저자는 감금 구조와 dott 개발 기술 지원 조르지오 Carlini 감사합니다. 원고의 철저한 개정 ORNELLA LoBrutto. 이러한 결과로 이어지는 연구는 유럽 연합 (EU)의 7 ​​번째 프레임 워크 프로그램에서 자금을받은 (ICT-FET FP7 / 2007-2013, FET 젊은 탐험가 계획) 보조금 협정 284772 뇌 활에서 (www.brainbowproject.eu).

실험 프로토콜은 DL 1,992분의 116에 따라와 제노바 (보호 해주는 대학의 지침에 의해 건강의 이탈리아 교육부 유럽 동물 관리 규정 (63분의 2,010 / EU)에 의해 승인되었다. N. 13130​​, 월 2011). 모든 노력이 프로젝트 동물의 수를 줄이기 위해 자신의 고통을 최소화 하였다. 재료 및 지지대 1. 준비 <ol><li> CNC (컴퓨터 수치 제어) 밀링 머신에 의해 PDMS (폴리 디메틸 실록산) 제약 조건을 구축 할 수있는 틀을 구축합니다. 이러한 몰드는 폴리 테트라 플루오로 에틸렌의 중앙 실린더 (PTFE), 폴리 카보네이트로 실현된다. PDMS가 강화되면이 물질은 마스크의 쉽게 추출 할 수 있습니다. 주 : 주형의 설계는 컴퓨터 지원 설계 (CAD)에 의해 수행하고 CNC 밀링 머신에 전달되었다. </li><li> PDMS 엘라스토머를 준비합니다. PDMS는 유기 폴리머이다. 그것은 두 가지 요소로 구성되어있다경화제 및 중합체. 1:10 부피비로 섞는다, 경화제의 일부 및 중합체의 아홉 부품. , 페트리 접시에서 두 성분을 넣어 진탕하고 기포를 제거하기 위해 10 분 동안 진공 챔버에서 믹스를 삽입한다. PDMS의 3g 하나의 제약 조건에 대한 충분하다. </li><li> PDMS 제약을 구축합니다. 성형기에 PDMS 재료를 넣고 120 ℃에서 30 분 동안 오븐에 넣어. PDMS 제약은 각각 22.0 및 3.0 mm의 외부 및 내부 직경 650 ㎛의 높이를 갖는 원통의 이상적인 형상을 갖는다. </li><li> 도금 하루 전에, 커플 실체 현미경 집게 의해 마이크로 전극 어레이 (다자간)의 활성 영역에 마스크 및 120 ° C의 오븐에서 PDMS 마스크 살균. </li><li> 원추형 바이알에 2 시간 동안 70 %의 에탄올 및 EV 회전 (42.3 ± 1.1 μm의 평균 직경의 인증을 40 ± 2 ㎛의 공칭 직경) 유리 마이크로 비드를 살균ERY 30 분 모든 마이크로 비드를 노출합니다. </li><li> 유리 병에서 에탄올 용액을 제거하고 마이크로 비드를 멸균 수로 2 회 씻어. </li><li> 0.05 ㎍ / ㎖의에 폴리 -D- 라이신과 라미닌 혼합액 24 μL와 (직경이 3.0 mm로 동일) PDMS 마스크로 구분 MEA 영역의 중앙 부분을 컨디셔닝. </li><li> 코트 0.05 μg의 /에서 접착 단백질, 라미닌 및 폴리 D 라이신과 마이크로 비드 용액과 안정하고 오래 지속되는 신경 네트워크를 획득하기 위해, 37 ℃ 배양기에 밤새두​​고 그들. </li><li> MEA의 유리 표면으로부터 유리 마이크로 비드에서 모두 접착 인자를 제거한다. 대기음 피펫 코팅 용액의 약 95 % 및 MEA 표면에 멸균의 작은 부피 - 24μl- 적용. 대기음은 다시 95 개 이상의 물 %와 세포를 도금하기 전에 1 시간 동안 층류 후드 아래 MEA 건조를 할 수 있습니다. 주 : 대신 마이크로 비드의 경우, 코팅 대기음용액 및 뉴런 도금한다 유리 표면을 컨디셔닝하기 위해, 멸균 수와 기본 배지와 같은로서의 B27 보충제와 두 번째 세척으로 제를 만든다. 마이크로 비드가 건조하게하지 마십시오. 유리 병 내부의 배양액에 부유을 남겨주세요. </li><li> 그들이 균일 한 층을 형성하는 자기 - 조립된다 멤브레인 삽입물 (구멍 직경 0.4 μm의)와 멀티 웰 플레이트에 피펫 -200 μl-, 처리 된 마이크로 비드 현탁액 (약 32,000)을 이용하여, 배포. </li><li> 매체의 0.5 ml의 멤브레인의 각 웰을 입력하고 인큐베이터에 넣어 (T = 37.0 O C, CO 2 = 5 %) 뉴런 (3.2) 도금 될 준비가 될 때까지. </li></ol> 2. 태아의 해부 및 조직의 해리 <ol><li> 일반 명암 일정에 따라 일정한 온도 (22 ± 1 ℃로)과 상대 습도 (50 %)에서 하우스 성인 여성 쥐 (2백~2백50그램) (빛에 오전 7시에서 오후 7시까지)에서동물 시설. 그 음식을 확인하고 물을 자유롭게 사용할 수 있습니다. </li><li> 3 % 이소 플루 란을 사용하여 개발 십팔일 (E18) 후 임신 여성 쥐를 마취. 그 후, 경부 탈구에 의해 동물을 희생. </li><li> 각 쥐의 배아에서 해마를 제거하고 칼슘과 마그네슘없이 얼음 차가운 행크의 균형 염 용액 2+에 배치합니다. 하루에 개발 해마와 피질 조직 (18)는 매우 부드럽고 작은 조각으로 절단 할 필요가 없습니다. 더 자세한 사항은 18, 19을 찾을 수있다. </li><li> 37 ℃에서 18-20 분 동안 DNase의 0.05 %를 함유하는 트립신 / 행크 용액의 0.125 %로 조직을 해리. </li><li> 파스퇴르 피펫으로 상층 액을 제거하고 5 분 동안 10 % 소 태아 혈청 (FBS)와 매체를 첨가하여 효소 소화를 멈춘다. </li><li> FBS로 배지를 제거하고 그 보충, 1 % L- 글루타민, 10 μg의 겐타 마이신 / ㎖와 배지로 한번 세척 하였다. 파스퇴르 핍 다시 매체를 제거두기가 보충은, 1 % L- 글루타민, 10 μg의 겐타 마이신 / ㎖와 함께 성장 배지의 소량 (500 μL)로 다시 채우고. </li><li> 세포의 유백색 현탁액 명백한까지 좁은 파스퇴르 피펫 기계적 조직 펠렛을 해리. 이 세포 현탁액을 원심 분리 할 필요가 없다. </li><li> 2.0 ㎖의 최종 부피를 얻기 위해 생장 배지로 세포 현탁액의 소량 희석. 혈구 챔버와 얻어진 세포의 농도를 계산합니다. / μL 600-700 세포의 원하는 세포 농도를 얻기 위해, 5 : 1에서 이러한 농도를 희석. </li></ol> 3. 셀 도금 <ol><li> PDMS 제약 조건에 의해 정의 된 MEA의 활성 영역 상에 약 2,000 세포 / mm 2의 밀도로 접시 세포는 2D 신경 네트워크를 생성한다. 참고 : 각 해마는 약 5 × 10 5 세포를 포함, (하나의 배아에 대한 1 × 6). 6 × 10의 총량을 얻기 위해 6 배아를 해부하다6 2 ㎖ 중의 세포 및 / μL 3,000 세포의 추정 농도. 원하는 세포 농도를 구하고 약 600-700 세포 / μL 판형 위해 5 : 1에서 이러한 농도를 희석. PDMS 제약으로 구분 MEA 영역은 약 7.065 mm (2) 및 도금 된 세포의 총 수의 경우 600 세포 / μL X 24 μL = 14,400 세포 / mm 2 2,038 세포의 최종 농도. </li><li> 37 ℃에서 가습 이산화탄소 분위기 (5 %)와 인큐베이터에 MEA 장치를 놓습니다. </li><li> 삼차원 배양 건설 예비 단계를 완료하기 위해 멀티 웰 플레이트 내부에 위치 된 마이크로 비드 단층의 표면 상 / μL 600-700 세포 (약 100,000 세포)의 세포 농도로 현탁액 160 μl를 배포한다. 37 ℃에서 가습 이산화탄소 분위기 (5 %)로 인큐베이터에서 멀티 웰 플레이트를 넣습니다. </li></ol> 4. 3D 신경 네트워크 구축 <ol><li> 6-8 시간 도금 후, 약 30 ~ 40 μL의 볼륨 설정 피펫을 사용하여 PDMS 제약에 의해 구분 된 지역 내에서 매우 신중하게 멀티 웰 플레이트에서 서스펜션 (신경 세포와 마이크로 비드)를 전송합니다. 각 전송 후에, 육각형 컴팩트 한 구조로 자기 조립에 마이크로 비드를 허용, 절반 잠시 기다립니다. </li><li> 모든 층들이 증착되고 자발적 조립되면, 매체의 약 300 μL PDMS 제약으로 구분 영역의 상부의 큰 방울 리필. </li><li> 인큐베이터 (T를 = 37.0 O C, CO 2 = 5 %)을 MEA에 연결된 3 차원 구조를 넣어 48 시간 동안 그 보충하여 성장 배지 배양의 최종 부피 (약 1ml)에 추가하기 전에. 참고 : 막 삽입 (30,000) 및 MEA 장치 (6000) 상에 단일 층에 구슬의 수와 멀티 웰 플레이트에 구슬의 수를 고려한다. 생성 된 3 차원 구조는 MI의 5 층으로 구성되고crobeads 세포. 3D 신경 네트워크는 기하학적으로 완전한 아니라고 고려하여, 결과적인 3D 구조는 5~8 층으로 구성 될 수있다. </li><li> 날은 도금 후, 조심스럽게 MEA 링 내부의 중간 (약 900 μL)의 최종 부피를 추가합니다. 4~5주 동안 37 ° C에서 CO 2의 가습 분위기 (5 %)의 3 차원 배양을 유지한다. 일주일에 한 번 매체의 절반을 교체합니다. </li></ol> 5. 공 초점 현미경 취득 <ol><li> 홀더 현미경의 단계로 생물학적 샘플을 수정합니다. 정확한 대역폭 방출 필터와 순서를 캡처 할 각 이미지에 대한 PMT의 화상, 이득 및 오프셋 (-0.3 %)을 획득하는 레이저 (아르곤 4백96에서 5백55까지 ㎚), 스캔 속도 (400 Hz에서) 설정할 포화를 방지하고 신호대 잡음비를 증가시키기 위해. 결과 섹션은도 4의 이미지를 획득하는 데 사용되는 값을보고한다. </li><li> Z -stack 순서, 전나무의 취득ST는, 상부 및 샘플의 하부 층의 Z 위치의 값을 선택하고 획득하게 스텝 사이즈를 선택한다. </li></ol>

<ol>
	<li>Van Pelt, J., Corner, M. A., Wolters, P. S., Rutten, W. L. C., Ramakers, G. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stability+and+developmental+changes+in+spontaneous+network+burst+firing+patterns+in+dissociated+rat+cerebral+cortex+cell+cultures+on+multi-electrode+arrays.">Long-term stability and developmental changes in spontaneous network burst firing patterns in dissociated rat cerebral cortex cell cultures on multi-electrode arrays.</a> Neurosci. Lett. 361, 86-89 (2004).</li><li>Rolston, J. D., Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precisely+timed+spatiotemporal+patterns+of+neural+activity+in+dissociated+cortical+cultures.">Precisely timed spatiotemporal patterns of neural activity in dissociated cortical cultures.</a> Neuroscienc. 148, 294-303 (2007).</li><li>Frey, U., Egert, U., Heer, F., Hafizovic, S., Hierlemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectronic+system+for+high-resolution+mapping+of+extracellular+electric+fields+applied+to+brain+slices.">Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices.</a> Biosens. Bioelectron. 24, 2191-2198 (2009).</li><li>Macis, E., Tedesco, M., Massobrio, P., Raiteri, R., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+microdrop+delivery+system+for+neuronal+network+patterning+on+microelectrode+arrays.">An automated microdrop delivery system for neuronal network patterning on microelectrode arrays.</a> J. Neurosci. Meth. 161, 88-95 (2007).</li><li>Kanagasabapathi, T. T., Ciliberti, D., Martinoia, S., Wadman, W. J., Decre, M. 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=Dual+compartment+neurofluidic+system+for+electrophysiological+measurements+in+physically+segregated+and+functionally+connected+neuronal+cell+culture.">Dual compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture.</a> Front. Neuroeng. 4, (2011).</li><li>Kanagasabapathi, T. T., 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=Functional+connectivity+and+dynamics+of+cortical-thalamic+networks+co-cultured+in+a+dual+compartment+device.">Functional connectivity and dynamics of cortical-thalamic networks co-cultured in a dual compartment device.</a> J. Neural. Eng. 9, (2012).</li><li>Cullen, D. K., Wolf, J. A., Vernekar, V., Vukasinovic, J., LaPlaca, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering+and+biohybridized+microsystems+for+neurobiological+investigation+in+vitro+(Part+1).">Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (Part 1).</a> Crit. Rev. Biomed. Eng. 39, 201-240 (2011).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+Bursting+in+Cortical+Cultures+with+Closed-Loop+Multi-Electrode+Stimulation.">Controlling Bursting in Cortical Cultures with Closed-Loop Multi-Electrode Stimulation.</a> J. Neurosci. 25, 680-688 (2005).</li><li>Shany, B., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+primary+hippocampal+neuronal+tissue+on+an+aragonite+crystalline+biomatrix.">Growth of primary hippocampal neuronal tissue on an aragonite crystalline biomatrix.</a> Tiss. Eng. 11, 585-596 (2005).</li><li>Schmidt, C. E., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering:+strategies+for+repair+and+regeneration.">Neural tissue engineering: strategies for repair and regeneration.</a> Ann. Rev. Biomed. Eng. 5, 293-347 (2003).</li><li>Ma, W., 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=CNS+stem+and+progenitor+cell+differentiation+into+functional+neuronal+circuits+in+three-dimensional+collagen+gels.">CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels.</a> Exp. Neurol. 190, 276-288 (2004).</li><li>Baranes, D., 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=Interconnected+network+of+ganglion-like+neural+cell+spheres+formed+on+hydrozoan+skeleton.">Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.</a> Tissue En. 13, 473-482 (2007).</li><li>Lee, J., Cuddihy, M. J., Kotov, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+matrices:+state+of+the+art.">Three-dimensional cell culture matrices: state of the art.</a> Tissue engineering. Part B, Review. 14, 61-86 (2008).</li><li>Schuz, A., Palm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+of+neurons+and+synapses+in+the+cerebral+cortex+of+the+mouse.">Density of neurons and synapses in the cerebral cortex of the mouse.</a> J. Comp. Neurol. 286, 442-455 (1989).</li><li>Pautot, S., Wyart, C., Isacoff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloid-guided+assembly+of+oriented+3D+neuronal+networks.">Colloid-guided assembly of oriented 3D neuronal networks.</a> Nat. Method. 5, 735-740 (2008).</li><li>Frega, M., Tedesco, M., Massobrio, P., Pesce, M., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+dynamics+of+3D+engineered+neuronal+cultures:+a+new+experimental+model+for+in-vitro+electrophysiology.">Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology.</a> Sci. Rep. 4, (2014).</li><li>Tang-Schomer, M. D., 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=Bioengineered+functional+brain-like+cortical+tissue.">Bioengineered functional brain-like cortical tissue.</a> Proc Natl Acad Sci U S. 111, 13811-13816 (2014).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cell. , (1998).</li><li>Hales, C. M., Rolston, J. D., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+culture,+record+and+stimulate+neuronal+networks+on+Micro-electrode+arrays+(MEAs).">How to culture, record and stimulate neuronal networks on Micro-electrode arrays (MEAs).</a> J. Vis. Exp. (39), (2010).</li><li>Jayakumar, R., Prabaharan, M., Nair, S. V., Tamura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+chitin+and+chitosan+nanofibers+in+biomedical+applications.">Novel chitin and chitosan nanofibers in biomedical applications.</a> Biotech. Adv. 28, 142-150 (2010).</li><li>Crompton, K. E., 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=Polylysine-functionalised+thermoresponsive+chitosan+hydrogel+for+neural+tissue+engineering.">Polylysine-functionalised thermoresponsive chitosan hydrogel for neural tissue engineering.</a> Biomaterial. 28, 441-449 (2007).</li><li>Frega, M., 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=3D+engineered+neural+networks+coupled+to+Micro-Electrode+Arrays:+Development+of+an+innovative+in-vitro+experimental+model+for+neurophysiological+studies.">3D engineered neural networks coupled to Micro-Electrode Arrays: Development of an innovative in-vitro experimental model for neurophysiological studies.</a> , 957-960 (2013).</li></ol>

The authors declare that they have no competing financial interests.

본 연구에서는 3 차원으로 구성 체외 플랫폼에서 새로운 실험 네트워크 전기 생리학에 대한 다자간가 제시되었다 결합의 연결을 문화를 설계. Z 방향을 따라 이동시킴으로써 행한다 신경 염성 생장을 허용하는 지지체로서 마이크로 비드의 사용은 평면 MEA와 일체화되도록 맞추어왔다. 이러한 방식으로, 유효하고 신뢰성있는 시험 관내 3D 모델에서 얻어진 마이크로 시스템 결과...

마이크로 전극 배열 (다자간)에 결합 된 생체 외 2 차원 신경망에서 신경 역학과 기본 연결 사이의 상호 작용을 연구하기 위해 채택 금 표준 실험 모델을 arethe. 개발하는 동안, 신경이 잘 표시 복잡한 네트워크를 다시 definedspatio-temporalpatternsof 활동 1, 2 (즉, 버스트, 네트워크 버스트, 임의의 급상승 활동). MEA의 네트워크 레벨에서 표현의 상세한 역학 조사를 허용 많은 위치에서 (수십 마이크로 전극으로부터 수천) 전기 생리 활성을 기록한다. 또한, 해리 배양의 사용은 설계 - 네트워크를 설계 할 possibile 만든다. 이것은 이런 식으로 이해하기 쉽게 기록 전기 생리 활성 간의 기능적 관계 및 셀 밀도 3, 4, 5의 모듈화 정도, 이종 신경 팝의 존재 등의 네트워크 조직의 파라미터등 ulations 6 그러나, 배양 된 세포에 해리 된 모든 시험 관내 시험은 2D 신경 네트워크에 기초한다. (7) 모든 방향으로 확대 할 수 평탄화되어 2D 모델 인 somata 성장 콘 (I) 및 축색 돌기 - 생장이 방법은 생체 내 (본질적으로 3 차원, 3D) 시스템에 대해 oversimplifications 리드. (II) 2D 체외 네트워크 전시는 네트워크 (8)의 신경 세포의 대부분을 포함하는 활동을 파열에 의해 지배 전기 생리 역학에 박힌. 최근, 다른 솔루션은 3D는 신경 네트워크를 해리 체외의 구성을 허용하도록 개발되었다. 일반적인 생각은 신경 세포는 3D 방식으로 성장할 수있는 발판을 만드는 구성되어 있습니다. 이러한 지지체는 고분자 겔 및 고체 다공성 매트릭스 9-13로 실현 될 수있다. 중합체의 기계적 특성을 이용함으로써, 내부 살전 세포를 포함 할 수있다neurosphere를 11의 3D 문화의 균일 한 블록을 정의하여 전자 구조. 이 방법의 주요 특징은 neurosphere를 9,12-의 강성, 기계적 특성이다. 그러나, 이들 물질은 기공률이 제한되어, 그들은 매트릭스 내부 세포 이동을 보장하지 않는다. 이러한 단점을 극복하기 위해, 가능한 해결책은 &#39;단위&#39;모듈로 매트릭스를 슬라이스로 구성되어 있습니다. 불행히도, 입자의 크기 및 형상은 다양 정규 층상 구조로 포장을 방해 할 수있다. 7에서 컬린과 동료 생리 활성 세포 외 기질 기반의 발판 내에서 뉴런 및 / 또는 성상 세포로 이루어진 3 차원의 연결 구조를 설계했다. 이러한 설계 신경 조직은 3 차원 미세 환경 내에서 신경 생물학적 반응을 연구하고 조작하는 체외 조사에있었습니다. 이 모델은 세포 외 기질 (ECM) 및 / 또는 하이드로 겔 지지체 (500-600 μm의 두께)에 걸쳐 분산 뉴런 및 아교 세포로 구성되었다. 이 공동으로/ mm 3 5,000 세포 - ndition, 최적의 세포 생존율 (90 % 이상)은 약 3,750의 최종 밀도로 세포를 플레이 팅에 의해 발견되었다. 그러한 밀도 값은 마우스 뇌 피질의 세포 밀도는 약 90,000 세포 / 14 mm 3 인 생체 조건에서 하나보다 훨씬 낮다는 것을 주목해야한다. 이러한 제한을 극복 Pautot 15은 셀 밀도와 네트워크 연결이 네트워크의 실시간 영상을 가능하게하면서 생체 조건에서 비슷하게 제어되는 3D 관내 시스템을 실현 동료에게. 실제로,이 방법은 배양 된 신경 세포는 실리카 마이크로 비드에 성장 할 수있는 해리 개념을 기반으로합니다. 이 구슬을 준수하는 신경 세포 기관 및 성숙, 성장, 확장, 다른 뉴런 시냅스 연락처를 정의하는 그들의 arborizations만큼 큰 성장 표면을 제공한다. 이 방법은 FO하는 단 분산 비드의 자발적인 조립 성질을 이용RM 차원이 다른 구슬에 신경 세포 사이의 제한된 연결과 다른 층에 신경 세포의 고유 한 하위 집합을 포함하는 육각형 배열을 계층. 이 방법으로 달성 세포 밀도는 / mm 3 약 75,000 세포이었다. 최근에, 우리는 다자간 16 Pautot의 방법을 적응 : 얻어진 결과는 3D 전기 생리 활동이 2D 네트워크에 의해 표현 된 것보다 활동의 폭 넓은 레퍼토리를 제시 것으로 나타났다. 3D 성숙한 문화가 향상된 동적을 전시하는 네트워크 버스트 임의 스파이크 활동이 공존하는 모두. 마찬가지로, 탕-Schomer 동료 17 개월 일부 체외에서 일차 피질 배양을 유지 실크 프로테인 계 다공성 지지체를 실현하고, 텅스텐 전극에 의해 전기 생리 활성을 기록했다. 이 연구에서, 실험 절차는 MEA를 결합 3D 신경 네트워크를 구축하는 것이 설명 될 것이다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25050-014</td>
			<td align="left">0.125% diluted 1:2 in HBSS w\o CA++, MG++</td>
		</tr>
		<tr>
			<td>DNAase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td>0.05% diluted&#160; in Hanks solution</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco Invitrogen</td>
			<td>21103049</td>
			<td>culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco Invitrogen</td>
			<td>17504044</td>
			<td>2% medium supplent</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F-2442</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Glutamax-I&#160;</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G.1272</td>
			<td>5mg/liter</td>
		</tr>
		<tr>
			<td>Poly-Dimethyl-Siloxane (PDMS)</td>
			<td>Corning Sigma</td>
			<td>481939</td>
			<td>curing agent and the polymer</td>
		</tr>
		<tr>
			<td>Micro-Electrode Arrays</td>
			<td>Multi Channel Systems (MCS)</td>
			<td>60MEA200/30-Ti-pr</td>
			<td>MEA with: Electrode grid 8x8; Electrode spacing and diameter 200 and 30 &#181;m, respectively; plastic ring without thread</td>
		</tr>
		<tr>
			<td>Microbead</td>
			<td>Distrilab-Duke Scientific</td>
			<td>9040</td>
			<td>&#160;1gr Glass Part.Size Stds 40 &#181;m</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Costar Sigma</td>
			<td>CLS 3413</td>
			<td>multiwell plates with membrane insert (6.5 mm diameter porous 0.4 &#181;m)</td>
		</tr>
		<tr>
			<td>HBSS w\o Ca++ ,Mg++&#160;</td>
			<td>Gibco Invitrogen</td>
			<td>14175-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Buffer Solution&#160;</td>
			<td>Sigma&#160;</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon</td>
			<td>Sigma</td>
			<td>430935-5G</td>
			<td>polytetrafluoroethylene</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Sprague Dawley</td>
			<td>Wistar Rat</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscopy Upright</td>
			<td>Leica</td>
			<td>TCS SP5 AOBS</td>
			<td></td>
		</tr>
		<tr>
			<td>20.0x0.50 WATER objective</td>
			<td>Leica</td>
			<td>Leica HCX APO L U-V-I&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>40.0x0.80 WATER objective</td>
			<td>Leica</td>
			<td>Leica HCX APO L U-V-I&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>25.0x0.95 WATER objective</td>
			<td>Leica</td>
			<td>HCX IRAPO L</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

.이 실험 절차에서 중요한 역할 3D 신경 네트워크의 성장을위한 기계적 지지체를 정의하는 마이크로 비즈에 의해 재생되는 1a 및 B 화면을 마이크로 비드의 배열 (40 ± 2 ㎛의 공칭 직경도]의 인증 평균 직경 평면에서 42.3 ± 1.1 μm의). 이러한 구조의 특수성은 마이크로 비드가 자발적으로 소형 육각형 형상 (그림 1B 및 C)를 정의하는 자기 조립이다. 이렇게 생성 ?...

Watch this Scientific Journal Video about Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model at JoVE.com

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 ...

interfacing-3d-engineered-neuronal-cultures-to-micro-electrode-arrays

Research

JoVE Journal

Neuroscience

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

9.9K 조회수.  University of Genova. 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.

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

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

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

배아 복부 Midbrain의 Organotypic 슬라이스 문화 : Dopaminergic의 연결을 개발 연구하는 시스템 체외에서</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker, 눈 돌림 분석에 대한 혁신적인 자동화 된 접근

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

소뇌 과립 신경 세포의 유전자 조작<em&gt; 체외</em&gt;와<em&gt; 생체</em&gt; 신경 세포의 형태와 마이그레이션을 연구하는

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

관통 마이크로 전극 배열을 펼친 해마 준비에 신경 활동 전파

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

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

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

생체 내 신경 회로 연결성 모방을 위한 체외 쐐기 슬라이스 준비

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...