우리는 건강 보조금 R01NS057476, P50NS010828, P01NS055104, R01EB000790, K99NS0670​​50, R01HL081273 및 R01EB007279와 미국 심장 협회 부여 0855772D 미국 국립 연구소의 지원을 인정하고 싶습니다.

1. 쥐 대뇌 피질의 vasculature에 pO2의보기 영상의 다양한 분야 <ol><li> 쥐 (250g -350 g)는 isoflurane과 함께 처음 anesthetized되며, 대퇴 동맥과 정맥은 심장 박동, 혈압 및 혈액 가스뿐만 아니라 정맥 주입을위한을 모니터 catheterized 있습니다. 체온은 ± 0.1 ° C.는 37 유지 기관 절개술이 수행되며, 쥐가 공기와 산소의 혼합물로 통풍이 있습니다. </li><li> 혈액 가스를 측정하기위한 혈액 샘플마다 30-45 분 촬영하고 환기와 마취는 정상적인 생리적 범위 내에서 혈액 가스를 유지하기 위해 조정됩니다. </li><li> 크기 정수리 뼈 4mm X 4mm의 휴일 두개골 창이 이미징을위한 준비가되어 있습니다. 뼈 및 두라 제거하고 두개골 창이 1.5 % 아가로 오스 가득한과 현미경 coverslip로 밀봉합니다. 이마 뼈에 추가 1mm이 버 구멍 KCl (~ 10 μl, 1 M)의 intracortical microinjection하여 CSD를 유도하는 데 사용됩니다. 익스 트림 치료는 대뇌 피질의 vasculature의 손상을 방지하기 위해 이동해야합니다. 초과 열, 기계적 압력, 또는 hypoxia의 짧은 기간은 중간 우주에 vasculature의 혈액 뇌 장벽과 염료의 원인 누설을 손상 수 있습니다. </li><li> 광학 불투명 소재의 마스크는 두개골 창의 주위에 배치됩니다. 마스크의 목적은 두라 사항의 가장자리에서 조직으로 유출 염색에서오고있다 인광 신호를 흡수하는 것입니다. 중간 공간에 염료는 측정을 망쳐놓 수있는 매우 밝은 인광의 원천입니다. 중간 공간에 색소의 증가 밝기가 낮은 산소 압력과 낮은 담금질 속도와 환경에서 염료의 구축의 결과입니다. 실험에 vasculature의 손상을 따라서 쉽게 실험 데이터가 거부되는 경우에는 밝고 인광 명소의 외관에 의해 인식됩니다. </li><li> 수술 완료 후 isoflurane이 중단되고 마취는 alphachloralose (50 MG / kg 정맥 볼러스 40 MG / (kg H) 주입에 의해 다음)으로 전환됩니다. </li><li> 동물은 환풍기, 혈압 및 온도 모니터 카트 전송됩니다. 실내 공기를 호흡​​하는 동안 동물은 빠르게 이미지 설정으로 이동 및 공기와 산소의 적절한 혼합으로 유량계에 reattached입니다. </li><li> 동물을 보유하고 stereotaxic 프레임은 목적에 따라 배치됩니다. 두개골 창이 목표 아래보기의 분야에서 중심과 초점 평면에 평행하게 위치합니다. </li><li> 펄스 레이저가 켜져 있는지 그리고 최소한의 펄스 에너지로 설정됩니다. 광학 빔은 10 개 이상의 MJ / cm 2이되고 조정됩니다 펄스 에너지 미터 및 샘플 전달 펄스 에너지쪽으로 이동합니다. ~ 60도 및 레이저 ISS의 경사 입사 각도와 두개골 창을 중심으로 빔 ISS가 비활성화. </li><li> 동물의 생리적 매개 변수가 정상적인 생리적 범위가 될 때까지 혈액 가스 측정 및 환기 및 마취의 조정이 수행됩니다. </li><li> 그 혈액 볼륨을 가정하면 인광 프로브 Oxyphor의 R3의 적절한 금액은 프로브 4 × 10 -5 M 혈액 농도를 달성하기위한 생리 1 ML에 용해되어 체중의 약 7 %입니다. 프로브 솔루션은 대퇴 정맥을 통해 주입합니다. </li><li> 염화칼륨 1 M 솔루션은 준비하고 ~ 1 UL은 CSD를 유도하기 위해 이마 뼈에 버 구멍 주사기로 주입됩니다. </li><li> 레이저가 켜져 및 이미징 즉시 KCl 용액의 주입 후에 시작됩니다. 인광의 이미징은 ~ 10 분 동안 수행됩니다. </li><li> 또 혈액 가스 측정 동물 생리학 매개 변수가 정상 범위 내에 아직도 확인이 수행됩니다. </li><li> 인광 수명은 Matlab의 통계 가중치와 비선형 평방 피팅을 사용하여 하나의 지수 부패를 피팅하여 모든 픽셀에 대해 얻을 수 있습니다. 인광 수명은 경험적 스턴 볼머 같은 관계 (보라 인프라)를 사용하여 pO2 값으로 변환됩니다. </li></ol> 2. 고해상도 두 광자 마우스의 대뇌 피질 마이크로 vasculature에 pO2 이미징 <ol><li> isofluorene과 대퇴 동맥은 심장 박동, 혈압 및 혈액 가스뿐만 아니라 염료의 관리를위한로 모니터 catheterized이다와 마우스는 anesthetized 있습니다. 체온이 37 유지 ± 0.1 ° C와 동물들이 자발적으로 공기와 산소의 혼합물을 호흡하고 있습니다. </li><li> 두개골 창이 원래 데이비드 Kleinfeld 및 Winfried Denk 개발한 기술에 따라 준비가되어 있습니다. 좋은주의 중간 공간으로 염색 가능한 누출을 방지하기 위해 vasculature의 손상을 방지하기 위해 이동해야합니다. </li><li> 혈액 가스를 측정하기위한 몇 가지 혈액 샘플은 모든 준비 절차 동안 촬영하고 환기 및 마취가 혈액 가스를 유지하기 위해 조정됩니다정상적인 생리적 범위 말이지. </li><li> 동물은 이미지 설정으로 이동됩니다. 동물을 보유하고 수정 stereotaxic 프레임은 목적에 따라 배치됩니다. 두개골 창이 올림푸스 4X 목표 아래보기의 분야에서 중심과 초점 평면에 평행하게 위치합니다. </li><li> 두개골 윈도우의 이미지가 eyepieces를 통해 디지털 카메라로 촬영한이며, 4X 목적은 올림푸스 20X 목표 (NA는 = 0.95)로 대체됩니다. </li><li> 동물의 생리적 매개 변수가 정상적인 생리적 범위까지 혈액 가스 측정 및 환기 및 마취의 조정이 수행됩니다. </li><li> 그 혈액 볼륨을 가정하면 체중의 약 7 %, 인광 프로브 PTP - C343의 적절한 금액 탐사선 ~ 15 μm의 혈액 농도를 달성하기 위해 호수의 0.2 ML에 녹아입니다. 프로브 솔루션은 대퇴 동맥을 통해 주입합니다. </li><li> 각 픽셀에 염료 여기 및 방출 수집 원하는 시간 간격을 설정하여 실험을 시작합니다. </li><li> 현재 깊이에서 vasculature 구조를 표시 인광 강도 부패의 속도가 느린 2 차원 래스터 스캔하는 조사 검사를 수집. 목적은 두개골 창에 수직 이동 (Z 축)과 인광 강도의 느린 2D 조사 스캔은 이미지 비행기에서 microvessels를 찾을 수 840 nm의에서 최소한의 레이저 파워를 사용하여 촬영하고 있습니다. </li><li> 그 깊이에 인광의 조사 검사를 기반으로 각각의 이미지 깊이에서, vasculature 이내에 포인트의 집합이 선택되며, 각각의 시점에서 인광 자연 붕괴의 기록은 평균의 미리 정의된 번호를 반복됩니다. </li><li> 원하는 평균 금액, 측정 간격, 그리고 실험 기간을 설정하고 측정을 시작합니다. PO 2 측정은 실험 기간 동안 지정된 측정 간격 선택한 위치에서 찾았습니다. </li><li> 측정하는 동안, 소프트웨어는 검류계 검사 거울의 위치를​​ 변경하여 선택한 위치에 여기 레이저를 다시 연결해줍니다. 검류계 스캐너, 전자 광학 변조기 및 기타 모든 장비의 제어는 LabView에서 맞춤 제작한 소프트웨어에 의해 수행됩니다. </li><li> 인광 수명은 Matlab의 비선형 평방 피팅을 사용하여 하나의 지수 부패를 피팅하여 선택한 모든 지점에 대해 얻을 수 있습니다. 인광 수명은 경험적 스턴 볼머 같은 관계 (보라 인프라)를 사용하여 pO2 값으로 변환됩니다. </li><li> 다양한 피질 깊이에서 데이터를 수집 후, 영상 microvasculature 구조 vasculature에 dextran - 복합 플루오레신 염료를 주입. </li><li> 4 채널 검출기에 녹색 채널을 사용하는 FITC 형광 반응의 두 광자 이미징을 수행하여 vasculature의 구조 이미지의 스택을 얻습니다. </li><li> 동물 실험 연구 애니멀 케어에 매사 추세츠 종합 병원 분과에 의해 명시된 지침과 규정에 따라 수행되었다. </li></ol> 3. 대표 결과 : <img src="/files/ftp_upload/1694/1694fig1.jpg" alt="그림 1" /> 이 그림의 왼쪽에있는 그림 1. 패널 (A)는 CSD 파의 도착 전에 산소 압력보기 이미지의 다양한 필드를 표시합니다. 오른쪽에있는 패널 (B)는 패널에 표시된 관심있는 지역 내의 CSD 전파 동안 평균 산소 압력의 시간적 진화 (A)를 보여줍니다. 그림 2 (AVI 동영상) :이 영화는 CSD 파의 전파하는 동안 전체 두개골 윈도우의 산소 압력의 시간적 변화를 보여줍니다. 스케일 바 수은 밀리미터 산소 압력을 나타냅니다. <img src="/files/ftp_upload/1694/1694fig3.jpg" alt="그림 3" /> 그림 3. 몇 군데 vasculature 스택의 3D 투영. 회색의 음영은 판매량 선박 마스크를 나타내는 구조 이미지를 기반으로 만들었습니다. 측정 PO 2 값을 색으로 표시됩니다. 스케일 바는 200 micrometers입니다. 자연 출판 그룹의 허가 Reprinted 7.

<ol>
	<li>Kleinfeld, D., Friedman, B., Lyden, P. D., Shih, A. Y., Chen, J., Xu, Z., Xu, X., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+occlusion+to+surface+and+deep+vessels+in+neocortex+via+linear+and+nonlinear+optical+absorption,+Animal+Models+of+Acute+Neurological+Injuries.">Targeted occlusion to surface and deep vessels in neocortex via linear and nonlinear optical absorption, Animal Models of Acute Neurological Injuries.</a> Contemporary Neuroscience Series. , (2007).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J Vis Exp. , (2008).</li><li>Lebedev, A. Y., Cheprakov, A. V., Sakadzic, S., Boas, D. A., Wilson, D. F., Vinogradov, S. A., A, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Phosphorescent+Probes+for+Oxygen+Imaging+in+Biological+Systems.">Dendritic Phosphorescent Probes for Oxygen Imaging in Biological Systems.</a> Applied Materials &#38; Interfaces. , (2009).</li><li>Finikova, O. S., Lebedev, A. Y., Aprelev, A., Troxler, T., Gao, F., Garnacho, C., Muro, S., Hochstrasser, R. M., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+microscopy+by+two-photon-excited+phosphorescence.">Oxygen microscopy by two-photon-excited phosphorescence.</a> Chemphyschem. 9, 1673-1679 (2008).</li><li>Sakad&#382;i&#263;, S., Yuan, S., Dilekoz, E., Ruvinskaya, S., Vinogradov, S. A., Ayata, C., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+cerebral+partial+pressure+of+oxygen+and+blood+flow+during+functional+activation+and+cortical+spreading+depression.">Simultaneous imaging of cerebral partial pressure of oxygen and blood flow during functional activation and cortical spreading depression.</a> Appl Opt. 48, D169-D177 (2009).</li><li>Yaseen, M. A., Srinivasan, V. J., Sakadzic, S., Wu, W., Ruvinskaya, S., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+monitoring+of+oxygen+tension+in+cortical+microvessels+with+confocal+microscopy.">Optical monitoring of oxygen tension in cortical microvessels with confocal microscopy.</a> Opt Express. 17, 22341-22350 (2009).</li><li>Sakadzic, S., Roussakis, E., Yaseen, M. A., Mandeville, E. T., Srinivasan, V. J., Arai, K., Ruvinskaya, S., Devor, A., Lo, E. H., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+high-resolution+measurement+of+partial+pressure+of+oxygen+in+cerebral+vasculature+and+tissue.">Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.</a> Nat Methods. 7, 755-759 (2010).</li></ol>

우리는 인광의 산소에 의존 담금질에 따라 대뇌 피질의 microvasculature의 pO​​2 측정의 두 어플 리케이션을 시연했다. CCD 이미징에 따라 첫 번째 방법은 2 광자 현미경을 기반으로 대뇌 피질의 microvasculature에서 산소의 부분 압력을 측정, pO2의 전망 모니터링의 다양한 분야를 제공하고 있지만 모세 해상도를 제공하고 깊이 이미징 수 있습니다. 두 가지 방법은 높은 속도와 높은 신호 대 잡음 측정을 제공합니다. 또한, pO2의 인광 수명 측정은 일반적으로 강도에 따라 대비 메커니즘을 가지고 다른 광학 이미징 기술에 대한 우려는 실험 기간 동안 조직의 광학 매개 변수의 변화에​​ 크게 구분하지 않습니다. 제시 악기 양적 산소의 동적 전달의 분석 및 정상과 질병 두뇌에 neurovascular 커플링의 더 나은 이해로 이어질 것입니다 뇌 조직 신진 대사를 허용

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<td>Glycopyrrolate</td>
<td>Reagent</td>
<td>American Regent Inc.</td>
<td>NDC 0517-4605-25</td>
<td>Used to control pharyngeal, tracheal, and bronchial secretions.</td>
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<tr>
<td>Lidocaine HCL</td>
<td>Reagent</td>
<td>Hospira Inc.</td>
<td>NDC 0409-4277-01</td>
<td>Used as the local anesthesia during surgeries.</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>Reagent</td>
<td>Baxter Healthcare Corp.</td>
<td>NDC 10019-360-40</td>
<td>Used as a general inhalation anesthetic drug during surgeries and as a general anesthesia during experiments with mice.</td>
</tr>
<tr>
<td>Alpha Chloralose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>C0128</td>
<td>Used as a general anesthesia during experiments with rats.</td>
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<tr>
<td>Fluorescein isothio-cyanate&#x2013;dextran</td>
<td>Reagent</td>
<td>Sigma</td>
<td>FD2000S</td>
<td>Administrated to create ~ 500 nM concentration in blood.</td>
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cerebral blood oxygenation measurement based on oxygen-dependent quenching of phosphorescence

대뇌 혈액과 조직 산소의 spatiotemporal 특성의 모니터링은 신경 신진 대사 - 혈관 관계의 이해를 위해 매우 중요합니다. 높은 공간 및 / 또는 시간적 해상도로보기 큰 분야의 pO2 동시에 모니터링 새로운 pO2 측정 modalities의 개발은 정상적인 두뇌의 기능에 더 큰 통찰력을 사용하고 같은 neurovascular 질병의 진단과 치료에 상당한 영향을 미칠 것이다 또한 뇌졸중, 알츠하이머 병, 그리고 머리 부상. 광학 이미징 modalities는 광 스펙트럼의 가시 성과 가까운 적외선 범위에서 헤모글로빈의 흡수를 기반으로 높은 해상도와 spatiotemporal pO2의 양적 이미징을 제공하기 위해 큰 잠재력을 보여주었다. 그러나 대뇌 혈액 산소의 multispectral 측정 높은 산란 뇌 조직을 통해 광자 마이 그 레이션에 의존합니다. 견적 및 실험 도중 역동적인 변화를 받아야 수 조직 광학 매개 변수의 모델링은 일반적으로 혈액 산소의 정확한 추정이 필요합니다. 한편, 인광의 산소에 의존 담금질에 따라 산소의 부분 압력 (pO2)의 평가는 크게 조직의 광학 매개 변수의 변화에​​ 의해 영향을 pO2의 절대 측정을 제공해서는 안됩니다. 산소에 민감한 염료를 사용하는 실험 시스템은 물론 생리 pO2 범위에서 정확한 산소 이미징 수있는 강력한 기술입니다 인광 담금질을 보여주는, 조직 문화의 산소 내용을 모니터링으로 perfused 조직의 생체내 연구에서 입증되었습니다. 여기 우리는 어떻게 인광 수명 이미징에 따라 대뇌 피질의 vasculature에 pO2 측정을 수행하는 두 개의 서로 다른 이미징 modalities과 함께 보여줍니다. 첫번째 예제에서 우리는 암흑의 대뇌 피질의 표면에 pO2의보기 영상의 다양한 분야를 제시한다. 이 영상 양상은 CCD 카메라와 레이저 펄스 녹색을 바탕으로 상대적으로 간단한 실험 설정을했습니다. Oxyphor의 R3의 염료 인광 수명에 따라 대뇌 피질의 확산 우울증을 모니터링의 예를 제시했다. 두번째 예제에서 우리는 고해상도 두 광자 마우스의 대뇌 피질 마이크로 vasculature에 pO2 이미징을 제시한다. 실험 설치 femtosecond 레이저, 전자 광학 변조기 및 광자 - 계산 사진 배율 튜브와 사용자 지정 구축 2 광자 현미경을 포함합니다. 우리는 향상된와 PTP - C343 염료 소설 2 광자 여기 크로스 섹션을 사용하여 영상의 예를 들어 모세 혈관을 포함하여 대뇌 피질의 microvasculature에 pO2 이질을 제시. 관련 기사 보려면 여기를 클릭하십시오 <a href="https://www-jove-com.remotexs.ntu.edu.sg/Details.php?ID=1731">생물 학적 시스템에서 산소 이미징에 대한 인광을 발하는 나노 프로브의 합성 및 교정합니다.</a>

Watch this Scientific Journal Video about Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence at JoVE.com

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

우리는 인광의 산소에 의존 담금질에 따라 대뇌 vasculature에서 산소의 부분 압력 (pO2)을 측정하는 실험 절차를 제시한다. 동물 준비 및 이미징 절차 쥐의 pO2 볼 때 CCD 기반 영상의 넓은 분야와 생쥐의 pO2의 2 광자 여기 기반 이미징 모두에게 설명했다.

인광의 산소에 의존 담금질을 바탕으로 대뇌 혈액 산소 측정

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

cerebral-blood-oxygenation-measurement-based-on-oxygen-dependent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.6K 조회수.  Massachusetts General Hospital and Harvard Medical School. 우리는 인광의 산소에 의존 담금질에 따라 대뇌 vasculature에서 산소의 부분 압력 (pO2)을 측정하는 실험 절차를 제시한다. 동물 준비 및 이미징 절차 쥐의 pO2 볼 때 CCD 기반 영상의 넓은 분야와 생쥐의 pO2의 2 광자 여기 기반 이미징 모두에게 설명했다.

비디오: Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

쥐 뇌 조각에서 뉴런의 Vibrodissociation는 시냅틱 전송 및 이미지 Presynaptic 터미널 공부

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

연구

신경과학

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

급성 Hippocampal 슬라이스에 Synaptically-evoked Astroglial와 Neuronal 응답의 듀얼 Electrophysiological 레코딩

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

의 라이브 영상<em&gt; 초파리</em&gt; 애벌레 Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

분리 및 대뇌 실질 세동맥의 캐 뉼러

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Microcoil 협와 가벼운 자궁내 Hypoperfusion의 쥐 모델

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

신선한 인간의 뇌 조직에서 뇌 모세 혈관의 절연

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

생체 내 뇌 단백질 합성의 지역 비율 측정을 위한 정량적 자가 방사선 측정 방법

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

가압된 설치류에 폭발을 이용한 Neurotrauma의 효과 중간 대뇌 동맥

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

통증과 가려움관련 행동을 동시에 측정하기 위한 뺨 주입 모델

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...