AgCl을을 사용하여실버 코팅 된 와이어는 절연 자극에 의​​해 생성 된 전기장을 생성한다. </li><li> 텅스텐 전극 (; 길이 7.62 cm 8 ° AC 테이퍼 팁 직경 127 μm의 저항 5 MΩ) 장소 시상 자극을하고, MEA 실 기준 전극을 배치합니다. 펄스 발생기에 의해 제어되는 격리 자극기를 사용하여 텅스텐 전극의 전류를 제공한다. </li></ol> 2. 뇌 조각 준비 <ol><li> 4-8주 세 남성 C57BL / 6J 마우스를 사용합니다. 집 에​​어컨 방에 ​​동물 음식과 물을 무료로 이용할 수로 (21 ~ 23 ° C, 50 % 습도 / 12 시간 / 암주기, 오전 8:00에 점등 12 시간). </li><li> 단계 1.1에서 제조 된 ACSF의 250 ML의 나누어지는을 가지고, 얼음이 들어있는 비커에 넣습니다. 동시에, 95 % O 2, 5 % CO 2로 구성되는 연속 가스를 공급한다. </li><li> 외과 <ol><li> 유리에서 4 % 이소 플루 란 동물을 마취약 3 분 동안 상자. 동물 (발가락 핀치에 대한 응답의 부족에 의해 표시) 마취의 수술 깊이에 도달하면, 얼음 조각으로 가득 얕은 트레이에 배치하고 가위를 사용하여 헤드를 제거합니다. </li><li> 두개골을 노출하고, 나머지 근육을 잘라. 다음으로, rongeurs를 사용하여 뇌에서 두개골의 지느러미 표면을 벗겨. rongeurs을 이용하여 두개골의 측면을 얻어 낸다. 75 % 에탄올 용액으로 수술 도구 모두 멸균. </li><li> 주걱을 사용하여 두뇌의 복부 표면을 따라 후각 전구 및 신경 연결을 절단, 뇌를 제거합니다. 잘린 후, 신속하게 얼음처럼 차가운 산소 ACSF 가득 비커에 두뇌를 전송합니다. </li></ol></li><li> 내측 시상 (MT) -ACC 뇌 조각의 준비 참고 : ACC (13)에 MT에서 경로를 포함 슬라이스를 준비합니다. <ol><li> 2.0 mm 각 반구의 중간 선 측 방향이 시상 인하와 함께 뇌 블록을 손으로 잘라피질 해부학을 표시합니다. 그런 다음 두 개의 각도 절단을합니다. 선조체에서 볼 수 섬유 기관의 첫 번째 크로스 컷 평행합니다. </li><li> thalamocingulate 경로에 복부와 평행 전방 접합면과 광학 기관 사이의 중간 지점으로 소뇌와 시각 피질 사이의 연결에서 두 번째 크로스 컷을 확인합니다. </li><li> 시아 노 아크릴 레이트 접착제와 각도 플레이트 (~ 120 °)로 뇌 블록을 연결하고 그냥 경로의 전환점 위에 상처를합니다. 플레이트를 펼쳐을 평평하게하고, vibratome의 실 단계에 그것을 접착제. </li><li> 내측 시상-ACC 뇌 조각 다음 (500 μm의 두께) 및 1 산소 ACSF로 녹화 실에 얼음처럼 차가운 산소 aCSF.Transfer 조각에서 그들을 몰입, 지속적인 관류 (12 ml / 분)에서 32 ° C로 유지합니다 시간. </li></ol></li></ol> 다중 전극 배열 녹화에 대한 관류 상공 회의소 3. 준비 <ol><li> 예비관류 상공 회의소 <ol><li> 다중 채널 시스템에서 MEA 프로브를 배치하고, 연동 펌프에 프로브를 연결하는 두 개의 분리 된 폴리에틸렌 튜브를 사용한다. MEA를 챔버와 챔버 밖으로 ACSF을 안내하는 다른 튜브에 ACSF을 안내하는 하나의 튜브를 사용합니다. 마지막으로, 연속적으로 따뜻한 (29-30 °의 C) 산화 된 ACSF (8 ml / 분)로 제조를 관류. </li></ol></li><li> MEA에 뇌 조각을 전송합니다. 젖은 면봉을 사용하여 MEA에 뇌 슬라이스를 누르십시오. 조심스럽게 ACC는 전극 위에 지향하기 위해 뇌 조각을 이동합니다. </li><li> 뇌 조각을 눌러 슬라이스 앵커 키트를 길게 다운을 사용합니다. 이 단계는 슬라이스와 전극 사이의 우수한 전기 접속을 보장한다. </li></ol> DCS에 의한 전기 분야의 4 세대 주 : 전계 방향의 정의는 ACC axodendritic의 축 방향을 기준으로 하였다. 수상 돌기 및 소마 구획의 방향이었다골지 염색 (12)을 이용하여 확인 하였다. <ol><li> 누산기에 인접 (양극으로 정의 됨) AgCl을 전극을 배치하고, ACC에 말단 (캐소드로 정의) 다른 전극을 배치합니다. MEA에 의한 두개의 전계 배향 (누산기 axodendritic 섬유에 평행 및 수직)에 의해 생성되는 자기장 강도를 기록 및 자극 장치를 이용하여 전계의 전류를 제공한다. </li><li> AgCl을 전극 (약 1.5 cm)의 거리를 수정 한 mA 0.5 내지 2를 DCS을 할 수있는 자극의 현재 강도를 조정합니다. </li></ol> 5. 전기에 의한 두피 시냅틱 응답 주 : 프로그램 가능한 전기 자극 발생기 직사각형 이상성 전류 펄스를 생성하는 MT의 전기 자극에 의​​한 ACC 시냅스 반응을 유도한다. <ol><li> 위의 반복 제 3 절. </li><li> 이동 단말에 텅스텐 전극을 배치하고, thal에 자극에서 펄스를 제공바이폴라 텅스텐 전극을 통해 조각의 아미드 영역입니다. </li><li> 에 ACC 응답을 이끌어 임계 값을 결정하기 위해 다양한 전류 강도를 사용합니다. 여기에 대부분의 조각에서 ACC에서 80 %의 최대 반응을 유도 200 마이크로 초의 ± 150 μA의 강도와 지속 시간을 사용합니다. </li><li> 최적의 응답 프로파일을 얻기 위해 MT-ACC 슬라이스에 (뇌량에 MT에서)를 thalamocingulate 경로를 따라 텅스텐 전극을 이동합니다. </li><li> ACC 응답의 10 ~ 20 스윕을 확인하고 소프트웨어를 사용 MT 자극에 의​​해 유발 ACC 모두 평균을 자동으로합니다. MT-ACC 경로에 의해 MT 자극으로부터 유도 된 ACC에서 시냅스 응답 ISS 결과입니다. </li></ol> 6. 전기에 의한 발작 같은 활동 (250 μM 4-AP) 및 bicuculline (5 μM) 발작 같은 활동은 4 아미노 피리딘의 응용 프로그램에 의해 유도되었다합니다. 이전 시간 제어 연구는 최대 안정적인 반응이 나타난 것을 보여 주었다약물 응용 프로그램 (14) 후 2 ~ 3 시간. <ol><li> 위의 반복 제 5 절. </li><li> 관류 액에 약물을 추가합니다. 4-AP (250 μM) 및 bicuculline (5 μM)를 사용합니다. 균일하게 약물을 혼합하고, 2 ~ 3 시간 동안 재관류를 계속합니다. </li><li> 간질 유사 활성을 촉진하기 위해, 또한 pH를 구배의 형성을 방지 할 수있는 상대적으로 빠른 혈류 속도 (8 ml / 분)에서 관류 펌프를 유지한다. </li><li> 이동 단말에서 텅스텐 전극을 배치하고 ACC 응답 프로파일을 얻기 위해 전기 자극 (150 μA, 200 마이크로 초 시간)를 제공합니다. </li><li> 10 ~ 20 스윕을 확인하고 응답을 평균. </li><li> 약을 씻어 신선한 ACSF와 관류 솔루션을 교체합니다. 단계를 반복 6.5. </li></ol> 유발 두피 응답에 DCS 7. 시험 효과 <ol><li> 반복 섹션 3과 4는 균일 한 전계가 M 내부에 배치되는 두 개의 평행 한 AgCl을 코팅은 배선 사이에 전류를 통과시킴으로써 생성된다 확인EA 실. 아무 문제가없는 경우, DCS는 0.5 2mA 사이에 유지됩니다. </li><li> DCS의 전원을 끄고 (150 μA, 200 마이크로 초 지속 시간 ±) 시상을 자극 텅스텐 전극을 배치합니다. 는 ACC에서 최대 시냅스 반응을 얻을 10-20 스윕을하고 응답을 평균합니다. </li><li> 동시에 DCS (2 MV / mm의 DCS 강도) 및 시상 자극 (350 μA, 200 마이크로 초 기간)를 켭니다. DCS 동안 시상 자극-유발 ACC 응답의 진폭의 변화를 평가합니다. </li><li> DCS의 전원을 끄고 관류 액에 4-AP (250 μM) 및 bicuculline (5 μM)를 추가합니다. 그런 다음 2 ~ 3 시간을 기다립니다. 약물은 뇌 슬라이스에 영향을 미칠 경우, 슬라이스는 대뇌 피질의 발작 반응을 생산하고 있습니다. </li><li> ACC 응답의 10 ~ 20 스윕을 확인하고 전기 유발 대뇌 피질의 발작 응답의 진폭과 지속 시간을 측정한다. </li><li> 단계 7.5 이후, 동시에합니다 (DCS (2 MV / mm DCS 강도) 및 시상 자극에 150 μA, 200 durati을 설정마이크로 초에). DCS 응용 프로그램 중 진폭과 유발 대뇌 피질의 발작 응답 시간의 변화를 평가합니다. </li><li> 약을 씻어 신선한 ACSF와 관류 솔루션을 교체하고 반복 7.2 및 7.3 단계를 반복합니다. </li><li> 다른 실험 조건에 데이터를 기록 데이터를 모두 수집하고, 기. 다른 실험 조건 하에서 대뇌 발작 응답의 진폭 및 지속 시간을 평가한다. </li></ol> 8. 데이터 분석 <ol><li> (예를 들어, MC 랙 소프트웨어)가 자동으로 기록 된 응답을 평균 소프트웨어를 사용하여 스프레드 시트에 원시 데이터를 내보낼 수 있습니다. 원시 데이터의 크기와 지속 시간을 분석하고 색상 수치를 생성합니다. </li><li> 진동 발작 이벤트를 검출하기 위해, 기준 값 및 표준 편차 (SD)를 측정하기 위해 소프트웨어를 사용한다. 임계 값과 잡음 레벨 3 SD를 설정합니다. 이 임계 값을 능가 발진 이벤트 기간 동안 피크의 진폭은 자동으로 감지됩니다에디션. </li><li> 학생의 t의 -test를 이용하여 통계 분석을 수행합니다. </li><li> 슬라이스의 수를 나타내는 N으로 고속 측정하고 평균 ± SE로서 텍스트의 변동 (ANOVA) 결과 일방향 분석 12 공부. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+direct-current+stimulation+against+cingulate+seizure-like+activity+induced+by+4-aminopyridine+and+bicuculline+in+an+in+vitro+mouse+model.">Treatment with direct-current stimulation against cingulate seizure-like activity induced by 4-aminopyridine and bicuculline in an in vitro mouse model.</a> Exp. Neurol. 265, 180-192 (2015).</li><li>Lee, C. M., Chang, W. C., Chang, K. B., Shyu, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+organization+and+input-specific+short-term+plasticity+in+anterior+cingulate+cortical+neurons+with+intact+thalamic+inputs.">Synaptic organization and input-specific short-term plasticity in anterior cingulate cortical neurons with intact thalamic inputs.</a> Eur. J. Neurosci. 25 (9), 2847-2861 (2007).</li><li>Chang, W. P., Shyu, B. C., Pandalai, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+thalamocingulate+pathway+in+the+regulation+of+cortical+seizure+activity.">Involvement of the thalamocingulate pathway in the regulation of cortical seizure activity.</a> Recent Research Developments in Neuroscience. 4, 1-27 (2013).</li><li>Brummer, S. B., Turner, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+considerations+for+safe+electrical+stimulation+of+the+nervous+system+with+platinum+electrodes.">Electrochemical considerations for safe electrical stimulation of the nervous system with platinum electrodes.</a> IEEE Trans. Biomed. Eng. 24 (1), 59-63 (1977).</li><li>Durand, D. M., Bikson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+and+control+of+epileptiform+activity+by+electrical+stimulation:+a+review.">Suppression and control of epileptiform activity by electrical stimulation: a review.</a> Proc. IEEE. 89 (7), 1065-1082 (2001).</li><li>Fritsch, B., 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=Direct+current+stimulation+promotes+BDNF-dependent+synaptic+plasticity:+potential+implications+for+motor+learning.">Direct current stimulation promotes BDNF-dependent synaptic plasticity: potential implications for motor learning.</a> Neuron. 66 (2), 198-204 (2010).</li></ol>

The authors declare that they have no competing interests.

본 연구에서 ACC 간질 유사 활성에 DCS 기간 및 배향의 영향을 시험 하였다. 마우스 뇌 조각에서 안정적인 데이터를 얻으려면 어떻게 MT-ACC 경로의 무결성을 유지하고,이 각도 복부 상처와 피질의 지느러미를 잘라 만든되는, 특히 단계 핵심 손상을 방지 할 수 있습니다. 또한, 뇌 조각을 준비하는 시간은 신선하고 강한 뇌를 유지할 수 짧은 시간이어야 뇌 슬라이스의 활성에 영향을 미칠 수있다. 이전...

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

Cathodal 두개 직류 자극 (tDCS)의 약제 내성에 발작 억제 효과를 유도한다. 효율적인 동작을 수행하기 위해, 상기 자극 파라미터 (예를 들면, 방향 전계 강도와 자극 시간)을 마우스 뇌 슬라이스 제제에서 검사 될 필요가있다. 테스트 및 마우스 뇌 슬라이스의 위치에 전극의 상대적 배향을 정렬이 가능하다. 본 발명의 방법은 앞쪽에 cingulate 피질 발작 같은 활동에 대한 DCS의 효과를 평가하기 thalamocingulate 경로를 유지합니다. 다 채널 어레이 레코딩 결과 cathodal DCS 크게 -4- 아미노 피리딘과 bicuculline 유도 발작 같은 활성의 자극 - 유발 반응의 크기 및 지속 시간을 감소 것으로 나타났다. 이 연구는 또한 15 분에서 cathodal DCS 응용 프로그램이 thalamocingulate 경로에 장기 우울증의 원인 것으로 나타났습니다. 본 연구는 thalamocingulat에 ​​DCS의 효과를 조사전자 시냅스 가소성 및 급성 발작 같은 활동. 현재의 방법은 시험 관내 마우스 모델에서 방향 전계 강도와 자극 시간을 포함하여 최적의 자극 파라미터를 테스트 할 수있다. 또한,이 방법은 두 셀룰러 네트워크 레벨에서 대뇌 발작 같은 활동 DCS의 효과를 평가할 수있다.

Thalamocingulate 조각과 MEA 녹화 시스템 설치 프로그램의 준비 생쥐에서 MT-ACC 슬라이스가 thalamocingulate 경로의 전기 생리 학적 특성을 탐구 할 수있는 특별한 제제 슬라이스이다.도 1a는 MT-ACC 슬라이스 제조 된 방식을 나타낸다. 마우스의 뇌를 신속하게 제거하고 (도 1A, A, B)의 차가운 ACSF 산소로 유지 하였...

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.

마우스 뇌 조각 준비에서 발작 같은 활동의 직류 자극 및 다중 전극 배열 기록

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

direct-current-stimulation-multi-electrode-array-recording-seizure

Research

JoVE Journal

Neuroscience

10.4K 조회수.  Academia Sinica. 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.

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

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

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

슬라이스 준비, Organotypic 조직 Culturing과 Suprachiasmatic 핵의 시계 유전자 활동의 루시페라제 녹음

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

연구

신경과학

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

Transcranial 직접 전류 자극의 전극 위치와 몬티지

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

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Organotypic 문화에의 연결을 눈사태의 다중 전극 어레이 레코딩

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

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

컴퓨터를 이용한 다중 전극 패치 클램프 시스템

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

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

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

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

발작과 같은 활동을 분석하고 운동을위한 저가의 방법<em&gt; 초파리</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

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

마우스의 전기 생리 녹음을위한 설계 및 초경량 무게의 제작, 조정 가능한 다중 전극 프로브

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

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

Transcranial 직류 자극 (tDCS) 마우스

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

The abundance of neuroimaging data and rapid development of machine learning has made it possible to investigate brain activation patterns. However, causal evidence of brain area activation leading to a behavior is often left missing. Transcranial direct current stimulation (tDCS), which can temporarily alter brain cortical excitability and activity, is a noninvasive neurophysiological tool used to study causal relationships in the human brain. High-definition transcranial direct current stimulation (HD-tDCS) is a noninvasive brain stimulation (NIBS) technique that produces a more focal current compared to conventional tDCS. Traditionally, the stimulation location has been roughly determined through the 10-20 EEG system, because determining precise stimulation points can be difficult. This protocol uses a 3D digitizer with HD-tDCS to increase accuracy in determination of stimulation points. The method is demonstrated using a 3D digitizer for more accurate localization of stimulation points in the right temporo-parietal junction (rTPJ).

Presented here is a protocol to achieve higher accuracy in determination of stimulation location combining a 3D digitizer with high-definition transcranial direct current stimulation.

고화질 경부 직접 전류 자극이 있는 3D 디지타이저를 사용한 자극 위치 측정

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

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

트랜스 척추 직접 전류 자극 중 유형 식별 쥐 척추 운동 신경의 생체 내 세포 내 기록

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

Simultaneous Application of Transcranial Direct Current Stimulation during Virtual Reality Exposure

Transcranial direct current stimulation (tDCS) is a form of non-invasive brain stimulation that changes the likelihood of neuronal firing through modulation of neural resting membranes. Compared to other techniques, tDCS is relatively safe, cost-effective, and can be administered while individuals are engaged in controlled, specific cognitive processes. This latter point is important as tDCS may predominantly affect intrinsically active neural regions. In an effort to test tDCS as a potential treatment for psychiatric illness, the protocol described here outlines a novel procedure that allows the simultaneous application of tDCS during exposure to trauma-related cues using virtual reality (tDCS+VR) for veterans with posttraumatic stress disorder (NCT03372460). In this double-blind protocol, participants are assigned to either receive 2 mA tDCS, or sham stimulation, for 25 minutes while passively watching three 8-minute standardized virtual reality drives through Iraq or Afghanistan, with virtual reality events increasing in intensity during each drive. Participants undergo six sessions of tDCS+VR over the course of 2-3 weeks, and psychophysiology (skin conductance reactivity) is measured throughout each session. This allows testing for within and between session changes in hyperarousal to virtual reality events and adjunctive effects of tDCS. Stimulation is delivered through a built-in rechargeable battery-driven tDCS device using a 1 (anode) x 1 (cathode) unilateral electrode set-up. Each electrode is placed in a 3 x 3 cm (current density 2.22 A/m2) reusable sponge pocket saturated with 0.9% normal saline. Sponges with electrodes are attached to the participant&#8217;s skull using a rubber headband with the electrodes placed such that they target regions within the ventromedial prefrontal cortex. The virtual reality headset is placed over the tDCS montage in such a way as to avoid electrode interference.

This manuscript outlines a novel protocol to allow the simultaneous application of transcranial direct current stimulation during exposure to warzone trauma-related cues using virtual reality for veterans with posttraumatic stress disorder.