저자는이 방법을 개발에서의 의견 도움 및 기술 지원을위한 로라 디아즈, 케빈 필립스, 윌라 A. Hsueh, 그리고 왕 C. 리를 감사드립니다.

1. 동물 준비 및 감기 치료 <ol><li> 실험실 쥐를 수용하기위한 승인 된 4 ° C 감기에 방을 찾아 검사한다. </li><li> 추운 방에서 하룻밤 사전 냉기 동물 케이지. 케이지는 피드와 침구없이 물이 병으로 조립된다. </li><li> 실험 하루 아침에, 장소 마우스 30 분 간격으로 미리 차가운 케이지의 각에 하나씩. 이 영상 실험실로 이송되기 전에 각 단독 갇힌 마우스는 거의 4 시간의 추운 방에서 숙박해야합니다. 쥐가 물이에 액세스 할 수 있도록 금식. </li><li> 이미징 랩에 한 번에 4 시간 게시물 감기 치료 운송에 하나의 동물은 매일 30 분. 이것은 얼음는 스티로폼 컨테이너를 입력하고 상자 안에 얼음 위에 미리 차가운 주택 케이지를 삽입함으로써 달성 될 수있다. 대충 스티로폼 상자에 덮개를 놓습니다. </li></ol> 2. 설치 Micro-PET/CT 이미징 워크 플로우 <p class=이 프로토콜 micro-PET/CT 영상에서 &quot;jove_content&quot;&gt;는 지멘스 Inveon 전용 PET (dPET) 시스템 및 Inveon의 Multimodality (MM) 고정 모드에서 시스템 (중부 표준시 / SPECT)로 달성된다. 동물이 처음 해부학 적 참조에 대한 컴퓨터 단층 촬영기로 스캔 MM 입구에서 배치 한 다음 정적 F18 PET 인수에 대한 dPET의 중심에 하락하고 있습니다. 호스트 컴퓨터가 자동으로 연속 작업을 수행 할 수 있도록하기 위해, 다음과 같은 &quot;워크 플로우&quot;가 전에 실제 이미지 세션에 Inveon 수집 직장 (법에) 소프트웨어 프로그램입니다. <ol><li> 중부 표준시 취득 : 몸 전체 CT 촬영의 경우는, 500에서 현재 UA, 80kV, 200 밀리 초에서 노출 시간 및 240 ° 회전 240 단계에서 전압을 설정합니다. X-선 검출기를 들어, 78mm 축 이미징 분야와 싱글 침대 모드로 &quot;낮은 시스템 배율&quot;에서 해상도를 선택합니다. 그 때문에 &#39;일반 콘 - 빔 재건 &quot;방법을 사용하여&quot;실시간 복구 &quot;를 선택으로 호스트 PC 회담작업을 시작하기위한 전용 실시간 재건 컴퓨터 (코브라). </li><li> PET 방출 취득 : &quot;시간에 의해 취득&quot;옵션에서 &quot;고정 스캔 시간&quot;에 대한 설정 600 초 (10 분). &quot;연구 동위 원소&quot;로 F-18를 선택하고 &quot;에너지 수준&quot;이라고 350-650 케빈를 사용합니다. </li><li> PET 방출 히스토그램 : 정적 검사를 달성 할 수있는 전체 기간에 대해 하나의 프레임으로 프로세스 데이터에 &quot;검정&quot;으로 설정 &quot;동적 프레임&quot;. 히스토그램 유형으로 &quot;3D&quot;를 선택하고 &quot;더 분산 보정 &#39;을 선택하지 않습니다. </li><li> PET 재건 : 재건 알고리즘으로 2D 순서 Subset이 기대 극대화 (OSEM2D)를 사용합니다. </li></ol> 3. FDG의 주입 <ol><li> 영상 실험실로 도착 ~ 30 분 예약 첫번째 주입하기 전에에 대한 지역 업체에서 18 F-FDG (10 MCI)의 임상 패키지를 주문하십시오. 방사성 물질이 포함 된 패키지를 받아 조사하는 연구소의 안전 절차를 따르십시오혹시 (RAM). </li><li> L-블록 테이블 탑 쉴드, 나누어지는 FDG와 살균 식염수에 dilutions을가 제공하는 보호를 갖추고 있습니다. FDG의 희석 활동 농도는 각 분사에 대한 200-300 μCi/100 UL에서 사용할 수 있습니다. 26G 2분의 1 인치 바늘로 1 ML의 주사기에 FDG 솔루션을 그리기, 그리고 선량 캘리브레이터을 가지고있는 전체의 주사기의 방사능을 측정합니다. </li><li> 단지 intraperitoneal (IP) 경로를 통해 FDG 솔루션 100 μl와 함께 추운 방 (단계 1.4 참조) 운송되는 동물을 주사. 분사 시간을 기록합니다. 선량 캘리브레이터로 다시 주사기의 잔류 방사능을 측정합니다. </li><li> 얼음 유지는 스티로폼 쿨러 내부의 차가운 케이지에 다시 동물을 놔. FDG의 이해를위한 1 시간에 추위 (~ 4 ° C)에서 동물을 길러. </li><li> 다음 수식에 의해 각 마우스의 주입 FDG 활동을 계산할 수 있습니다 : 주사 전에 주사기에 주입 활동 (μCi) = 활동- 주입 후 주사기 활동 </li></ol> 4. Micro-PET/CT 이미징 <ol><li> micro-PET/CT 영상은 추위 치료 후 FDG 분사 또는 5 시간 후 1 시간을 시작합니다. 산소의 Isoflurane 3%로 마취 유도 챔버에 동물을 놔. </li><li> 일단 마취가 유도되고, 동물의 머리가 지속적으로 2 L / 분의 유량에서 (2 %) Isoflurane 제공하는 콘 얼굴 마스크 내에서 휴식과 마이크로-CT 펠릿 (동물 침대)에 이동합니다. 전기 가열 패드 (BioVet, M2M 이미징 주식회사)는 몸의 온도를 유지하기 위해 동물에서 배치됩니다. </li><li> MM 스캐너의 입구에 동물을 슬라이드하면, 도구 모음에서 &quot;레이저&quot;아이콘을 활성화하고, 동물의 가슴은 수평 및 수직 레이저 라인 십자가 위에 오도록 침대를 이동하려면 터치 패드 컨트롤을 사용합니다. &#39;레이저 정렬&#39;창에서 optio로 &quot;PET 인수가 워크 플로에 포함&quot;CT 촬영으로 &quot;처음 스캔 유형&quot;을 선택하고,N. </li><li> &quot;스카우트보기 &#39;창을 열고 X-레이 방사선 스카우트보기를 취득. 중부 표준시의 전망의 중앙 필드는 마우스 본체 (간)의 중심부에 위치한 있도록 동물 침대의 위치를​​ 조정하는 법에 사용합니다. 이 단계 필요한 경우를 반복합니다. </li><li> 2 단계에서 설립 된 &quot;워크 플로우&quot;를 시작합니다. 옵션이 팝업되면, CT 재건에 사용될 수있는 적절한 3D PET-CT 변환 매트릭스 파일을 선택하고 더 감쇠 보정과 PET 재건을위한 최근에 만든 정상화 파일을 선택합니다. 법에는 중부 표준시와 PET 스캔 순서로 프로그램을 시작합니다. </li><li> 전체 워크 플로우가 완료되면, 이는 20-25 분 걸릴 수 있습니다, 간단히 획득 중부 표준시와 ASIPro VM, 마이크로 PET 분석 소프트웨어 법에와 공동으로 설치되어있는 PET 이미지의 품질을 평가합니다. 아카이브는 영상 데이터 저장 장치에 데이터 나 추가 분석을 위해 게시물을 영상 분석 워크 스테이션 (5 단계 참조) 네트워크를 통해 데이터를 전송합니다. </li><li> ReleaSE는 이미징 시스템에서 동물과 상온에서의 복구를위한 정상적인 음식과 물 공급과 깨끗한 주택 케이지에 넣습니다. 시스템은 현재 대기열에있는 다음 동물에 대한 준비가되어 있습니다. 후 영상 동물의 관리 및 취급이 &quot;RAM으로 주입 실험 동물의 취급&quot;에 관한 연구소의 규정을 준수해야합니다. 또한 연구소의 관련 RAM 폐기물 처리 규정에 따라 바늘 / 주사기, 흡수성 패드, 장갑, 모든 침구를 사용하고 대변가 방사성 폐기물로 간주되어야하며 처리된다는 점에 유의하십시오. </li></ol> 5. 후 영상 분석 <ol><li> 수동으로 열기 Inveon 연구 직장 (IRW) 소프트웨어 (지멘스)과 중부 표준시와 PET 데이터 세트를 모두 가져옵니다. &quot;등록&quot;에서 &quot;일반 분석&quot;기능과 도구를 사용하여 창 및 &quot;검토&quot;창 아래에 공동 등록 중부 표준시와 PET 이미지는 중부 표준시와 모든 3 차원의 PET 이미지 사이의 완벽한 정렬을해야합니다. </li><li>공동 등록 중부 표준시 이미지가 제공하는 참조로 &quot;관심 영역 (ROI) 정량화&quot;창에서, 목, 성인 쥐에서 가장 지배적 인 냉 inducible의 BAT의 interscapular 지역에서 BAT를 확인하고, 관심 볼륨을 그리는 (VOI) PET 데이터 세트 이상 BAT의. &quot;정량화 유형 &#39;으로&quot;Voxel 강도 &quot;를 선택하고 BQ / ML로 VOI 내에서 평균 방사능을 기록합니다. BQ / ML에 카운트 / 초 변환 교정 계수는 이전에 알려진 방사능과 팬텀을 검색하여 설립되었습니다. </li><li> BAT에서 FDG의 이해는 붕괴 보정과 그램 조직 당 비율 주입 용량 (% ID / g)으로 정량화되어 있습니다. 주입 용량 단계 3.5의 결과 그러나, becquerel (BQ) 단위 (1 μCi = 37,000 BQ)로 변환, 우리는 조직의 1 ML은 1g에 동일 있다고 가정합니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

본 연구에서는 micro-PET/CT-based 이미징 방법은 단순히 감기 치료 18 F-FDG 상업적으로 이용 가능한 중 하나 주입을 필요로 성인 쥐 BAT 활동을 검출하기 위해 개발되었습니다. 전체 절차는 치료 및 모든 동물 취급 이미징 때까지 매 30 분마다 시작 이미징 순서에 따라 하루에 수행 할 수 있습니다. 에 설명 된 실험 조건 하에서, 10 마우스 (5 마우스 2 그룹)의 총은 하나의 영상 시스템과 같은 날에 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> Micro-PET/CT 이미징 시스템 </td><td> 지멘스 의료 솔루션 미국, 주식회사 </td><td> Inveon 전용 PET 시스템 및 Inveon Multimodality 중부 표준시 / SPECT 시스템 (도킹) </td><td></td></tr><tr><td> Propranolol </td><td> 시그마 </td><td> P0884 </td><td></td></tr><tr><td> BRL 37344 </td><td> 시그마 </td><td> B169 </td><td></td></tr><tr><td> 18 F-FDG </td><td> Cyclotope 주식회사 </td><td></td><td></td></tr><tr><td> C57BL/6J 남성 마우스 </td><td> 잭슨 연구소 </td><td> 000664 </td><td> 3-4개월 이전 </td></tr></tbody></table>

functional imaging of brown fat in mice with 18f-fdg micro-pet/ct

갈색 지방 조직 (BAT)는 별개의 위치와 풍부한 vascularization과 미토콘드리아의 높은 밀도로 인해 갈색 - 붉은 색으로 흰색 지방 조직 (와트)에서 다릅니다. BAT는 에너지 지출 및 비 떨고 thermogenesis 신생아 포유 동물에서뿐만 아니라 성인 1 중요한 역할을 담당하고 있습니다. BAT로 인한 thermogenesis가 높은 β 아드레날린 수용체 2, 3을 통해 주로, 교감 신경계에 의해 조절된다. 최근 연구는 인간의 성인 BAT 활동이 부정적인 신체 질량 지수 (BMI) 및 기타 당뇨병 매개 변수 4-6과 상관 아르 것으로 나타났습니다. BAT 따라서 anti-obesity/anti-diabetes 요법은 에너지 밸런스 6-8의 변조에 초점을위한 잠재적 인 타겟으로 제안되었습니다. 여러 추위 도전 기반의 양전자 방출 단층 촬영 (PET) 방법은 인간의 BAT 9-13을 검출하기 위해 설립 있지만, 이미징 및 quantif에 대한 표준화 된 프로토콜은 기본적으로 없습니다같은 마우스와 같은 작은 동물 모델에서 BAT의 ication. 여기 쥐 BAT의 기능 평가를위한 강력한 PET / CT 이미징 방법을 설명합니다. 간단히, 성인 C57BL/6J 마우스는 18 F-Fluorodeoxyglucose (FDG) 중 하나 복용량을받은 전에 4 시간 기간 동안 금식 조건에서 처리 추위했다. 마우스는 하나의 추가 시간 후 FDG 주입을 위해 추위에 남아, 그리고 작은 동물 전용 micro-PET/CT 시스템을 스캔했다. 획득 PET 이미지는 해부학 적 참조에 대한 중부 표준시 이미지와 함께 공동 등록 BAT 활동을 제시 할 수있는 interscapular BAT 지역의 FDG의 이해를 분석했다. 이 추위에 치료 표준화 및 이미징 프로토콜 약리 개입하는 동안 테스트 BAT 활동을 통해 검증 된, 예를 들어, β-adrenoceptor 14, 15 propranolol 길항제, 또는 β3 작용제 BRL37344 16 향상된 BAT 활성화의 치료에 의해 ​​억압 BAT 활성화. 방법 DES여기 cribed는 BAT의 활동을 조절하거나, 유전자 / 다양한 잠복기 및 기본 연구 BAT 개발 및 규제에 관련된 경로를 확인하기 위해 약물 / 화합물에 대한 화면으로 적용 할 수 있습니다.

마우스 BAT의 micro-PET/CT 영상의 예는 그림 1에 표시됩니다. 중부 표준시 영상은 해부학 적 정보를 제공하지만, PET 영상은 몸 전체에 걸쳐 18 F-FDG의 호응을 배포하고 수량을 인코딩합니다. 이 영상 자료는 (1C) 융​​합, 또는 최대 강도 투사 (MIP, 1D)와 같은 3D 기능을 시연 (1A 및 1B) 개별적으로 볼 수 있습니다. 3D 이미징 도구, 관심 볼륨 (VOI)의 도움으로, 여기 interscapular BAT 지역은 <s...

Watch this Scientific Journal Video about Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT at JoVE.com

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

마우스 갈색 지방 조직 (BAT)의 기능 영상의 방식은 BAT에서 18F-Fluorodeoxyglucose의 냉 자극 이해 (FDG)가 아닌 invasively 표준화 micro-PET/CT 프로토콜로 과세되는 설명되어 있습니다. 이 방법은 마우스 모델에서 BAT 활동의 차이를 감지 할 강력하고 민감하다.

FDG micro-PET/CT와 마우스의 브라운 지방의 기능 영상

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

functional-imaging-of-brown-fat-in-mice-with-18f-fdg-micro-petct

Research

JoVE Journal

Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

19.0K 조회수.  The Methodist Hospital Research Institute, Houston. 마우스 갈색 지방 조직 (BAT)의 기능 영상의 방식은 BAT에서 18F-Fluorodeoxyglucose의 냉 자극 이해 (FDG)가 아닌 invasively 표준화 micro-PET/CT 프로토콜로 과세되는 설명되어 있습니다. 이 방법은 마우스 모델에서 BAT 활동의 차이를 감지 할 강력하고 민감하다.

비디오: Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

마우스에 대한 진한 / 라이트 전환 테스트

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

연구

생물학

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

이런 산화물로 hESCs과 hMSCs을 라벨링하는 MR 영상과 생체내 추적 비침습에 대한 Nanoparticles

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

광학 이미징과 비침습 감지를위한 형광 염료와 함께 줄기 세포를 라벨링

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

강화, Eyetracking 및 생리 감시 기능 이미징

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

마우스에 대한 고가 플러스 메이즈

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

조립, 로딩 및 분석 초원 심 분리기 샘플 셀 정렬

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

마우스의 전통 NASA Hindlimb 하역 모델 Alternant 방법

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

쎄의 후유증을 매핑하는 기능 영상과 함께 인간 청각 컴퓨터에 접속 자극을 버스트

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

비 트랜스제닉 무척추 동물의 단일 신경 해상도를 가진 네트워크 역학을 기록하기위한 포토다이오드 기반 광학 이미징

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.