Name: collection of frozen rodent brain regions for downstream analyses
Uploaded: 2020-04-23T13:00:00
Description: Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

이 작업은 NIH, DA043982 및 DA046196에 의해 지원되었습니다.

이 연구에서 사용된 동물은 인디애나 대학의 기관 동물 관리 및 사용 위원회 (IACUC) 및 국립 보건원 (NIH) 지침에 의해 명시된 대로 윤리적이고 인도적인 방식으로 취급되었습니다.
참고: 모든 공구와 표면은 작업을 시작하기 전에 뉴클레아제18을 제거하기 위해 적절한 용매로 세척해야 합니다.
1. 뇌 저장
<ol><li>안락사된 성인 CD1 야생형 ?...

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F., 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=Cannabinoid+Exposure+via+Lactation+in+Rats+Disrupts+Perinatal+Programming+of+the+Gamma-Aminobutyric+Acid+Trajectory+and+Select+Early-Life+Behaviors.">Cannabinoid Exposure via Lactation in Rats Disrupts Perinatal Programming of the Gamma-Aminobutyric Acid Trajectory and Select Early-Life Behaviors.</a> Biological Psychiatry. , (2019).</li><li>Choi, D. W., Rothman, 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=The+role+of+glutamate+neurotoxicity+in+hypoxic-ischemic+neuronal+death.">The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death.</a> Annual Review of Neuroscience. 13 (1), 171-182 (1990).</li><li>Guhaniyogi, J., Brewer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mRNA+stability+in+mammalian+cells.">Regulation of mRNA stability in mammalian cells.</a> Gene. 265 (1-2), 11-23 (2001).</li><li>Sampaio-Silva, F., Magalh&#227;es, T., Carvalho, F., Dinis-Oliveira, R. J., Silvestre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+RNA+degradation+for+estimation+of+post+mortem+[corrected]+interval.">Profiling of RNA degradation for estimation of post mortem [corrected] interval.</a> PloS One. 8 (2), 56507 (2013).</li><li>Yamagata, K., 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=Signs+of+biological+activities+of+28,000-year-old+mammoth+nuclei+in+mouse+oocytes+visualized+by+live-cell+imaging.">Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging.</a> Scientific Reports. 9 (1), 4050 (2019).</li><li>Allen Brain Atlas: Developing Mouse Brain. Allen Institute Available from: <a target="_blank" href="https://developingmouse.brain-map.org/static/atlas">https://developingmouse.brain-map.org/static/atlas</a> (2008)</li></ol>

이 작품은 핵산과 단백질의 분해를 제한하면서 뇌의 작은 특정 영역을 분리하는 기술을 설명합니다. 뇌 조직의 손상은 유기체가 죽으면 빠르게 발생합니다. 이것은 부분적으로 세포 외 글루타민산염의 급속한 축적과21발생하는흥분독성에 기인한다. 메신저 RNA는 특히저하에 취약하다 22,,23. 단백질과 핵산의 분해는24년</su...

이 연구는 알렌 마우스 브레인 아틀라스1과같은 참조를 사용하여 고품질의 핵산 또는 단백질을 추출하기 위한 냉동 뇌 영역의 해부를 가이드로 보여줍니다. 이 기술에서, 뇌는 플래시 냉동 및 동결 된 상태에서 유지되는 동안 나중에 단면및 해부를 위해 -80 °C에서 저장됩니다. 이 과정을 통해 연구원은 한 세션에서 많은 수의 뇌를 수확하고 나중에 여러 뇌 영역의 정확한 수집을 위해 해부 할 수 있습니다.
유전자 및 단백질 발현과 관련된 질문에 답할 때 관심 있는 뇌 영역(ROI)의 정확한 수집이 종종 필요합니다. 약리학, 전기 생리학 및 광유전학은 관찰된 행동2,,3,,4를뒷받침하는 분자 변화를 해명하는 것을 돕기 위하여 야생형 또는 유전자 변형 설치류에 사용될 수 있는 동안, 전사체 및 단백질에 있는 유도한 변경의 측정은 수시로 이 사실 인정을 지원하기 위하여 이용됩니다. 정량적 역전사 중합체 연쇄 반응(RT-qPCR), 서부 블로팅, RNAseq5,MAPSeq6 및 HPLC7과 같은 기술은 견고하고 상대적으로 비용이 저렴하여 많은 실험실이 작은 뇌 영역2,,4,,5,,6내에서 유도된 분자 변화를 연구할 수 있도록 합니다.
뇌 영역에서 핵산 또는 단백질을 추출하고 정제하는 방법에는 여러 가지가 있다8,,9,10,,911,,12. 많은 실험실은 수확9,,13의시간에 얼음에 뇌를 냉각하고 절단하여 뇌 영역을 수확 . 이러한 접근법은 고품질 의 핵산 및 단백질을 초래할 수 있지만, 조직의 미세 환경 내의 분해가 이들 온도에서 일어날 수 있기 때문에 다소 시간 제한적이다. 이것은 한 자리에 많은 수의 동물 이나 ROI를 해부 하려고 할 때 특히 사실 일 수 있습니다. 샘플을 동결상태로 유지하는 것은 비교적 순수한 샘플을 수집하기 위한 노력의 일환으로 각 섹션의 양쪽에 있는 랜드마크를 신중하게 비교할 수 있는 연구자의 시간을 제공하면서 비순한 표적 분자를 유지하는 데 도움이 됩니다. 레이저 포획은 뇌 영역에서 RNA 또는 단백질 분석을 위한 조직을 수집하는 또 다른 방법10. 이 절차는 매우 작고 불규칙한 모양의 ROI가 식별되고 분리될 수 있다는 점에서 기계적 해부보다 우수하다. 그러나, 레이저 포획은 고가의 장비 및 시약의 사용에 의해 제한되며, 시간이 많이 소요되며 또한 시료 열화에 더 취약할 수 있다.
냉동 된 조직에 마이크로 펀치 해부는 새로운 것이 아닙니다. 미클로스 팔코비트와 다른 사람에 의해 초기 논문은 세부사항 14,,15의기본 기술을 설명합니다 . 이 프레젠테이션은 주로 효율성을 용이하게하고 필요한 장비의 비용을 줄이기 위해 몇 가지 개선과 함께, 원래의 작업을 다음과 같습니다. 예를 들어, 뇌 섹션은 저온 저온 이기보다는 얼어 붙은 뇌 블록에서 만들어집니다. 이렇게 하면 ROI 샘플을 수집하는 데 필요한 섹션 수가 줄어듭니다. 이 방법은 또한 절연 된 상자 내에서 드라이 아이스에 앉아 냉동 유리 판에 샘플을 해부. 이것은 작동할 벤치에서 서브 동결 단계를 생성합니다. 이러한 방식으로 해부된 섹션은 쉽게 조작할 수 있으므로 연구원은 원하는 ROI 외부 영역의 오염을 제한하기 위해 각 섹션의 양쪽을 참조와 비교할 수 있습니다.
이 프로토콜의 장점은 1) 뇌가 과정 전반에 걸쳐 동결 된 상태로 유지되어 단백질과 핵산을 보존하고 연구원에게 ROI를 신중하게 수확할 시간을 주며 2) 필요한 시약이 저렴하고 대부분의 분자 생물학 실험실에서 발견된다는 것입니다. 이 과정에서 전체 뇌는 뇌 매트릭스에서 0.5-1.0 mm로 단면화되고 드라이 아이스로 지속적으로 냉각되는 냉동 유리 판에 놓입니다. 알렌 브레인 아틀라스에서 발견 된 랜드 마크1 또는 다른 뇌 지도판16,,17 관심 영역을 식별하는 데 사용됩니다, 다음 차가운 펀치 또는 메스중 하나를 사용하여 해부되는. 조직이 결코 해동되지 않았기 때문에, 이러한 방식으로 수확된 부위는 다운스트림 분석을 위한 고품질 RNA 및 단백질을 제공한다.

<table>
	<tbody>
		<tr>
			<td>0.5 mm Mouse coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 505C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>0.5 mm Rat coronal brain matrice</td>
			<td>Braintree Scientific</td>
			<td>BS-SS 705C</td>
			<td>Cutting block</td>
		</tr>
		<tr>
			<td>1.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-01</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Dot Scientific</td>
			<td>229443</td>
			<td>For storing frozen ROIs</td>
		</tr>
		<tr>
			<td>1.5 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-02</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mm Biopsy Punch with plunger</td>
			<td>Electron Microscopy Sciences</td>
			<td>69031-03</td>
			<td></td>
		</tr>
		<tr>
			<td>4-12% NuPage gel</td>
			<td>Invitrogen</td>
			<td>NPO323BOX</td>
			<td>protein gradient gel</td>
		</tr>
		<tr>
			<td>Bioanalyzer System</td>
			<td>Agilent</td>
			<td>2100</td>
			<td>RNA analysis system</td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>Millipore Sigma</td>
			<td> 
			D8938</td>
			<td>Glass tissue homogenizer</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Lite</td>
			<td>Dolan-Jenner Industries Inc.</td>
			<td>Model 180</td>
			<td>Cool lamp</td>
		</tr>
		<tr>
			<td>Glass plates</td>
			<td>LabRepCo</td>
			<td>11074010</td>
			<td></td>
		</tr>
		<tr>
			<td>HALT</td>
			<td>ThermoFisher</td>
			<td>78440</td>
			<td>protease inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Low profile blades</td>
			<td>Sakura Finetek USA Inc.</td>
			<td>4689</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-actin antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>JLA20</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Scientific</td>
			<td>2000C</td>
			<td>Used in initial RNA purity analysis</td>
		</tr>
		<tr>
			<td>No. 15 surgical blade</td>
			<td>Surgical Design Inc</td>
			<td>17467673</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking buffer</td>
			<td>LiCor Biosciences</td>
			<td>927-40000</td>
			<td>Western blocking reagent</td>
		</tr>
		<tr>
			<td>Omni Tissue Master 125</td>
			<td>VWR</td>
			<td>10046-866</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr>
			<td>rabbit anti-KCC2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>94725S</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>RNA Plus Micro Kit</td>
			<td>Qiagen</td>
			<td>73034</td>
			<td>Used to extract RNA from small tissue samples</td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Excelta Corp.</td>
			<td>16050103</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard razor blades</td>
			<td>American Line</td>
			<td>66-0362</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>15596026</td>
			<td>Used to extract RNA from tissue</td>
		</tr>
	</tbody>
</table>

collection of frozen rodent brain regions for downstream analyses

신경생물학에 대한 우리의 이해가 진행됨에 따라, 분자 분석은 종종 내측 전두엽 피질 (mPFC) 또는 핵 accumbens와 같은 작은 두뇌 지역에 실행됩니다. 이 작업의 과제는 검사할 미세 환경을 유지하면서 올바른 영역을 해부하는 것입니다. 이 백서에서는 대부분의 랩에서 쉽게 사용할 수 있는 리소스를 사용하는 간단하고 저렴한 방법을 설명합니다. 이 방법은 과정을 통해 동결 조직을 유지하여 핵산과 단백질을 보존합니다. 뇌는 뇌 매트릭스를 사용하여 0.5-1.0 mm 섹션으로 절단되고 냉동 유리 판에 배열됩니다. 각 섹션 내의 랜드마크는 알렌 마우스 브레인 아틀라스와 같은 참조와 비교되며, 지역은 차가운 메스 또는 생검 펀치를 사용하여 해부됩니다. 그런 다음 조직을 사용할 때까지 -80°C에서 보관합니다. 이 과정을 통해 쥐 및 마우스 mPFC, 핵 accumbens, 등쪽 및 복부 해마 및 그밖 지구는 qRT-PCR 및 서양 분석학을 사용하여 성공적으로 분석되었습니다. 이 방법은 명확한 랜드 마크에 의해 식별 될 수있는 뇌 영역으로 제한됩니다.

이 방법을 검증하기 위해, 내측 전두엽 피질을 성인 CD1 야생형 수컷 마우스 및 RNA로부터 수집하여 추출하고 특징화하였다. RNA는 모세관 전기동에 의해 분석되었다. 분해된 RNA는 28S 및 18S 리보좀 밴드의 강도에서 손실을 나타내고 또한 25 및 200 뉴클레오티드 사이의 얼룩으로서 분해 생성물을나타낸다(그림 5A,샘플 1). 고품질 RNA는 낮은 분자량 영역에서 신호가 거의 또는 전혀 ...

Watch this Scientific Journal Video about Collection of Frozen Rodent Brain Regions for Downstream Analyses at JoVE.com

Collection of Frozen Rodent Brain Regions for Downstream Analyses

이 절차는 저렴하고 일반적으로 사용할 수있는 도구를 사용하여 고품질의 단백질과 RNA를 얻기 위해 이산 냉동 뇌 영역의 수집을 설명합니다.

다운스트림 분석을 위한 냉동 설치류 뇌 영역 모음

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

이 절차는 저렴하고 일반적으로 이용 가능한 공구를 사용하여 고품질 단백질 과 RNA를 얻기 위하여 이산 냉동 두뇌 지구의 집합을 기술합니다. 뇌 영역은 해부를 통해 수확에서 얼어 붙은 유지되기 때문에, 표적 분자는 보존되고 연구원은 주의 깊게 해부하고 관심있는 지역을 저장하는 시간이있다. 관심 영역을 식별하는 것은 처음에는 어려울 수 있습니다.일반적인 뇌 랜드마크를 사용하여 나타나고 원하는 지역과 관련하여 섹션을 통해 후퇴하면 식별이 명확해집니다. 안락사 성인 CD-1 야생 형 마우스에서 뇌를 제거 한 후, 플래시는 액체 질소 또는 isopentane에서 60 초 동안 조직을 동결. 드라이 아이스로 미리 식히고 섭씨 영하 80도에 보관하십시오.조직을 해부하기 24 시간 전에 해동 된 냉동고 팩 더미에 깨끗한 뇌 매트릭스를 놓습니다. 그리고 면도기 슬롯의 바닥과 팩의 상단 사이에 약 반 센티미터를 남길 수 있도록 두 개의 냉동고 팩 사이에 매트릭스에 측면을 샌드위치. 상단에서 약 5센티미터의 얼음으로 절연 된 상자를 채우어 해부에 얼어 붙은 유리 판을 설정합니다.그런 다음 2.5센티미터의 드라이 아이스 층을 위에 놓습니다. 그리고 검은 색 플라스틱 시트로 덮습니다. 플라스틱 위에 유리 판을 놓습니다.그리고 접시의 테두리 주위에 드라이 아이스. 냉동고에서 냉동 뇌 매트릭스를 제거하고 뇌, 피질 측면을 매트릭스에 삽입합니다. 조직이 10 분 동안 상자의 온도에 평형되도록 합니다.이 시간 동안 뚜껑을 열어 두는 것입니다. 차가운 집게를 사용하여 매트릭스에서 뇌의 위치를 조정하여 시상 부비동과 횡방향 부비동이 블록의 수직 홈과 정렬되도록 합니다. 뇌가 제자리에 있으면 차가운 면도날을 중앙 근처에 놓고 약 1mm를 조직으로 누릅니다.다음으로, 뇌의 양쪽 끝에 차가운 칼날 하나를 놓고 매트릭스로 모든 방법을 누릅니다. 그런 다음 장밋빛 끝에 차가운 블레이드를 추가하여 한 번에 하나씩 슬롯에 넣고 1 밀리미터를 조직으로 부드럽게 누르기 시작합니다. 1밀리미터 간격으로 블레이드를 계속 추가하여 caudal 끝으로 작업합니다.모든 블레이드가 제자리에 있을 때 손가락, 손바닥 또는 무딘 물체로 위를 누르고 좌우로 천천히 흔들어 조직을 통해 이동합니다. 블레이드가 슬롯의 바닥에 도달하면 블레이드 그룹의 각 면을 잡고 앞뒤로 흔들어 행렬에서 해방하십시오. 블레이드 그룹을 풀어낸 후 유리 판에 장밋빛 면을 올려 놓고, 드라이 아이스를 스택 옆이나 위에 올려 놓고 시료를 더 쉽게 분리할 수 있습니다.그런 다음 날카로운 가장자리로 스택을 놓고 엄지와 손가락 사이의 스택을 이동하여 블레이드를 분리합니다. 유리 판의 단면을 로스트랄에서 caudal으로 정렬하고 손가락 사이를 구부리거나 두 번째 차가운 블레이드로 분리하여 블레이드에서 조직을 분리합니다. 때때로, 조직은 칼날의 양쪽에 충실할 수 있고 소달 오리엔테이션에 장밋을 유지하기 위하여 주의해야 합니다.해부를 시작하기 전에, 알렌 마우스 뇌 아틀라스 또는 다른 참조를 열고 관심 영역을 식별하는 데 필요한 랜드 마크를 찾을 수 있습니다. 차가운 집게 나 블레이드를 사용하여 섹션을 뒤집습니다. 또한 관심 영역이 섹션 전체에 걸쳐 일관성이 있는지 확인합니다.깨끗한 메스나 펀치로 단면으로 잘라내어 금속을 부드럽게 밀어 넣습니다. 컷을 만들기 위해 앞뒤로 흔들어. 관심 있는 지역을 수확한 후, 미리 냉각된 1.5mm 튜브에 넣고 섭씨 영하 80도에 보관하십시오.조직을 처리하려면, 단백질 추출을 위한 차가운 RIPA 버퍼 또는 RNA 추출을 위한 용매를 포함하는 guanidinium을 추가하고 유리 다운 또는 기계균질화로 즉시 균질화하십시오. 이러한 방법을 검증하기 위해, 내측 전두엽 피질은 성인 CD-1 야생형 남성 마우스로부터 수집되었고, RNA 및 단백질이 특징이었다. 모세관 전기포고증으로 분석될 때, 열화된 RNA는 28S와 18S 리보소말 대역의 강도에 있는 손실뿐만 아니라 25와 200뉴클레오티드 사이의 얼룩을 표시합니다.고품질 RNA는 낮은 분자량 영역에서 거의 또는 전혀 신호를 뚜렷한 리보소말 밴드를 보여줍니다. 냉동 해부 방법을 사용하여 얻은 RNA는 갓 수확된 조직에서 제조된 RNA와 비교하였다. 두 방법 모두 높은 무결성 수와 강한 리보소말 밴딩RNA를 생성합니다.이 방법이 나중에 분석을 위해 해부된 조직의 미세 환경을 보존하는 데 사용될 수 있는지 확인하기 위해 RNA는 몇 주 동안 저장되었던 해부된 내측 전두엽 피질로부터 추출되었다. 모든 샘플은 8개 이상의 뚜렷한 리보소말 밴딩 및 RNA 무결성 번호를 가진 고품질 RNA를 생산했습니다. 냉동 해부 시료의 단백질 무결성을 확인하기 위해 단백질을 수집하여 높은 분자량 표적 KCC2에 대한 항체를 가진 니트로셀룰로오스 및 프로브로 이송되었으며, 저분자 단백질 액틴을 하였다.모든 경우에 밴드는 눈에 띄는 고장 제품없이 날카롭고 뚜렷했다. 이 섹션의 미세 환경을 보존하고 뇌 아틀라스 이미지와 비교섹션 형태를 유지하기 때문에 프로세스 전반에 걸쳐 조직을 동결 유지하는 것이 중요합니다. 이러한 방식으로 수집된 관심 영역은 음의 80도에서 몇 달 동안 저장될 수 있으며 RT-qPCR, RNA-Seq, 서부 블롯 분석 및 HPLC를 포함한 많은 다운스트림 응용 분야에서 활용할 수 있습니다.이 방법은 THC 및 기타 카나비노이드에 노출 된 주산기 설치류의 변연 시스템 내에서 분자 변화를 탐구하는 데 사용되어 이러한 약물이 뇌 발달에 어떻게 영향을 미칠 수 있는지 더 잘 이해합니다.

collection-of-frozen-rodent-brain-regions-for-downstream-analyses

Research

JoVE Journal

Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

쥐 두뇌에 동시 fMRI와 전기 생리학

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

연구

신경과학

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

로던트에서 뉴런과 비 인간 메이트 뇌 조각의 DiOLISTIC 라벨링

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

총 RNA의 분리 및 다운 스트림 차세대 시퀀싱 및 유전자 발현 프로 파일링에 대한 개별 마우스 뇌 영역의 Microdissection 아닌 레이저 캡처 현미경 접근법

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

망막 섹션 Morphometric 분석

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

성인 쥐의 뇌의 기존 및 비 인습적 인 지역에서 신경 줄기 세포 성장

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

설치류 중추 신경계의 생체 Optogenetic 자극

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

청각 차별 학습 한 후 마우스 뇌 영역의 높은 해상도 양적 시냅틱 프로테옴 프로파일

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

쥐 두뇌에 있는 Microinjections에 대 한 개인화 된 바늘

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

외상 성 부상에 의해 영향을 하는 뇌 영역의 정위 적 아틀라스 유도 레이저 캡처 기정

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

시 냅 스 밀도 Hippocampal 설치류 두뇌 분할 영역에서의 평가

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...