우리는 기술 지원을 왕위 (바오로) 후진타오, 레이첼 Sinit에게 감사의 말씀을 전합니다. 이 연구는 NIH 보조금 (PN2의 EY016525)에 의해 및 다운 증후군 연구 및 치료 재단과 래리 L. Hillblom 재단에서 자금을 지원합니다.

수술과 동물의 절차는 관리에 대한 NIH 지침 및 실험 동물의 사용에 따라 엄격하게 수행된다. 동물의 사용과 관련된 모든 실험은 UCSD 기관 동물 관리 및 사용위원회에 의해 승인됩니다. 1 플라스미드 복제, 모노 - 비오틴 BDNF의 발현과 정제 (mBtBDNF) 참고 : HEK293FT 셀 (10)의 한 pcDNA3.1 벡터와 같이 발현에 사전 proBDNFavi과 피라의 cDNA를 구축합니다. mBtBDNF 성숙 및 생물학적 활성 monobiotinylated 신경 성장 인자 (mBtNGF) (10)의 제조 이전에 발행 방법에있어서의 Ni-NTA 비드를 사용하여 정제 하였다. 미세 유체 챔버의 2 준비 신경 미세 배양 장치는 유체 적 신경 세포체에서 축삭을 분리하는 것을 가능하게한다. 바로 각 해부하기 전에 갓 코팅 된 커버와 챔버를 조립합니다. 사용되는 미세 유체 챔버이 프로토콜에서 상업적으로 (재료 및 장비의 표 참조) 구입. 핸드 워시와 5-6x까지 다시 상업적으로 구입 한 실. <ol><li> 손세탁 1 % Alconox에서 미세 유체 챔버. 30 분마다 밀리-Q 물로 3 회 씻어. 70 % 에탄올에 다시 손 세척 챔버. </li><li> 층류 후드에서 파라 필름에 건조 챔버를 배치. 방출 및 UV 하에서 20 분 동안 챔버의 양측 소독. 쇼핑몰 실온에서 파라 필름으로 밀봉 멸균 15cm 접시에 멸균 챔버. </li><li> 유리 용기에 24 X 40mm 1 위 유리 커버 슬립을 놓습니다. 밤새 회에 35 % 염산 산에서 커버 슬립을 적 십니다. 다음 날, 30 분 물 각각에 대한 커버 슬립을 세 번 헹군다. </li><li> 100 % 에탄올로 각각의 커버 슬립을 침지하고 분젠 버너 위에 불타는하여 커버 슬립을 하나씩 소독. 멸균 페트리 접시에 건조 된 커버를 저장하고 코팅까지 실온에서 보관하십시오. </li><li> OU를 놓는다15cm 배양 접시에 커버 슬립을 t. 코트 0.01 % 폴리-L-라이신 (PLL)의 0.7 ml의 각각과 커버 슬립을 실온에서 배양 후드. </li><li> 1 시간 후, 커버 슬립에게 멸균 수로 3 회 씻어. 6cm의 배양 접시에 진공과 장소 각 coverslip에 건조 된 커버. </li><li> 미세 유체 챔버를 조립하기 위해, 마이크로 그루브를 만지지 않도록주의 PLL 코팅 된 커버 슬립 위에 하단에 마이크로 그루브면이 챔버를 배치합니다. 부드럽게 챔버는 단단히 밀봉 된 보장하기 위해 피펫 팁과 챔버를 눌렀습니다. </li></ol> 챔버에 3의 연결을 문화의 해부 및 플레이트 <ol><li> 1 % 펜 / 패 혈성 및 10 mM의 HEPES 2 ㎖의 해부 버퍼 (HBSS, 아니 칼슘, 아니 마그네슘을 포함하는 15 ML 원뿔 튜브에 해부 두 E17-E18 쥐의 해마 (뇌의)를 배치합니다. 조직을 5 ㎖로 3 배 씻어 해부 최대한 해부 버퍼를 제거합니다. 때마다 버퍼와 신선한 디의 900 μl를 추가ssection 버퍼입니다. </li><li> , 조직을 소화 1X 작업 농도를 확인하기 위해 해부 버퍼에 10 배 트립신 (2.5 %)의 100 μl를 추가합니다. 37 ° C의 물을 욕조에 원뿔 튜브를 놓습니다. 소화 10 분 후, 1 ㎎ / ㎖의 주위에 최종 농도 10 ㎎ / ㎖의 DNase I의 100 μl를 추가합니다. </li><li> 화재 연마 파스퇴르 유리 피펫을 사용하여 부드럽게 아래로 5-10x 피펫 팅에 의해 조직을 씹다. 오른쪽 분쇄 한 후, (10 % FBS, 2 mM의 GlutaMax, 2 % B27와 Neurobasal) 평판 배지 2 ㎖의 트립신으로 급랭. </li><li> 바닥에 정착하기 위해 조직의 파편을 할 수 있도록 5 분 동안 후드 샘플을 둡니다. 조심스럽게 펠릿 세포 5 분 동안 200 XG에 깨끗한 무균 15 ML 원뿔 튜브와 원심 분리기로 상층 액 2 ㎖를 제거합니다. 50 ㎕의 도금 미디어에서 펠렛을 재현 탁 </li><li> 혈구와 세포를 계산합니다. 로드 미세 유체 챔버의 한 구획에 15 ~ 20 ㎕의 세포 현탁액 (~ 40,000 세포). 챔버를 배치배양기에서 10 분 세포가 커버 슬립에 첨부 할 수 있도록합니다. 10 분 후, 챔버의 두 구획을 채우기 위해 더 도금 미디어를 추가 할 수 있습니다. </li><li> 해부의 두 번째 날, 완전히 세포체와 축삭 구획 모두 (2 mM의 GlutaMax, 2 % B27와 Neurobasal) 유지 보수 매체와 도금 미디어를 교체합니다. 해마 신경 세포의 축삭은 3 일에 마이크로 그루브를 건너 일 5-7 사이의 축삭 구획에 도달하기 시작합니다. 이 기간 동안, 보수 신선한 매체 매 24 ~ 48 시간 배양하여 매체의 절반을 대체. </li></ol> QD-BDNF의 4 축삭 전송 <ol><li> , QD-BDNF 축삭 수송의 영상을 살고있는 마이크로 유체 챔버의 세포체와 축삭 구획 모두에서 BDNF 고갈에 앞서 철저히하여 2 시간 동안 BDNF-무료, 무 혈청 Neurobasal 매체 매 30 분 간격으로 두 구획을 씻어. </li><li> BDNF 고갈 동안, QD-BDNF 접합체를 준비합니다. 모노 - 바이오틴 BD의 50 nm의 혼합NF의 neurobasal 매체에서 50 nM의 스트렙 타비 딘 - 컨쥬 게이트 QD655와 다이머 및 60 분 동안 얼음 위에서 배양한다. </li><li> 축삭 함에서 용지를 제거하고 37 ° C에서 4 시간 동안 0.25 nm의 최종 농도로 300 ㎕의 QD-BDNF를 추가합니다. 세포체 납부에 QD-BDNF의 확산을 최소화하기 위해, 항상 축삭 구획보다 세포체 함의 매체의 높은 수준을 유지하는 것이 매우 중요하다. 라이브 영상 전에 배양 한 후 언 바운드 QD-BDNF를 씻으십시오. </li><li> 100X 오일 대물 렌즈가 장착 된 거꾸로 현미경을 사용하여 QD-BDNF 수송의 라이브 영상을 수행하십시오. 범위 및 일정한 온도 (37 ° C) 및 CO 2 (5 %)에 부착 된 환경 챔버를 따뜻하게. QD655 신호를 시각화하기 위해 텍사스 레드 여기 / 방출 큐브 집합을 사용합니다. </li><li> 획득 및 CCD 카메라를 사용하여 2 분의 1에 대한 전체 프레임 / 초의 속도로 중간 축삭 내의 저속 이미지를 캡처. 더 축삭과 함께 사용 마이크로 그루브 그쪽으로t는 QD 따라서 침투에 대한 제어와 같은 신호가 없다. </li><li> 어떤 화상 해석 소프트웨어 또는 NIH ImageJ에 BDNF를 사용 수송 분석. </li></ol>

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관심 없음 충돌 선언하지 않습니다.

본 연구에서 우리는 생물학적 활성을 생산하는 신규 한 방법의 개발을보고, BDNF의 축삭 수송을 추적하는 데 사용될 수 BDNF (mBtBDNF)를 monobiotinylated. 양자점 스트렙에 단백질 컨쥬 게이트, 및 마이크로 유체 챔버를 사용함으로써, 상기 방법은 하나는 실시간 및 시공간 해상도와 단일 분자 감도 차와 뉴런의 BDNF의 축삭 전송을 검출 할 수있다. 여기에서 사용 된 도구는 건강과 질병에서 신경 세포에서 B...

신경 세포는 고도의 그 긴 종종 고도로 정교한 프로세스 확립 및 신경 회로의 구조와 기능을 유지하기위한 기본이되는 셀을 편광된다. 축삭과 시냅스에서화물을 운반하는 중요한 역할을한다. 소마 세포에서 합성 된 단백질은 신경 세포 소기관 및 기능을 지원하는 시냅스 전 말단에 도달하는 축삭을 통해 전송 될 필요가있다. 대응하여, 신호는 말단 축색 돌기가 형질 도입 될 필요가 수신 및 소마로 반송. 이러한 프로세스는 신경 세포의 생존, 분화 및 유지 관리에 필수적이다. 일부 신경 축삭에서 그 전송을 통해 거리보다 1000 배 세포체의 직경을 수행해야에서 가능성 용이 심지어 작은 결손이 현저하고 신경 회로 기능에 영향을 미칠 수 있다는 것을 구상한다. 뇌 유래 신경 영양 인자 (BDNF), 성장 인자의 뉴로 트로 가족의 일원은, 엄마의 존재해마, 대뇌 피질과 기저 전뇌를 포함하여 뉴욕의 뇌 영역. BDNF는인지 회로에 참여하는 신경 세포의 생존, 분화 및 기능을 지원하여인지와 기억 형성에 중요한 역할을한다. BDNF는 그것이 미토 겐 활성화 단백질 키나아제 / 세포 외 신호 - 조절 된 단백질 키나제 (MAPK / ERK), 포스파티딜 이노시톨 3 - 키나제 포함 TrkB-매개 신호 전달 경로를 활성화 축삭 말단, 수용체, 티로신 키나아제 TrkB에 바인딩 (PI3K) 및 포스 포 리파제 C-감마 (PLCγ). 이러한 신호 전달 경로에 참여하는 단백질은 다음 역행 신경 소마로 이송하는 엔도 좀 1-6 신호 BDNF / TrkB를 형성하는 세포 내 이입 기공 구조에 포장됩니다. 마이크로 유체 문화 실 정상 조건에서뿐만 아니라 부상과 질병 7,8의 설정에서 축삭 생물학 연구를위한 매우 유용한 플랫폼이다. 분리 축삭으로세포 기관에서, 장치는 축삭 8-10에 구체적으로 교통을 연구하는 일을 허용했다. 본 연구에 사용 된 450 μm의 마이크로 그루브 장벽 PDMS 기반의 미세 유체 플랫폼은 상용 (재료 표 참조)을 구입 하였다. BDNF 전송을 조사하기 위해, 우리는 monobiotinylated BDNF (mBtBDNF)를 생산하는 새로운 기술을 개발했다. 우리는 (또한 AviTag라고도 함) 비오틴 수용체 펩타이드, AP를 이용했다. 그것은 특히 대장균 효소 리가 비오틴, 피라 의해 바이오틴에 결찰 될 수 리신 잔기를 포함하는 15 개의 아미노산 서열이다. 우리는 PCR (도 1a)에 의해 마우스 cDNA를 미리 proBDNF의 C-말단에 융합 AviTag. 구조체는 포유류 발현 벡터, 한 pcDNA3.1-myc의 자기 벡터에 클로닝 하였다. 또한 한 pcDNA3.1-myc의 자기 벡터에 피라 세균의 DNA를 클로닝. 두 플라스미드 모두 단백질을 일시적으로 발현하는 HEK293FT 세포로 공동 - 형질 감염시켰다. 피라는 비오의 결찰 촉매특히 라이신이 일에서 BDNF의 C-말단에 AviTag 내에 상주에서 : 1의 비율로 BDNF의 단량체를 monobiotinylated 생성한다. 비오틴은 ~ 18 kDa의 분자량의 BDNF와 성숙 회수 및 Ni-수지 (도 1C)를 사용하여 미디어에서 정제 하였다. (그림 1D)를 발현 수준에 의해 수정되지 않은 BDNF 감지 할 수없는 심판으로 BDNF의 바이오 티 닐화는 완전했다. 스트렙 타비 딘 결합 된 양자점, QD 655는 QD-BDNF를 만들기 위해 mBtBDNF 라벨을 사용 하였다. mBtBDNF이 인산화 TrkB (그림 1E)를 활성화하고 재조합 인간 BDNF (rhBDNF)의 정도에 신경 돌기의 파생물 (그림 1 층)을 자극 할 수 있다는 AviTag의 존재는 BDNF의 활동을 방해하지 않았다. QD-BDNF는 QD-BDNF는 생리 활성 (그림 1G) 인 것을 나타내는, 해마 축삭에서 TrkB와 colocalized 것으로 나타 면역 염색. BDNF 전송을 연구하기 위해, QD-BDNF는 원위부 축삭 구획에 추가쥐 E18 해마 신경 세포 (그림 2A)를 포함하는 마이크로 유체 문화. 축삭 내 QD-BDNF 역행 전송은 적색 형광 태그 (지원 영상 S1, S2)의 실시간 라이브 영상에 의해 체포됐다. 생성 kymograph를 분석함으로써, QD-BDNF는 약 1.06 μM / 초 (도 3a)의 이동 속도로 역행 수송 될 것으로 관찰되었다. GFP 또는 mCherry - 태그 BDNF는 BDNF의 축삭 이동을 추적하는 데 사용되었다. 주요 단점은 단일 분자 연구에 충분히 밝은하지 수 있다는 점을들 수있다. 또한, 선행 성 모두와 역행 BDNF 움직임의 존재 어려운 역행 수송 BDNF는 뉴로 트로 / 수용체 복합체에 참여할지 여부를 평가하기 위해 만든다. 이 비디오에서는, 우리는 차 신경 세포를 사용하여 미세 유체 챔버에서 QD-BDNF의 라이브 인신 매매를 검사하는 데 사용되는 기술을 보여줍니다. ultrabrightness 및 양자점 살려 우수한 광 안정성에스 것이 가능 BDNF 수송의 장기 추적을 수행 할 수 있습니다. 이 기술은 AD, HD, 및 다른 신경 퇴행성 질환에서 축삭의 기능 연구를 향상시키기 위해 이용 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Name</td>
			<td style="width:205px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase&#160;</td>
			<td>Invitrogen</td>
			<td>11708021</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EcoRI&#160;</td>
			<td>Fermentas</td>
			<td>FD0274</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>Fermentas</td>
			<td>FD0054</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEK293FT cells</td>
			<td>Invitrogen</td>
			<td>R70007</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM-high glucose media</td>
			<td>Mediatech</td>
			<td>10-013-CV</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">d-biotin&#160;</td>
			<td>Sigma</td>
			<td>B4639</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TurboFect&#160;</td>
			<td>Fermentas</td>
			<td>R0531</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF&#160;&#160;</td>
			<td>Sigma</td>
			<td>P7626</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">aprotinin</td>
			<td>Sigma</td>
			<td>A6279</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA resins</td>
			<td>Qiagen</td>
			<td>30250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>&#160;S8820</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">silver staining kit&#160;</td>
			<td>G-Biosciences</td>
			<td>786-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant BDNF</td>
			<td>Genentech</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic chambers</td>
			<td>Xona</td>
			<td>SND450</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24x40 mm No. 1 glass coverslips&#160;</td>
			<td>VWR</td>
			<td>48393-060</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-Lysine&#160;</td>
			<td>Cultrex</td>
			<td>3438-100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco</td>
			<td>14185-052</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Neurobasal&#160;</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GlutaMax&#160;</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27&#160;&#160;</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QD655-streptavidin conjugates</td>
			<td>Invitrogen</td>
			<td>&#160;Q10121MP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Avi tag antibody</td>
			<td>GenScript</td>
			<td>A00674</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">streptavidin-agarose beads&#160;</td>
			<td>Life Technology</td>
			<td>&#160;SA100-04</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma</td>
			<td>T6399</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HRP-streptavidin&#160;</td>
			<td>Thermo Scientific</td>
			<td>N100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-pTrkB antibody</td>
			<td colspan="2">a generous gift from Dr M. Chao of NYU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-TrkB antibody</td>
			<td>BD Science</td>
			<td>610101</td>
		</tr>
	</tbody>
</table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNF는 신경 세포의 생존, 분화 및 기능의 몇 가지 측면에서 중요한 역할을한다. 축색 돌기의 구조 및 기능 적자는 점점 알츠하이머 병 (AD)와 헌팅턴 병 (HD)을 포함한 신경 퇴행성 질환의 초기 기능으로 볼 수 있습니다. 아직까지 불분명 축삭 손상이 유발되는 메커니즘 (들)입니다. 우리는 생물학적 활성을 생산하는 신규 한 방법의 개발을보고, BDNF의 축삭 수송을 추적하는 데 사용될 수 BDNF (mBtBDNF)는 monobiotinylated. 양자점 표지 BDNF (QD-BDNF)가 mBtBDNF에 양자점 655 공역에 의해 제작되었다. 미세 유동 장치는 신경 세포체에서 축삭을 분리하는데 사용되었다. 축삭 구획 QD-BDNF의 추가 축삭에서 BDNF 수송의 라이브 영상을 허용했다. 우리는 QD-BDNF는 약 1.06 μm의 / sec의 이동 속도에서 거의 일시 정지와 역행 본질적으로 독점적으로 이동 있음을 보여 주었다. 이 시스템은 저를 조사하는 데 사용할 수 있습니다AD 또는 HD에서 중단 축삭의 기능뿐만 아니라 다른 퇴행성 질환의 chanisms.

생산 및 생물 학적 활성 모노 - 바이오틴 BDNF의 정화 AviTag 순서 (GGGLNDIFEAQKIEWHE)와 융합 BDNF의 발현 벡터는 이전에 발행 된 프로토콜 (10)에 따라 작성되었습니다. 전체 길이 융합 단백질의 분자량은 ~ 32 kDa의이 (것으로 예측 하였다 <a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ) 18 kDa의 (도 1a)의 예측 된 분자량을 가진 성...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF, 신경 영양 인자의 축삭 수송은 여러 신경 인구의 생존과 기능을 위해 중요하다. 일부 퇴행성 질환은 축삭 구조와 기능의 장애로 표시됩니다. 우리는 차 신경 세포를 사용하여 미세 유체 챔버에서 QD-BDNF의 라이브 인신 매매를 검사하는 데 사용되는 기술을 보여 주었다.

양자의 축삭 교통 수단의 실시간 이미징 차 뉴런에서 BDNF를 도트 표지

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

real-time-imaging-axonal-transport-quantum-dot-labeled-bdnf-primary

Research

JoVE Journal

Neuroscience

15.0K 조회수.  University of California, San Diego.  BDNF, 신경 영양 인자의 축삭 수송은 여러 신경 인구의 생존과 기능을 위해 중요하다. 일부 퇴행성 질환은 축삭 구조와 기능의 장애로 표시됩니다. 우리는 차 신경 세포를 사용하여 미세 유체 챔버에서 QD-BDNF의 라이브 인신 매매를 검사하는 데 사용되는 기술을 보여 주었다. 

비디오: Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

의 Organotypic 슬라이스 문화 GFP - 표현 주변 신경 가지의 실시간 이미징을위한 마우스 배아를

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

연구

신경과학

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Axonal 개발 제어하기 위해 뉴런의 기계 조작

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation 및 Mitochondrial 교통 : 긴 기간이 이상 Hippocampal 뉴런의 라이브 영상

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

구조적 조명 현미경을 사용하여 쥐 차 해마 신경 세포의 돌기 쪽 이미징

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

<em&gt; 전의 VIVO</em&gt; 레이저에 의한 척수 손상 모델은 실시간으로 축삭 변성의 메커니즘을 평가하기 위해

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

관리의 실시간 fMRI Neurofeedback 훈련에 대 한 프로토콜

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

모터 뉴런과 유사한 NSC-34 세포 내에서 실시간으로 핵 세포질 수송을 측정하는 분석

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

배아 운동 뉴런의 Axonal 투영 패턴의 시각화<em&gt; Drosophila</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

뇌 슬라이스와 기본 세포 배양에서 피라미드 뉴런의 탄도 라벨링

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

미세 유체 챔버 시스템을 사용하여 모터 뉴런 배양에서 세포기관의 축축한 수송

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...