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><tbody><tr><td>Ascorbic Acid</td><td>Sigma Aldrich</td><td>A4403</td><td>Make 4 mg/mL aliquots</td></tr><tr><td>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><td>Beakers - glass - 100 mL</td><td>VWR</td><td>13912-160</td><td></td></tr><tr><td>Beakers - glass - 500 mL</td><td>VWR</td><td>10754-956</td><td></td></tr><tr><td>Blunt-tipped thumb forceps</td><td>World Precision Instruments</td><td>500365-G</td><td></td></tr><tr><td>Cryogenic vial - sterile, 2 mL</td><td>Fisher Scientific</td><td>10-500-26</td><td></td></tr><tr><td>Cryogenic vial - sterile, 5 mL</td><td>Fisher Scientific</td><td>10-500-27</td><td></td></tr><tr><td>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><td>DNase</td><td>Worthington Biochemical</td><td>LK003172</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>04-355-451</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>ATCC</td><td>30-2030</td><td></td></tr><tr><td>Fine-tipped thumb forceps</td><td>World Precision Instruments</td><td>501324-G</td><td></td></tr><tr><td>Glass Pipets</td><td>VWR</td><td>14673-010</td><td></td></tr><tr><td>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><td>Hemocytometer chip</td><td>Fisher Scientific</td><td>22-600-101</td><td></td></tr><tr><td>Hibernate EB complete</td><td>BrainBits</td><td>D00118</td><td>Ambient CO&lt;sup&gt;2&lt;/sup&gt; cell storage media</td></tr><tr><td>Horse Serum</td><td>Atlanta Biologicals</td><td>S12195H</td><td></td></tr><tr><td>Laminin</td><td>Sigma Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>MCS filter amplifier</td><td>MultiChannel System</td><td>FA60SBC</td><td></td></tr><tr><td>MCS headstage/pre-amplifier</td><td>MultiChannel System</td><td>MEA1060-INV</td><td></td></tr><tr><td>MCS microelectrode array</td><td>MultiChannel System</td><td>60MEA200/10iR-ITO</td><td></td></tr><tr><td>MCS power supply</td><td>MultiChannel System</td><td>PS20W</td><td></td></tr><tr><td>MCS signal divider</td><td>MultiChannel System</td><td>SDMEA</td><td></td></tr><tr><td>MCS stimulus generator</td><td>MultiChannel System</td><td>STG4002</td><td></td></tr><tr><td>MCS temperature controller</td><td>MultiChannel System</td><td>TC02</td><td></td></tr><tr><td>Media storage bottle - glass 500 mL</td><td>VWR</td><td>10754-818</td><td>Autoclave</td></tr><tr><td>Microcentrifuge tubes - 0.4 mL</td><td>Thermo Fisher</td><td>3485</td><td>Autoclave</td></tr><tr><td>Microcentrifuge tubes - 2 mL</td><td>Thermo Fisher</td><td>3434ECONO</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 10 &amp;mu;L</td><td>VWR</td><td>37001-166</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 1,000 &amp;mu;L&amp;nbsp;</td><td>VWR</td><td>13503-464</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 200 &amp;mu;L&amp;nbsp;</td><td>VWR</td><td>37001-596</td><td>Autoclave</td></tr><tr><td>Micropipetter - 10 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641170N</td><td></td></tr><tr><td>Micropipetter - 1,000 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641210N</td><td></td></tr><tr><td>Micropipetter - 200 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641230N</td><td></td></tr><tr><td>Papain - 0.22 Filtered</td><td>Worthington Biochemical</td><td>LK003178</td><td></td></tr><tr><td>Penicillin-Streptomycin (Pen Strep)</td><td>Thermo Fisher</td><td>15070063</td><td></td></tr><tr><td>Petri dishes -100 mm disposable</td><td>Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Petri dishes -100 mm glass</td><td>VWR</td><td>10754-788</td><td></td></tr><tr><td>Petri dishes -35 mm&amp;nbsp;</td><td>Fisher Scientific</td><td>08-757-100A</td><td></td></tr><tr><td>Phosphate buffer saline (PBS) (1x)</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Plasma Cleaning System</td><td>Plasma Etch, Inc.</td><td>PE-50</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide (PDL)</td><td>Sigma Aldrich</td><td>P6407-5MG</td><td></td></tr><tr><td>Polyethylene conical (centrifuge) tube -15 mL</td><td>Fisher Scientific</td><td>05-527-90</td><td></td></tr><tr><td>Polypropylene conical (centrifuge) tube -50 mL</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Procedure masks</td><td>Imco</td><td>1530-imc</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma Aldrich</td><td>P4562-5G</td><td></td></tr><tr><td>Scalpel blades</td><td>World Precision Instruments</td><td>&amp;nbsp;500237-G</td><td></td></tr><tr><td>Scissors - iris</td><td>World Precision Instruments</td><td>14111-G</td><td></td></tr><tr><td>Scissors - large</td><td>World Precision Instruments</td><td>14214</td><td></td></tr><tr><td>Scissors - small surgical</td><td>World Precision Instruments</td><td>&amp;nbsp;501733-G</td><td></td></tr><tr><td>Serological pipette - sterile, 1 mL&amp;nbsp;</td><td>VWR</td><td>89130-892</td><td></td></tr><tr><td>Serological pipette - sterile, 2 mL&amp;nbsp;</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>Serological pipette - sterile, 25 mL&amp;nbsp;</td><td>VWR</td><td>89130-900</td><td></td></tr><tr><td>Serological pipette - sterile, 50 mL&amp;nbsp;</td><td>VWR</td><td>89130-902</td><td></td></tr><tr><td>Software: Matlab GUI</td><td>Peixoto Lab</td><td>npeixoto@gmu.edu</td><td>Used to analyze .mat files.&amp;nbsp; Available for free upon request.&amp;nbsp; Contact npeixoto@gmu.edu to request a copy.</td></tr><tr><td>Software: MCS MC_Rack</td><td>MultiChannel System</td><td>N/A</td><td>Used for data acquisition</td></tr><tr><td>Software: NeuroExplorer</td><td>Plexon&amp;nbsp;</td><td>http://www.plexon.com/products/neuroexplorer</td><td>Used to convert .nex files to .mat</td></tr><tr><td>Software: Offline Sorter</td><td>Plexon&amp;nbsp;</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><td>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><td>Software Manual: NeuroExplorer</td><td>Plexon&amp;nbsp;</td><td></td><td>https://www.neuroexplorer.com/downloads/Nex3Manual.pdf</td></tr><tr><td>Software Manual: Offline Sorter</td><td>Plexon&amp;nbsp;</td><td></td><td>www.plexon.com/system/files/downloads/Offline%20Sorter%20v2.8%20Manual.pdf</td></tr><tr><td>Spatula - small double-ended</td><td>World Precision Instruments</td><td>&amp;nbsp;503440</td><td></td></tr><tr><td>Stericup 0.22 &amp;micro;m pore filter - 250 mL</td><td>Millipore</td><td>SCGVU02RE</td><td></td></tr><tr><td>Transfer pipette - large bore&amp;nbsp;</td><td>Thermo Fisher</td><td>335-1S</td><td></td></tr><tr><td>Transfer pipette - small bore&amp;nbsp;</td><td>Thermo Fisher</td><td>242-1S</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma Aldrich</td><td>T8154-20ML</td><td></td></tr><tr><td>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

Nanyang Technological University

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

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 ...

연구

신경과학

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

내 분 비 방해 다중 전극 어레이 사용 하 여 척추 신경 네트워크 기능 개발에 화합물의 효과의 평가

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

발생학

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

어떻게 문화, 기록 및 미세 전극 배열에의 연결을 네트워크를 자극 (MEAs)에

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

생물학

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

줄기의 네트워크 문화 생물 신경 독소의 기능 평가 중추 신경계 신경 세포가 유래

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

뇌 조직에서 측정 세포 외 이온의 신호에 대한 두 번 질주과 동심 미소 전극

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

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 ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

전기 단일 및 두 개 차원 문화에 자기장을 사용하여 신경 세포의 외부인가 전압

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

마이크로전극 어레이를 이용한 척추 노셉티브 회로에서의 네트워크 활동 기록

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

마이크로 스케일 신경 세포 배양 장치를 위한 3D 프린팅의 응용

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

저자 스포트라이트: 뇌 기능 분석을 위한 모듈식 뉴런 네트워크

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

2광자 중합 마이크로 스케일 신경 세포 배양 장치의 3D 프린팅

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

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

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이 비디오의 챕터

Organotypic 문화에의 연결을 눈사태의 다중 전극 어레이 레코딩

내 분 비 방해 다중 전극 어레이 사용 하 여 척추 신경 네트워크 기능 개발에 화합물의 효과의 평가

어떻게 문화, 기록 및 미세 전극 배열에의 연결을 네트워크를 자극 (MEAs)에

줄기의 네트워크 문화 생물 신경 독소의 기능 평가 중추 신경계 신경 세포가 유래

뇌 조직에서 측정 세포 외 이온의 신호에 대한 두 번 질주과 동심 미소 전극

마이크로 전극 배열에 3D 엔지니어링의 연결을 문화의 인터페이스 : 혁신을

전기 단일 및 두 개 차원 문화에 자기장을 사용하여 신경 세포의 외부인가 전압

마이크로전극 어레이를 이용한 척추 노셉티브 회로에서의 네트워크 활동 기록

2광자 중합 마이크로 스케일 신경 세포 배양 장치의 3D 프린팅

2광자 중합 마이크로 스케일 신경 세포 배양 장치의 3D 프린팅