이 작품은 국립 과학 재단 보조금 CMMI - 1300007에 의해 재정 지원되었다. 우리는 조지 메이슨 대학교 (George Mason University)에서 이러한 프로토콜의 설계 및 문화 유지에 도움을 주신 이전 연구원들에게 감사드립니다. Joseph J. Pancrazio 박사, Hamid Charkhkar 박사, Gretchen Knaack 박사 , Franz Hamilton 박사, Michael Maquera 박사, Robert Graham 박사.

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저자는 공개 할 것이 없습니다.

이 프로토콜에 설명 된 단계는 초보자가 MEA에서 자신의 연결 문화를 판명하고 네트워크 활동을 기록 할 수 있도록 충분한 세부 정보를 제공합니다. 이 프로토콜은 문화가 전극 배열 위에 세포의 양탄자 층을 형성하고, 건강을 유지하고 몇 달 동안 오염 물질이 없도록 적절하게 부착하는 것을 보장합니다. 프로토콜의 모든 부분을 준수하는 것이 가장 좋지만 성공적인 결과에 중요한 단계가 프로세스 전반에 걸쳐 있습니다. 배양 물이 오염되는 것을 막기 위해서는 전체 공정에 걸쳐 무균 기술을 사용해야합니다. 프로토콜에 설명 된대로 새로운 MEA를 친수성으로 만들어야합니다. 그렇지 않으면 세포 접착력이 떨어집니다. 가혹한 피펫 팅을 피하고 해리 중에 공기 방울이 형성되면 손상된 세포의 수가 줄어들어 더 높고 더 건강한 수율로 이어질 것입니다. 첫 번째 이후 DMEM 5/5에서 DMEM +로 전환먹이는 것도 중요합니다. DMEM 5/5에는 말 혈청이 포함되어있어 지속적으로 사용하면 신경 교세포가 배양을 지배하게되어 문화가 건강 해 보일지라도 열악한 신경 세포 활동을 초래합니다. 예정대로 배양액을 공급하고 적절한 배양 조건에서 배양하는 것도 중요합니다. MEA에서 세포 배양 판을 만드는 것은 최적의 결과가 아닌 결과를 가져올 수있는 많은 변수를 포함합니다. 목표가 세포의 완벽한 &quot;양탄자&quot;이지만, 위에 언급 된 중요한 단계를 해결하지 못하면 세포 성숙이 불량 해지거나 오염 될 수 있습니다. 가난한 세포 성숙과는 다른 세포 접착력이 나쁘다는 것도 관심사입니다. 이것은 도금 이전의 불량한 MEA 준비 또는 오래된 매체의 사용을 포함하는 여러 요인들로 인해 발생할 수 있습니다. 안정화 된 형태의 L- 글루타민과 혈청이없는 신경 세포 배양액을 함유 한 오래된 배지 ( 물질 표)가 사용되면, 세포는 처음에 붙지 만 약 2 주 후에 떠 다니게됩니다. 세균 오염이 지속적으로 문제가된다면 암피실린이나 펜 렙틴과 같은 항생제를 배지에 첨가 할 수 있습니다. 곰팡이 오염을 치료할 수있는 살균제도 있습니다. 이것들은 문화적 결과에 영향을 줄 수있는보다 일반적인 변수입니다. 시간과 경험 후에 만 ​​만날 수있는 많은 다른 것들이 있습니다. 유리 미세 전극의 사용과 비교하여,이 기법은 네트워크 역학 및 약리학 적 반응을 연구하는데 탁월합니다. 그것은 많은 다른 시공간 자극 패턴의 사용을 가능하게하고 동시에 여러 영역에서의 신경 반응을 기록 할 수있게합니다. 이전 그룹은 여기에 설명 된 것과 유사한 프로토콜을 사용하여 흥미로운 결과를 보여주었습니다 18 . 문화가 몇 주 또는 몇 달 동안 지속되고 동일한 문화가 재사용 될 수 있기 때문에이 기술은 또한동일한 네트워크에서 여러 실험을 반복합니다. 그러나이 기술에는 한계가 있습니다. MEA는 비 침습적입니다. 따라서 패치 클램핑이나 피펫을 사용한 세포 내 기록과는 달리 세포 외 활동 만 기록 할 수 있습니다. 또한, 배열의 각 전극은 여러 개의 세포로 덮여 있기 때문에 단일 뉴런의 활동을 해결할 수 없습니다. 반대로 이것들은 시험 관내 배양 물이기 때문에 뇌에서의 네트워크의 구조적 특성을 완전히 재현 할 수는 없습니다. 또한, 세포가 pH 균형을 유지할 수 있도록 CO 2 대기를 제공하는 메커니즘없이 한 번에 30 분 이내에 만 활동을 기록 할 수 있습니다. 일단이 기술이 숙달되면, 전기 자극이 있거나없는 약리학 적 조작이 탐구 될 수 있습니다. 뉴런 네트워크에서 학습과 기억 형성을 탐색하기위한 새로운 프로토콜을 설계하고 테스트 할 수 있습니다.hippocampal 또는 척수 네트워크에 대한 rotoci. 네트워크의 자극과 훈련을위한 프로토콜은 이미 발표되었으며, 그 중 일부는 Eytan et al.이 제안한 &quot;선택적 적응&quot;과 같은 생체 내 프로토콜로 추가 개발 되었다. 19 . 여러 프로토콜이 테스트되었습니다. 그러나 2005 년 루아로 (Ruaro)가 제안한 파상풍 치료법을 수정 한 결과 만이 여기에 제시되어있다.

Micro-electrode arrays (MEAs)는 약물 독성, 차세대 맞춤 의학을위한 디자인 패러다임을 연구하고, 연결 문화에서 네트워크 역학을 연구하는 데 사용할 수 있습니다 1 . 단일 세포에서의 활동 만 기록 할 수있는 패치 클램핑 (patch-clamping)과 단일 장소에서 전극을 둘러싸고있는 뉴런의 세포 외 반응을 기록 할 수있는 유리 피펫으로 필드 기록 (record recording)과 같은 전통적인 방법과 대조적으로 MEA는 동시에 각 전극을 개별적으로 배치하는 힘든 작업을하지 않고도 세포 배양의 여러 사이트에서 기록 할 수 있습니다. 이를 통해 해당 문화권 내에서 네트워크를 형성하는 세포 그룹 간의 동적 상호 작용을 연구 할 수 있습니다. 또한, 네트워크 소성 패턴 2 , 3 , 4 , 5 및 네트워크 제어 6 에 대한 전기 자극의 효과 &lt;/ sup&gt;는 잘 설명되어 있으며, 동일한 실험 설정 내에서 다양한 전기 자극 및 제어 구성을 쉽게 적용 할 수 있으므로 광범위한 시공간 역학을 탐구 할 수 있습니다. 이러한 시험 관내 연구에 대한 주요 역학 중 하나는 배양 된 네트워크가 학습 7 , 8 , 9 , 10 , 11 , 12 , 13을 나타내는 속성을 나타내는 정도였습니다. Peixoto Lab은 이전에 Ruaro et al. 에 설명 된대로 고주파 훈련 신호의 영향을 조사했습니다 . 14 , microelectrode 배열에 도금 마우스 뉴런의 네트워크에. 이 실험에서 네트워크는 응답의 증가g 전기 자극에 의해 유발 된 훈련. 증가 된 반응은 자극 인식을 통한 학습의 한 형태로 간주되어 네트워크가 특정 자극 ( 예 : 훈련) 프로토콜을 적용한 후 자극의 변화에 ​​일관된 방식으로 응답했습니다. 이 프로토콜은 MEA에서 연결 세포를 배양하고 도금 된 접시의 95 % 이상에서 성공적으로 기록하고, 자극 패턴, 단일 단위 활동 정렬, 플롯 히스토그램에 응답하도록 네트워크를 훈련하는 프로토콜을 확립하고 결과를 해석하는 방법을 보여줍니다. 그러한 실험. 신경 세포 배양의 자극과 기록을위한 독점 시스템 ( Table of Materials 참조)의 사용뿐만 아니라 소프트웨어 패키지 ( Table of Materials 참조)를 사용하여 신경 단위를 분류 할 수 있습니다. 맞춤형 그래픽 사용자 인터페이스 (재료 표 참조)를 사용하여 포스트 S를 시각화합니다.시간 간격 히스토그램, 인터 - 버스트 간격 및 버스트 지속 시간뿐만 아니라 훈련 프로토콜 전후에 자극에 대한 세포 반응을 비교할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="1135">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Ascorbic Acid</td>
			<td style="width:204px;">Sigma Aldrich</td>
			<td style="width:155px;">A4403</td>
			<td style="width:356px;">Make 4mg/mL aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">B-27</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>Serum-free supplement for neural cell culture. Make 1 mL aliquots and freeze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Beakers - glass - 100 mL</td>
			<td>VWR</td>
			<td>13912-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Beakers - glass - 500 mL</td>
			<td>VWR</td>
			<td>10754-956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Blunt-tipped thumb forceps</td>
			<td>World Precision Instruments</td>
			<td>500365-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Cryogenic vial - sterile, 2 mL</td>
			<td>Fisher Scientific</td>
			<td>10-500-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Cryogenic vial - sterile, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>10-500-27</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">DMEM with Glutamax</td>
			<td>Gibco Life Technology</td>
			<td>10569-010</td>
			<td>Cell culture media that contains a stabilized form of L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">DNase</td>
			<td>Worthington Biochemical</td>
			<td>LK003172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Fetal bovine serum (FBS)</td>
			<td>ATCC</td>
			<td>30-2030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Fine-tipped thumb forceps</td>
			<td>World Precision Instruments</td>
			<td style="width:155px;">501324-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Glass Pipets</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">GlutaMAX -- I (100X)</td>
			<td>Gibco Life Technology</td>
			<td>35050-061</td>
			<td>A stabilized form of L-glutamine used as a suplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Hemocytometer chip</td>
			<td>Fisher Scientific</td>
			<td>22-600-101</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">Hibernate EB complete</td>
			<td>BrainBits</td>
			<td>D00118</td>
			<td>Ambient CO2 cell storage media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Horse Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S12195H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS filter amplifier</td>
			<td>MultiChannel System</td>
			<td>FA60SBC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS headstage/pre-amplifier</td>
			<td>MultiChannel System</td>
			<td>MEA1060-INV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">MCS microelectrode array</td>
			<td>MultiChannel System</td>
			<td>60MEA200/10iR-ITO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS power supply</td>
			<td>MultiChannel System</td>
			<td>PS20W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS signal divider</td>
			<td>MultiChannel System</td>
			<td>SDMEA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS stimulus generator</td>
			<td>MultiChannel System</td>
			<td>STG4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS temperature controller</td>
			<td>MultiChannel System</td>
			<td>TC02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Media storage bottle - glass 500 mL</td>
			<td>VWR</td>
			<td>10754-818</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Microcentrifuge tubes - 0.4 mL</td>
			<td>Thermo Fisher</td>
			<td>3485</td>
			<td>Autoclave</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:420px;">Microcentrifuge tubes - 2 mL</td>
			<td>Thermo Fisher</td>
			<td>3434ECONO</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 10 uL&#160;</td>
			<td>VWR</td>
			<td>37001-166</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 1000 uL&#160;</td>
			<td>VWR</td>
			<td>13503-464</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 200 uL&#160;</td>
			<td>VWR</td>
			<td>37001-596</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 10 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641170N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 1000 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641210N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 200 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641230N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Papain - 0.22 Filtered</td>
			<td>Worthington Biochemical</td>
			<td>LK003178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Penicillin-Streptomycin (Pen Strep)</td>
			<td>Thermo Fisher</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -100mm disposable</td>
			<td>Fisher Scientific</td>
			<td>08-757-100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -100mm glass</td>
			<td>VWR</td>
			<td>10754-788</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -35 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Phosphate buffer saline (PBS) (1X)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Plasma Cleaning System</td>
			<td>Plasma Etch, Inc.</td>
			<td>PE-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Poly-D-lysine hydrobromide (PDL)</td>
			<td>Sigma Aldrich</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Polyethylene conical (centrifuge) tube -15 mL</td>
			<td>Fisher Scientific</td>
			<td>05-527-90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Polypropylene conical (centrifuge) tube -50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Procedure masks</td>
			<td>Imco</td>
			<td>1530-imc</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>P4562-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scalpel blades</td>
			<td>World Precision Instruments</td>
			<td>&#160;500237-G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">Scissors - iris</td>
			<td>World Precision Instruments</td>
			<td>14111-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scissors - large</td>
			<td>World Precision Instruments</td>
			<td>14214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scissors - small surgical</td>
			<td>World Precision Instruments</td>
			<td>&#160;501733-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 1 mL&#160;</td>
			<td>VWR</td>
			<td>89130-892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 2 mL&#160;</td>
			<td>VWR</td>
			<td>89130-894</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 25 mL&#160;</td>
			<td>VWR</td>
			<td>89130-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 50 mL&#160;</td>
			<td>VWR</td>
			<td>89130-902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: Matlab GUI</td>
			<td>Peixoto Lab</td>
			<td>npeixoto@gmu.edu</td>
			<td>Used to analyze .mat files.&#160; Available for free upon request.&#160; Contact npeixoto@gmu.edu to request a copy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: MCS MC_Rack</td>
			<td>MultiChannel System</td>
			<td>N/A</td>
			<td>Used for data acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: NeuroExplorer</td>
			<td>Plexon&#160;</td>
			<td>http://www.plexon.com/products/neuroexplorer</td>
			<td>Used to convert .nex files to .mat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: Offline Sorter</td>
			<td>Plexon&#160;</td>
			<td>http://www.plexon.com/products/offline-sorter</td>
			<td>Used to sort neural spikes and convert data files to .plx and then to .nex</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software Manual: MCS MC_Rack</td>
			<td>MultiChannel System</td>
			<td></td>
			<td>https://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/manuals/MC_Rack_Manual.pdf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software Manual: NeuroExplorer</td>
			<td>Plexon&#160;</td>
			<td></td>
			<td>https://www.neuroexplorer.com/downloads/Nex3Manual.pdf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software Manual: Offline Sorter</td>
			<td>Plexon&#160;</td>
			<td></td>
			<td>www.plexon.com/system/files/downloads/Offline%20Sorter%20v2.8%20Manual.pdf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Spatula - small double-ended</td>
			<td>World Precision Instruments</td>
			<td>&#160;503440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Stericup 0.22 &#181;m pore filter - 250 mL</td>
			<td>Millipore</td>
			<td>SCGVU02RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Transfer pipette - large bore&#160;</td>
			<td>Thermo Fisher</td>
			<td>335-1S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Transfer pipette - small bore&#160;</td>
			<td>Thermo Fisher</td>
			<td>242-1S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Trypan blue</td>
			<td>Sigma Aldrich</td>
			<td>T8154-20ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Whatman filter paper</td>
			<td>Whatman</td>
			<td>1442 150</td>
			<td>Cut into 8 pie wedges and autoclave in a glass Petri dish</td>
		</tr>
	</tbody>
</table>

time-dependent increase in the network response to the stimulation of neuronal cell cultures on micro-electrode arrays

마이크로 전극 배열 (MEAs)은 약물 독성, 차세대 맞춤 의학을위한 디자인 패러다임을 연구하고, 연결 문화에서 네트워크 역학을 연구하는데 사용될 수 있습니다. 단일 세포에서 활동 만 기록 할 수있는 패치 클램핑과 같은 기존의 방법과 달리 MEA는 각 전극을 개별적으로 배치하는 힘든 작업을 수행하지 않고도 네트워크의 여러 사이트에서 동시에 기록 할 수 있습니다. 더욱이, 수많은 제어 및 자극 구성을 동일한 실험 설정 내에서 쉽게 적용 할 수 있으므로 광범위한 역학을 탐구 할 수 있습니다. 이러한 시험 관내 연구에 대한 주요 역학 중 하나는 배양 된 네트워크가 학습을 나타내는 특성을 나타내는 범위였습니다. MEA에서 배양 된 마우스의 신경 세포는 전기적 자극에 의해 유도 된 훈련 후 반응의 증가를 나타낸다. 이 프로토콜은 MEA에서 신경 세포를 배양하는 방법을 보여줍니다. 성공적으로 다시도금 된 접시의 95 % 이상에서 코드; 자극 패턴에 반응하도록 네트워크를 훈련시키는 프로토콜을 수립한다. 그러한 실험의 결과를 정렬, 플롯 및 해석합니다. 신경 세포 배양을 자극하고 기록하기위한 독점 시스템의 사용이 입증되었습니다. 소프트웨어 패키지는 또한 연결 단위를 정렬하는 데 사용됩니다. 맞춤형 그래픽 사용자 인터페이스는 자극 후 시간 히스토그램, 인터 버스트 간격 및 버스트 지속 시간을 시각화하고 훈련 프로토콜 전후의 자극에 대한 세포 반응을 비교하는 데 사용됩니다. 마지막으로,이 연구 노력의 대표 결과 및 향후 방향에 대해 논의한다.

Watch this Scientific Journal Video about Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays at JoVE.com

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

다중 전극 어레이에서 배양 된 마우스 신경 세포는 전기적 자극 후 반응의 증가를 나타낸다. 이 프로토콜은 뉴런을 배양하는 방법, 활동을 기록하는 방법, 자극 패턴에 반응하도록 네트워크를 훈련시키는 프로토콜을 수립하는 방법을 보여줍니다.

미세 전극 배열에서 신경 세포 배양의 자극에 대한 네트워크 반응의 시간 의존적 증가

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

time-dependent-increase-network-response-to-stimulation-neuronal-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.8K 조회수.  George Mason University.  다중 전극 어레이에서 배양 된 마우스 신경 세포는 전기적 자극 후 반응의 증가를 나타낸다. 이 프로토콜은 뉴런을 배양하는 방법, 활동을 기록하는 방법, 자극 패턴에 반응하도록 네트워크를 훈련시키는 프로토콜을 수립하는 방법을 보여줍니다. 

비디오: Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

기본 모드 네트워크를 검사하기 위해 Transcranial 자석 자극과 fMRI를 결합

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

연구

신경과학

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS 장치 : FDA를 실시는 우울증 치료 프로토콜을 승인

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

동물 행동하자, 깨어있는 단일 단위 레코딩을위한 Microwires의 조직 된 배열을 주입하기위한 절차

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

단일 셀의 정밀도와의 연결을 공동 문화의 준비

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

Zebrafish의 애벌레에서 비주얼 응답의 전위도 분석

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

마이크로 전극 배열에 3D 엔지니어링의 연결을 문화의 인터페이스 : 혁신을<em&gt; 체외</em&gt; 실험 모델

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

결합 된 방법론을 활용하여 축삭 분기 및 신경 세포 형태 형성 동안 플라즈마 멤브레인 배달의 역할을 정의합니다

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...