우리는 동영상 촬영 준비를 구성하는 그녀의 뛰어난 기술 지원 및 마헤 쿠마르 Teli에 대한 브리타 Kaltwasser 감사드립니다.

1. 동물 <ol><li> 실험은 실험실 동물의 보호 및 사용에 대한 NIH 가이드 라인에 따라 실시하고 지방 자치 단체에 의해 승인되었습니다. 모든 실험 C57Bl6 / J 야생 타입 마우스, ApolipoproteinE - / - (ApoE KO) 및 (12-16주 오래된 26~30그램의 체중, 실험 집단 당 5-6 동물) SV129 마우스가 사용되었습니다. </li></ol> 2. 컬러 라텍스와 뇌 혈관의 얼룩 <ol><li> EP 관 1시 20분 비율에 라텍스 화합물의 0.5 ML (Pebeo, 프랑스)와 25 μl 카본 블랙 잉크 (Herlitz, 독일)의 혼합물을 준비하고 물을 욕조에 37 ° C에서 혼합을 따뜻하게. 동물을 anaesthetizing 전에 28 30g의의 바늘로 2 ML의 주사기에 혼합를 수집합니다. </li><li> 멸균 정상적인 생리의 1 ML에 papavarine 염산염 분말의 50 MG를 해산. 인슐린 주사기의 솔루션을 수집합니다. 다른 멸균 인슐린 주사기에서 바늘을 중지. 20 C의 끝에이 바늘을 삽입합니다m 길이 PE10 튜브. 지금 vasodilatation과 혈관의 적절한 충전을 보장하기 위해 넙 다리 정맥을 통해 주입 할 수 papavarine 하이드로 클로라이드 솔루션을 포함하는 인슐린 주사기의 바늘에이 PE10 튜브를 연결합니다. </li><li> 또 다른 두명의 인슐린 주사기에게 생리의 각 함유 한 ML을 준비합니다. 인슐린 주사기는 정확한 위치에 주입 유체의 원활한 전송을 할 수 있습니다. 그러나, 라텍스, 대체 이동식 넓은 바늘 1 ML 주사기의 높은 점도로 인해 사용해야합니다. </li><li> 운영 단계에 가까운 모든 주사기와 살균 해부 악기를보세요. 운영 단계 주위에 수술 포목상 시트를 확산. 작업 단계는 충분히 조명해야하고 필요한 경우 운영 현미경을 사용할 수 있습니다. </li><li> 이제 C57Bl6 / J 마우스의 체중을 측정 한 후 5%이 70 % N 2 O 30 % O 2의 isoflurane 기화로 마취를 유도. 기화 isoflurane의 1 %로 마취를 계속합니다. 적절한 positionin 후사지의 그 뒤로 고정에 마우스 g, 마취의 깊이를 신고 할 수 있습니다. 그런 다음 왼쪽 넙 다리 정맥을 통해 피부를 잘라 정맥을 찾습니다. PE10 튜브에 삽입 바늘의 날카로운 끝을 넣고 천천히 분 당 100 μl (50 밀리그램 / kg 체중)의 속도로 papavarine 하이드로 클로라이드 솔루션을 주입. </li><li> 분사 후, 복강과 관통 가로막을 잘라. 을 손상하거나 멍이없이 마음을 노출 갈비뼈를 잘라. 동맥 포셉으로 내림차순 흉부 대동맥을 클립. 정맥 피가 사라져 있도록 오른쪽 심방을 통해 날카로운 상처를합니다. 18-20 초 이상 약간의 압력을 수동으로 색 라텍스를 주입 한 다음, 왼쪽 심실에 생리 2 ML를 삽입. 미리 채워진 주사기 다른 후 하나를 사용하십시오. 모든 거품을 주입하지 마십시오. </li><li> 라텍스의 주입 후, 5 분의 동물을 두십시오. 동물 목을 벨 조심스럽게 전체 뇌를 제거합니다. 선박 건축과 corte을 해치다하지를 잘 가져가X 두개골 뼈와 meninges를 제​​거하는 동안. 사진을 가져 4% PFA 6-8 ML을 포함하는 60mm 세포 배양 접시의 뇌를 놓습니다. 투명한 접시 아래에 측정 통치자를 놓습니다. 운영 현미경에 부착 된 카메라로 등쪽와 뇌의 복부 표면의 25x 확대 사진을 찍습니다. 사진은 원래 거리를 수량화하기 위해 페트리 접시와 현미경 대물 사이에 90 ° 각도로 촬영해야합니다. </li><li> 또 다른 4-5 동물의 절차를 반복합니다. </li></ol> 3. 카본 블랙 잉크의 혼합과 뇌 혈관의 얼룩 <ol><li> 우리 프로토콜 라텍스 기반의 대뇌 혈관 착색의 결과를 비교하려면, 1.5 ML EP 관 펠리칸 Scribtol Schwarz 잉크 (CB2) 900 μl를 Herlitz Stempelfarbe 잉크 (CB1) 100 μl의 혼합물을 준비하고 있습니다. 1시 9분 비율 CB1과 CB2 2 ML 혼합물의 총 볼륨을 두 EP 튜브를 준비합니다. 두 개의 인슐린 주사기 (1 혼합물을 수집ML 각). 37 ° C.까지 따뜻하게 주사기를 모자하고 물 욕조에 넣어 또 다른 두명의 인슐린 주사기에 생리 2 ML를 수집하고,뿐만 아니라 물 목욕에 배치합니다. </li><li> 동물을 마취하고 papavarine 하이드로 클로라이드의 주입을 제외하고 위에서 설명한 것과 같은 절차를 반복합니다. 왼쪽 심실에 두 ML salaine의 주입 후, 카본 블랙 잉크 대신 색 라텍스의 2 ML 혼합물을 주입. ML 당 18-20 초 이상 약간의 압력 손으로 주사를 수행합니다. 주사하기 전에 내림차순 흉부 대동맥을 클립. </li><li> 동물을 희생 뇌를 제거하고 사진을 찍어. </li><li> 부동 뇌는 완전히 submerges 때까지 뇌의 장기 보존의 경우 (5 % 15 % 다음 다음 30 %) 점차 증가 농도 자당과 부화에 의해 PFA는 하루 아침에 다음 4퍼센트에 뇌를 넣어. 색 라텍스와 perfused 동물의 두뇌는 같은 방법으로 보존 할 수 있습니다. </li><li> 을 수행또 다른 4-5 동물의 절차. 다른 유전 배경이나 변종으로 인한 뇌성 혈관 해부학의 차이를 비교하려면 각각 ApoE KO와 SV129 마우스의 동등한 수의 프로토콜을 반복합니다. </li><li> 깊은 마취 (수술에 대한 자세한 설명은 본 문서의 범위를 넘어 아래에있는 MCA의 intraluminal 가림의 표준 프로토콜에 따라 45 분 또는 90 분에 과도 초점 뇌성 국소 빈혈을 유발, 허혈성 조건 하에서 혈관 해부학을 공부한다 리더는 Doeppner 외., 2010)라고합니다. 경색 볼륨을 식별 할 수 reperfusion 기간 (1 일 ~ 5 일) 계획의 끝에서 CB1 + CB2 잉크 만 사용하여 혈관 착색을 수행하고 5-10 분 동안 37 ° C에서 2% TTC 솔루션을 가지고있는 전체의 뇌를 더럽 히다 . TTC는 mitochondrial 효소 (특히 호박산 탈수소 효소)에 의해 붉은 색의 formazan으로 감소된다. TTC의 착색 후, infarcted 조직 unstai는 붉은 색의 metabolically 활성 조직 얼룩 남아있는 동안네드는와 때문에 역기능과 변성 mitochondrial 효소에 흰색 나타납니다. 위에서 설명한 것과 같은 방식으로 이미지를 수집합니다. </li></ol> 4. 뇌 혈관 지역의 연구 <ol><li> 대뇌 혈관, + CB2 잉크가 ImageJ 소프트웨어 (영상 처리 및 분석에 사용되는 자바 기반의 공개 소프트웨어)를 분석 할 수 색 라텍스 또는 CB1과 뇌 중 스테인드의 등 표면의 이미지의 총 해부학을 분석 할 수 있습니다. CB1 + CB2 살포가 복부와 등쪽 표면 모두에있는 모든 선박을 착색 반면 색 라텍스와 perfused 뇌는, 등쪽 표면에있는 선박 착색의 변수도 표시 될 수 있습니다. </li><li> ImageJ 소프트웨어와 이미지 파일을 엽니 다. ACA와 MCA 사이 anastomotic 점을 확인합니다. anastomotic 포인트는 혈관 직경이 좁은 또는 각각 ACA의 가장 가까운 분기 포인트와 MCA 지점 사이의 반 거리, 2 사이트로 정의됩니다. <s까지&gt; 분할 줄 도구를 그리기의 도움과 플러그인 &quot;점선&quot;로 anastomotic 점을 표시하여 가상의 선 (anastomotic 선)을 그립니다. </li><li> mm의 규모를 설정합니다. anastomotic 라인과 직선 그리기 도구 및 &quot;측정&quot;도구를 사용하여 뇌의 전두엽 극에 꼬리 4mm의 중간 선 사이의 거리를 측정합니다. caudally 정면 극에서 6mm에서 동일한 작업을 수행. 동물 당 3-4 이미지를 분석하고 평균 값을 계산합니다. 뇌 혈관 해부학의 변화를 확인하기 위해 동물의 서로 다른 그룹 사이의 값을 비교합니다. C57Bl6 / J 동물에서 ACA와 MCA 사이의 anastomotic 라인은 caudally 정면 극에서 6mm에서보다 4mm에 가까운 중간 선을 추적 할 수 있습니다. ApoE KO 마우스는이 점에 큰 차이를 제공하지 않습니다. 한편, SV129 마우스에 anastomotic 라인은 정면 극에서 caudally 추가 및 4mm의 중간 선 모두 병렬 및 6mm 거짓말을합니다. </li><li> 혈관 해부학을 분석해서N 허혈성 조건은 위에서 언급 한 동일한 계산을 수행합니다. 각각 metabolically 활성 조직 및 괴사 흠없는 조직을 대표하는 빨간색과 흰색 컬러 조직 마진로 경색 테두리를 식별합니다. 4mm에서와 뇌의 전두엽 극에서 꼬리 6mm의 중간 선에서 경색 테두리의 거리를 측정합니다. 동물 당 3-4 이미지에서 평균 값을 수집합니다. 다른 국소 빈혈 reperfusion 및 기간 사이의 결과를 비교합니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

이 특정 압력 2,3을 의미 할 특정 장치를 포함하지 않으므로 수동으로 분사하여 CB1 + CB2의 재관류는 집중 훈련없이 성공적으로 수행 할 수 있습니다. 우리 프로토콜의 재관류의 결과의 이질성도 무시할 수 있습니다. 20 허혈성 동물의 아웃 만 1 16 비 허혈성 동물에서 동물과 3가 완전 재관류을 보여 왔습니다. 이러한 경우에 혈관 폐색으로 이어지는 생리 재관류 동안 거품의 설립은 실패한 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 명 </td><td> 카탈로그 번호 </td></tr><tr><td> Scribtol Schwarz (CB2) </td><td> 펠리칸, 독일 </td><td> 221 135 </td></tr><tr><td> Stempelfarbe (CB1) </td><td> Herlitz PBS AG, 독일 </td><td> 10417202 </td></tr><tr><td> Gedeo 라텍스 </td><td> Pebeo, 프랑스 </td><td> 13042B </td></tr></tbody></table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

대뇌 혈관의 해부학 적 구조는 뇌 혈류 역학의 주요 결정자뿐만 아니라 허혈성 모욕에 따라 부상의 심각성입니다. 대뇌 vasculature 동적으로 다양한 pathophysiological 상태에 응답하며 긴장 사이 유전자 조작 조건에 따라 상당한 차이를 전시하고 있습니다. 기본적으로 intracranial 선박 착색에 대한 신뢰할 수있는 기술은 허혈성 뇌졸중의 pathogenesis을 연구하기 위해 필수적입니다. 최근까지 다양한 기술의 집합은 낮은 점도의 수지, araldite F의 주입을 포함하여 대뇌 vasculature, 젤라틴 2 또는 검은 색 3 탄소없는 다양한 염료 1 (즉, 카민 붉은 색, 인도 잉크) 또는 라텍스과 혼합.를 시각적으로 고용되었습니다 오름 대동맥을 통해 흰색 라텍스 화합물의 재관류가 먼저 코일과 Jokelainen 3보고되었습니다. 마에다 외. 2 carbo을 추가하여 프로토콜을 수정 한뇌의 생리 재관류 후 혈관의 개선 대비 시각화를위한 라텍스 화합물로 N 검은 색 잉크. 그러나, 비효율적 인 재관류와 혈관의 불충분 한 충전이 자주 라텍스 화합물 4의 높은 점도로 인해 발생합니다. 따라서, 우리는 재현 방식으로 5 대뇌 vasculature을 시각화 두 상업적으로 이용 가능한 카본 블랙 잉크 (CB1과 CB2)의 혼합물을 사용하여 간단하고 비용 효율적인 기술을 설명하고 있습니다. 우리는 라텍스 재관류 5 비교에서 더 높은 밀도에 훨씬 작 뇌성 선박의 얼룩에 마우스 결과에 CB1 + CB2와 그 세례를 보여 주었다. 여기, 우리는 앞 (ACA)와 다른 유전 적 배경을 가진 쥐 선박 변화를 공부하는 중 뇌동맥 (MCA) 사이 anastomotic 점을 식별하기위한 프로토콜을 설명합니다. 마지막으로, 우리는 CB1를 조합하여 마우스의 과도 초점 대뇌 국소 빈혈 모델에서의 기술의 가능성을 보여줍니다 +허혈성 부상의 다양한 각도 TTC의 착색과 CB2로 인한 선박 착색.

여기에 설명 된 프로토콜 쥐 대뇌 vasculature의 기존의 라텍스 기반의 시각화의 기술 한계를 극복. 그림 1A는 색 라텍스의 다음과 같은 재관류를 보여줍니다, 복부 표면에 만 대형 선박은 전체 등쪽 표면이 흠없는 떠나는, 스테인드 있습니다. 결과는 매우 변수입니다. 뇌 (데이터가 표시되지 않음)의 등 표면에 표시 ACA 및 MCA의 부분적인 착색 여섯번 쇼 하나 밖에 동물. 반대로, CB1 + 같은 ?...

Watch this Scientific Journal Video about Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels at JoVE.com

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

쥐 뇌 혈관 해부학 분석 실험 뇌졸중 연구에 중요한 역할을한다. 이러한 맥락에서, 색 라텍스와 혈관 재관류는 몇 년을위한 표준 도구로 간주되었습니다. 그러나,이 기술은 재현성을 저해 별개의 기술적 한계를 의미합니다. 여기, 우리는 재현 방식으로 뇌성 혈관을 시각화 할 수있는 간단한 방법을 설명합니다. 고 대비 시각화와 뇌성 혈관의 충분한 충전의 왼쪽 심실 심근 결과를 통해이 상업적으로 이용 가능한 카본 블랙 잉크 혼합물의 주입. 우리는 성공적으로 다른 유전 적 배경을 가진 쥐 대뇌 혈관 지역 사이 anastomotic 포인트를 확인하기 위해이 기술을 적용했습니다. 쥐 경색 볼륨을 관찰하고 분석 할 수있는 널리 사용되는 도구 - 우리는 마침내 우리 선박 착색이 소설과 간단한 방법은 triphenyltetrazolium 염화 (TTC) 염색과 결합 할 수 있다는 증거를 제공합니다.

카본 블랙 잉크의 혈관 재관류은 뇌 혈관의 신뢰성 시각화 수 있습니다

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

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Research

JoVE Journal

Neuroscience

15.3K 조회수.  University of Duisburg-Essen Medical School. 쥐 뇌 혈관 해부학 분석 실험 뇌졸중 연구에 중요한 역할을한다. 이러한 맥락에서, 색 라텍스와 혈관 재관류는 몇 년을위한 표준 도구로 간주되었습니다. 그러나,이 기술은 재현성을 저해 별개의 기술적 한계를 의미합니다. 여기, 우리는 재현 방식으로 뇌성 혈관을 시각화 할 수있는 간단한 방법을 설명합니다. 고 대비 시각화와 뇌성 혈관의 충분한 충전의 왼?...

비디오: Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

쥐 중간 대뇌 동맥의 폐색의 Endovascular 봉합사에 의해 초점 대뇌 국소 빈혈 모델

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

연구

신경과학

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

동물 커뮤니케이션 연구를위한 자동화된 대화형 비디오 재생

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

사용하는 마우스 대뇌 피질과 해마의 시각화 및 Dendrites와 쪽이의 유전자 조작<em&gt; utero에서</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

신생아 설치류에서 분리 뇌간 - 척수 준비에 전기 생리학은 신경 호흡 네트워크 출력 녹화를 할 수 있습니다

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

마우스 뇌 혈관의 정화

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

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 ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

신생아 뇌에 바이러스 성 입자와 형광 마이크로 비즈의 뇌실과 혈관 내 주입

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

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 ...