Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

1 준비 <ol><li> 37 ° C에서 5 % CO 2 세트 조직 문화 인큐베이터에서 실시간 세포 분석기의 역을 놓습니다. 멸균 조건 하에서 조직 배양 후드 내의 모든 세포 배양 처리 및 약리 치료를 수행한다. </li><li> NOTE : 표준 배양 조건에 비해 서로 다른 온도 설정을 필요로하는 신경 세포 라인, 그에 따라 조정되어야한다. 약하게 부착 세포주를 들어, E-96 플레이트의 웰의 바닥에 금 미소 전극과 세포의 상호 작용을 용이하게하기 위해 코팅제를 사용한다. </li><li> (적당한 용매에 신경 보호제 또는 두번째 메신저 경로 억제제 - 디메틸 설폭 사이드 또는 멸균) 1000 배 스톡 세포 배양 치료를위한 약물 화합물의 솔루션을 준비하고 -20 ° C에 저장합니다. 다음과 같은 치료에 차량과 같은 용매를 사용합니다. </li><li> DMEM / F1 문화 인간 신경 모세포종 SK-N-SH 세포이 매체는, 약 75 %의 밀도로 175cm 2면과 세포 배양 플라스크에서 10 % 태아 소 혈청 (FBS) (증식 배지)로 보충. 세포 증식 및 세포 독성에 대한 응답 속도의 관점에서 실험 사이의 일관성을 확인하기 위해 (약 10 유로의) 범위 내에서 세포 통로 번호를 유지합니다. 하부 통로 번호가 바람직하다. </li><li> PBS에 부착 SK-N-SH 세포 층을 세척하고, 37 ℃에서 5 분 동안 0.05 % 트립신-EDTA 용액으로를 Trypsinize. 원심 분리기는 5 분 동안 170 × g에서 세포를 트립신을 제거합니다. 10 ml의 증식 배지에서 세포를 재현 탁. 홀 셀 카운터 셀 카운트 및 증식 배지로 희석하여 세포를 300,000 세포 / ml의 세포 수를 조정한다. </li></ol> 2 도금 및 SK-N-SH 세포의 증식 <ol><li> E-플레이트 (96)의 각 웰에 100 ㎕의 증식 매체를 추가하고 평형을 RT에서 조직 문화 후드에서 30 분 정도 둡니다. E-괞 찮아 삽입37 ° C에서의 CO 2 배양기에서 실시간 세포 분석기 역에서 테 96. </li><li> 실시간 셀 분석기 소프트웨어를 시작. 레이아웃 페이지에서 우물 실험에 포함 된 선택하고 편집 상자에 셀 타입, 휴대폰 번호, 이름 및 세포 치료에 사용되는 화학 물질의 농도에 대한 정보를 입력합니다. 참고 :이 프로토콜의 목적을 위해 최소 4 회 반복 각각의 치료를 위해 사용하는 것이 좋습니다. </li><li> 스케줄 소프트웨어 페이지 실험에서, 세포 지수와 각 단계의 스위프 사이의 간격을 측정 스위프의 번호를 선택하여 포함 &quot;단계&quot;를 결정한다. 1 단계는 배경에 소정의 측정되기 때문에, 셀 인덱스 96 시간 동안 15 분마다 측정하고, 2 단계로 설정 &quot;하는 단계를 추가&quot;를 선택한다. 참고 : 치료를 들어,있는 빠른 반응 속도와 효과가 예상되는 짧은 간격으로 스윕을 설정합니다. </li><li> 자동으로 미리 정해진 단계를 시작하려면 시작을 클릭 한및 매체의 배경 임피던스를 측정한다. 세포를 웰에 첨가하면이 값이 자동으로 각 데이터 포인트에서 소프트웨어에 의해 감산된다. </li><li> 증식 배지에서 300,000 세포 / ml의 1.5 이전 단계에서 제조 됨) 세포 현탁액을 사용한다. / 웰 세포 독성 / 신경 연구와 잘 세포 증식 연구를위한 15,000 세포 / 30,000 세포. E-플레이트를 꺼내 세포 독성 / 신경 연구를 위해 잘 당 100 ㎕의 세포 현탁액을 추가하고 세포 증식 연구를 위해 잘 당 50 μL 세포 현탁액 50 ㎕의 증식 매체를 추가 할 수 있습니다. 물론 당 도금 셀의 수는 경험적으로 각 세포주에 대해 최적화 될 필요가있다. </li><li> 조심스럽게 세포의 경우에도 도금 E-플레이트 96 소용돌이 친다. 세포가 웰의 바닥에 균일하게 정착 할 수 있도록 RT에서 조직 배양 후드에서 30 분 동안 E-플레이트 (96)를 떠난다. </li><li> 실시간 세포 분석 국에 E-플레이트 (96)를 삽입하고 일정 페이지에 2 단계를 시작연령. 플롯 페이지 및 소프트웨어 셀 인덱스 페이지 원시 셀 색인 데이터 셀 곡선을 보면 실험 내내 셀 인덱스 값을 모니터한다. </li></ol> 3 혈청 박탈 <ol><li> 24 시간 후 세포를 도금 실험을 일시 중지하고, 실시간 셀 분석기 역 E-플레이트 (96)를 제거한다. DMEM / F12의 무 혈청 배지로 1000 배 주식에서 두번째 메신저 억제제를 희석 - 200에 최종 농도 ×. 각각의 경로를 억제하기 위해, 두번째 메신저 경로의 1 μL 억제제가 30 분 세포 독성을 시작하기 전에 신경 독성 / 신경에서의 참여를 판정하는과 세포를 치료. 역에 E-플레이트 (96)를 돌려 실험 휴대 지수 측정을 다시 시작합니다. </li><li> 30 분 후 다시 실험을 일시 중지합니다. 조심스럽게 부착 된 세포층을 방해하지 않도록주의하면서 피펫 (또는 멀티 채널 피펫)와 E-플레이트에서 완전히 96 우물을 확산 매체를 제거웰의 코너 피펫 팁의 끝을 지시함으로써. 200 μL / 무 혈청 DMEM / F12 배지 (혈청 박탈 매체)의도를 추가합니다. 세포의 기계적 교반을 최소화하는 세포 배양 배지의 신속한 교환을 보장한다. </li><li> 화합물을 희석하는 최종 농도 × 200 무 혈청 DMEM / F12 매체와 1000 배의 재고 자신의 신경에 미치는 영향을 평가한다. 1 ㎕의 화합물을 추가하는 것은 자신의 신경 보호 효과와 실험 계획에 따라 개별 우물에서 두번째 메신저 경로의 1 μL 억제제 테스트합니다. </li><li> 확산 연구에 대한 세포에서 매체를 변경하지 않습니다. 물론 당 1 μL로 처리 증식 배지에서 문화를 계속 확산 매체의 최종 농도 × 200에 1000 배의 재고에서 테스트 할 화합물을 희석. </li><li> 이전에 설정 한 96 시간 동안 셀 지수 측정과 매 15 분을 계속 실험을 다시 시작합니다. </li></ol> (4) 데이터(분석) <ol><li> 신경 독성은 / 신경 데이터는 플롯 페이지의 &quot;시간을 정상화&quot; &quot;표준화 된 셀 인덱스&quot;를 선택하여 실험 간의 편차를 줄이고하기 위해 약물 치료 또는 매체 변경 전 마지막 포인트로 셀 인덱스를 정상화. </li><li> 관심의 실험 조건에 대한 플롯 페이지의 우물을 선택하고 정규화 된 셀 인덱스 곡선을 그릴 &quot;추가&quot;를 클릭합니다. 또는 시간의 함수로 정규화 된 셀 인덱스 곡선으로 두번째 메신저 억제의 신경 독성 / 신경 보호 효과의 반응 속도를 관찰한다. </li><li> 증식에 대한 신경 보호 효과 / 신경 독성 효과에 관여되는지 여부를 평가하기 위해 시간의 함수로서 증식 배지에서 시험 약물 치료 용 세포 지수 곡선을 관찰한다. </li><li> 결과의 통계적인 평가를 개시 엑셀 파일로 셀 색인 소프트웨어 페이지의 모든 셀 인덱스 시점에 대한 정보를 내보내는 실험 <br/&gt; NOTE : 통계 분석에서 초기 단계의 p 값, 웰치 t-테스트를​​ 사용하여 개질 MATLAB 프로그램 세트 semilogarithmically 플롯으로 (보조 코드 파일로 제공되는)의 시간 척도에 대하여 반전 축을 이용한다. 이러한 프로그램은, 전체 실시간 셀 분석기 실험의 시간 과정을 통해 P-값의 검출을 허용하고, 따라서보다 상세한 통계적 분석 시점의 선택을 돕기. 프로그램에 대한 자세한 내용은 보충 자료를 참조하십시오. </li><li> 특정 시점에서 다른 처리 조건 하에서 셀 인덱스 값의 차이의 통계적 분석을 위해, 반출 엑셀 파일에서 각각의 시점에서 셀을 선택 인덱스 값. 통계 소프트웨어를 이용하여 사후 시험 뒤에 분산의 단방향 또는 양방향 분석 (ANOVA)으로 선택한 시점에서 셀 인덱스 값들을 분석한다. </li></ol>

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현재 비디오 기사에 대한 출판 비용은 &quot;ACEA 생명 과학&quot;에 의해 후원되었다.

현재 프로토콜은 연속적 및 라벨없는 조건에서 신경 세포주에서 화합물의 신경 / 신경 독성 효과를 평가하기 위해 상기 효과에 관련된 제 메신저 경로에 대한 통찰력을 얻기 위해 실시간 셀 분석기의 유틸리티를 제공한다. 세포 독성 및 세포 증식에​​ 대한 약물의 효과를 연구하기 위해 실시간 셀 분석기 효용은 일반적으로 인식 되더라도, 단지 몇 연구는 신경 관련 세포 ?...

신경 세포의 사멸이 많은 뇌 관련 질환의 병태 생리 1에서 중요한 역할을한다. 체외 독성 분석의 신뢰성 및 높은 처리량의 가용성은 신경 독성의 메커니즘에 대한 통찰력을 얻기 위해 약물 개발이의 치료 후보로 신경 분자를 선택하기 위해 매우 중요합니다. 그러나 가장 널리 운동 해상도를 허용하지 않는 하나의 시간 시점에서 신경 독성 / 신경을 평가 체외 신경 독성 assays.They 사용에 많은 제한이있다; 종종 신호 전달 경로를 방해하고 같은 세포 집단에서 추가 연구를 제한하고, 종종 노동 집약적이며, 많은 경우에 기계적인 통찰력을 제공하지 않습니다 수 있습니다 라벨 또는 프로브를 사용합니다. 본 연구에서 우리는 실시간 미만 신경 세포주에서 세포 독성 및 신경 보호를 결정하기 위해 실시간 임피던스 기반 세포 분석기의 유용성을 증명 라벨없는조건 효과에 관련된 제 메신저 경로의 분석을 통해 하류 메커니즘에 대한 통찰력을 제공하고. 이전의 연구는 표준 기법 3,4,5,6 비해 세포 독성은 물론 세포주에서 세포 증식에 미치는 영향을 결정하기 위해 실시간 세포 분석의 유효성을 확인했다. 예를 들어, 상관 관계는 기저 증식 조건 및 HeLa 세포에서 3 개의 다른 독성 패러다임 후 여러 시점에서 표준 세포 생존 WST-1 분석의 판독 및 셀 인덱스 값 사이에서 관찰되었다. 셀 지수 측정에 의해 평가하고 표준 적 사용 sulforhodamine의 B (SRB) 분석 4시 미세 소관 안정 파클리탁셀과 자극 A549 및 MDA-MB-231 세포의 증식과 세포 독성은 매우 유사한 값을 나타내었다. 불후의 해마 신경 세포의 신경 세포 라인에서 HT-22 세포 지수 측정은 자신의 능력 t에 대해 검증 하였다O 널리 사용되는 3 - (4,5 - 디메틸 -2 - 일)에 대해 세포 증식, 글루타메이트 독성 세포 보호 및 2,5 - 디 페닐 테트라 졸륨 브로마이드 (MTT) 분석 (5)를 검출한다. 같은 연구에서, MTT 분석 결과 및 셀 인덱스 측정은 또한 팬 카스파 제 억제제 QVD (5)에 의해 성장 인자 결핍 및 세포 독성의 복구 후 신경 전구 세포의 증식, 세포 독성을 측정하는 상관 관계. Vandetanib (혈관 내피 성장 인자 수용체 및 표피 성장 인자 수용체 억제제)으로 NIH 3T3 세포에서 유도 된 세포 독성 세포 인덱스 값 또는 중성 적색 흡수 분석법 (6)로 측정 유사한 결과를 보였다. 우리가 최근 세로토닌 2A의 신경 보호 효과를 평가하기 위해 실시간 세포 분석 시스템을 사용 하였다 (5-HT 2A) 수용체 작용제, (±) -2,5 - 디메 톡시 4 - iodoamphetamine 염산염 신경 세포주 (DOI) ( SK-N-SH 세포)와 세코의 참​​여에 대한 검사관찰 된 신경 보호 (7) 상에 화학적 억제의 효과를 모니터링을 통해 차 메신저 경로. 흥미롭게도, 5-HT의 2A 수용체는 일반적으로 별개의 두번째 메신저 경로 팔 모두를 활성화시킬 수있다 (DOI 및 lisuride 각각 같은) 환각 및 nonhallucinogenic 모두 효능을 가지고 있습니다. 제시된 기술의 이점은, 일의 과정에서 세포 생존에 대한 정보를 실시간으로 수집하는 신경에​​ 대한 증식 효과 가능한 공헌을 평가하고 최적 시간을 선택하는 제 메신저 경로가 관여 묘사 할 수 있다는 점이다 같은 세포 집단에 대한 자세한 엔드 포인트의 연구. 현재 프로토콜의 워크 플로우의 개략도는 그림 1에 제시되어있다.

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Name of Material/ Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:457px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">xCELLigence&#160; RTCA SP system bundle</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 00380601030</td>
			<td>consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">E-plate 96</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 05232368001</td>
			<td>for culturing the cells, inserted in the RTCA SP Station</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">SK-N-SH cells</td>
			<td style="width:161px;">ATCC - (in partnership with LGC Standards)</td>
			<td style="width:119px;">HTB-11</td>
			<td>can be replaced by another adherent neuronal cell line of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM/F12&#160;</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8437</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">fetal bovine serum</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">16140-063</td>
			<td>supplements proliferation, but not serum deprivation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">tisue culture flask T175</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">83.1812.302&#160;</td>
			<td>for culturing cells, which will be later plated on the E-Plate 96</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">0.05% Trypsin-EDTA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">25300-054</td>
			<td>for trypsinization of cells cultured in tissue culture flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Scepter 2.0 cell counter</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:119px;">PHCC20060</td>
			<td>automated cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">phosphate-buffered saline</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">10010-015</td>
			<td>for washing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">(&#177;)-DOI hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D101</td>
			<td>5-HT2A agonist for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">LY-294,002 hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">L9908</td>
			<td>PI3-K inhibitor for cell culture treatment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">lisuride maleate</td>
			<td style="width:161px;">Tocris Bioscience</td>
			<td align="right" style="width:119px;">4052</td>
			<td>compound with 5-HT2A agonistic activity for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dimethyl sulfoxide</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4540</td>
			<td>for dissolving LY-294,002 and lisuride maleate</td>
		</tr>
	</tbody>
</table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

많은 뇌 관련 질환은 병태 생리에 관여하는 신경 세포의 죽음이있다. 약물과 신경 / 신경 독성의 분자 메커니즘에 대한 통찰력을 확보하고 잠재적으로 신약 개발을 촉진 할 수 있습니다 도움이 될 관련 다운 스트림 경로의 신경 또는 신경 독성 효과를 연구하기 위해 체외 모델에서 개선. 그러나, 기존의 많은 시험 관내 독성 분석법은 중요한 제한이 - 가장 안 시간 코스 및 효과의 동력학을 관찰 할 수 있도록, 하나의 시점에서 신경 보호 및 신경 독성을 평가한다. 또한, 실시간으로 신경 보호에 관여하는 신호 전달 경로 다운 스트림에 대한 정보를 수집 할 수있는 기회는 매우 중요하다. 현재의 프로토콜에서는 라벨없는 실시간 condit하에 신경 세포주에서 세로토닌 2A (5-HT의 2A) 수용체 작용제의 신경 보호 효과를 결정하기 위해 실시간 임피던스 기반 세포 분석기의 사용을 설명임피던스 측정을 이용하여 이온. 더욱이, 우리는 제 메신저 경로의 억제제는 신경 보호 효과에 관여하는 분자 스트림을 묘사하기 위해 사용될 수 있음을 보여준다. 우리는 또한 세포 증식에​​ 영향이 관찰 된 신경 보호 효과에 기여 여부를 확인하려면이 기술의 유틸리티를 설명합니다. 시스템은, 웰의 바닥 표면에 금 미세 전극 배열을 교대 셀 센서로서 포함 E-플레이트라고도 특수 마이크로 플레이트를 이용한다. 임피던스 읽기가 부착 세포, 세포 생존, 형태, 및 접착 숫자로 변경된다. 셀 인덱스라는 무 차원 파라미터는 전기 임피던스 측정으로부터 도출되고, 상기 셀의 상태를 나타내는 데 사용된다. 전반적으로, 실시간 임피던스 기반 세포 분석기는 더 기여 실시간, 신경 보호 및 신경 독성의 라벨없는 평가, 및 제 메신저 경로 개입의 평가를 허용치료 후보를 선택하기위한 시험 관내에서 잠재 신경 화합물의 상세하고 높은 처리량 평가.

<html> 혈청 부족은 실시간 셀 분석기로 연속적으로 모니터링 할 수있다 셀 인덱스 값에서 감소하는 리드 세포 신경 독성 자극 제시된 기법 및 특정 신경 독성 자극과 연구 세포 유형에 의존하는 역학과 실시간으로 모니터링 할 수있다 셀 인덱스 값의 저하를 초래할.도 2의 증가를 보여 SK-N-SH 세포는 고원 (녹색 선)과 박탈 매체 (빨간 선) 혈청하는 세포?...

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

신경 세포주에 신경 독성과 신경에 관여하는 분자 진학의 윤곽을하는 도구로 실시간 임피던스 기반의 세포 분석기

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

real-time-impedance-based-cell-analyzer-as-tool-to-delineate

Research

JoVE Journal

Neuroscience

11.3K 조회수.  University of Zürich. Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

비디오: Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

여러 스케일에서 디지털의 연결 형태론의 자동 숄 분석

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

연구

신경과학

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포에서 인간의 연결을 Progenitors과 뉴런의 효율 유도

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

동시 Electroencephalography, 설치류 대뇌 피질의 젖산 농도와 Neuronal 활동 Optogenetic 조작의 실시간 측정

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. 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 ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

신경 발사 환율의 조작에 의해 성상 세포 수용체의 가소성을 유도

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

나노 입자 신경 독성을 결정하는 대리 바이오 센서로 미세 아교 세포

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

자궁 내 일렉트로는 신경 세포의 부분 집단의 흥분 및 단일 세포의 연결을 연구에 접근

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hippocampal 슬라이스 문화 연구 Neuroprotection 및 종양 세포의 침입을 모델로

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...