이 작품은 로렌스 G. Rahme에 건강 (NIH) 부여 AI063433의 국립 연구소, 로날드 G. 톰프킨스 (A. 아리아 Tzika의 원장 건강의 국립 연구소 연구소 (NIH) 센터 그랜트 (P50GM021700)에 의해 부분적으로 지원되었다 A. 아리아 Tzika 어린이 연구 부여 (# 8893)에 대한 NMR 코어) 및 슈라 이너 병원. 우리는 Dionyssios Mintzopoulos 박사 감사 이 프로토콜 및 Ovidiu C. Andronesi 박사를 개발의 초기 단계에서 도움을 TOBSY 펄스 시퀀스를 지원.

1 부 : HRMAS 측정 준비 Drosophila <ol><li> 표준 flylab 절차 1을 사용, 새로 3 일간 파리 eclosing 신선한 플라이 음식을 포함하는 튜브를 비행을 전송 수집합니다. 파리 방금 전에 실험 5~8일 이전 될 수 있도록 오일에 대한 수집 파리 알을 품다. 단 하나의 성별은 (보통 남성) 실험에 사용됩니다. </li><li> 건강을 사용, 손상 야생 유형은 예를 들어 충격이나 유전자 파리 1 개조 치료와 비교 날아. </li><li> 이전 실험 비행 식품 24 시간 조각 (~ 0.2ml)을 포함 2ml 튜브에 하나의 파리를 놓습니다. 이러한 튜브는 불꽃 - 온수 인슐린 바늘을 사용하여 만든 튜브 컵에 구멍을 부담. </li><li> 고정밀 밸런스를 사용하여 비행 삽입 전후 바로 튜브를 저울질하고 후자에서 첫 번째 가치를 빼서 각 비행기의 무게를 정의합니다. 한 남자 비행은 일반적으로 0.7-1 MG 무게. </li></ol> 2 부 : HRMAS의 로터 준비. <ol><li> 플라이 내부를 마취하는 분도 위해 얼음에 하나의 튜브를 놓으십시오. </li><li> 얼음에 위치 알루미늄 호일의 레이어에 파리를 틀어서 플라이 부상을 피하면서 전체 비행 삽입을 보장하기 위해 부드러운 브러시를 사용하여 NMR의 회전자의 삽입의 semispherical 빈 공간으로 부드럽게 날아 밀어. </li><li> 회전자의 회전자 삽입하고 아래 (figure1 참조) 사이의 파리를 찾습니다 지르코늄 산화물 (ZrO 2) 로터 튜브 (4mm 직경 50 μl)에 삽입 놓습니다. </li><li> 나사와 삽입을 닫고 비행 및 TSP 표준 용액 (TSP 사이의 접촉을 방지하기 위해 parafilm (figure1 참조)로 커버 : δ trimethylsilyl - 프로피 - 2 ,2,3,3 - D4 산, MW = 172, = 0.00 PPM, 진정제를 맞았을 물에 - D 2 O, 공명 화학 변화와 부량 모두에 대해 참고로 그 함수)에서 50 MM. </li><li> 회전자 삽입 위에 TSP 표준 용액의 8 μl를 추가합니다. (그림 1 참조). </li><li> 안전하고 (그림 1 참조) 최고의 모자와 로터에 설정된 전체를 조입니다. </li></ol> 파트 3 : HRMAS 데이터 수집 <ol><li> HRMAS 프로브에 날개를 소개하고 마법의 각도 54.7 ° (그림 1 참조)에서 위치를. </li><li> MAS의 공압 장치와 함께 BTO - 2000 단위로 4 ° C에서 온도를 설정합니다. 파리는 마취를 유지하는 분광계 4 ° C의 동안에 보관됩니다. </li><li> 2 kHz에서 매스에서 HRMAS 1 H MRS 회전 속도를 설정합니다. </li><li> MAS 속도 ​​컨트롤러 (회전 주파수)에 의해 2.0 ± 0.001 kHz에서에서 MAS 회전 주파수를 안정화. </li><li> 자석 준비 : 조정 및 최적의 성능과 심 스펙트럼의 최적의 품질에 대한 자석 코일을 일치합니다. </li><li> 에 T 2 필터로 작동 [N - 인수 - - (τ τ - 180 °) 90 °], 카 - 퍼셀 - Meiboom - 길 (CPMG) 스핀 에코 펄스 시퀀스 2, 동기 로터를 사용하여 1 차원 1 H 스펙트럼을 취득 모든 샘플 한 차원 데이터를 획득하기 위해 스펙트럼 확대 1 H NMR 단일 플라이 스펙트럼을 제거합니다. MAS 회전 주파수 (2 kHz에서)에 간 펄스 지연 (τ = 500μs) 동기화. 32,768 (32K) 데이터 지점과 256에 과도의 개수를 설정합니다. 수집 시간 9 분. </li><li> 단열 펄스 3 TOBSY 순서를 사용하는 모든 샘플에서 2 차원 (2D) 1 H - 1 H NMR HRMAS 단일 플라이 스펙트럼을 습득. 인수 매개 변수는 다음과 같습니다 2K 데이터 포인트를 직접 치수 (11 PPM 스펙트럼 폭), 1 S 완화 지연 중에 물을 미리 채도, 증가 당 8 스캔,​​ 128 단위, 2의 총 반복 시간, 45 MS 믹싱 시간, 총 인수 29 분 시간. </li></ol> 4 부 : 데이터 처리 / 분석 <ol><li> MestReC 소프트웨어 (Mestrelab 연구 www.mestrec.com)를 사용하여 표본의 MRspectra를 분석 </li><li> 0.5 Hz의 라인 확대 apodization 기능을 사용하여 이전 푸리에 변환에 대한 모든 HRMAS 1 H의 FIDs에 적용됩니다. </li><li> δ = 0.0 PPM (외부 규격)에서 TSP와 관련하여 MR 스펙트럼을 참조. </li><li> 수동 스펙트럼 위상 및 사전 피크 면적 계산에 대한 기준의 광범위한 구성 요소를 뺄 휘태커 기준 견적 도구 (Traffic Estimator)를 적용합니다. </li><li> MestReC 소프트웨어를 사용하여 최대 영역을 예상하고있다. 존중 스케일 최고점은 각 인수 스펙트럼 (TSP 피크 높이 = 1) TSP 수 있습니다. 사이 그룹 비율로 내에서 - 그룹을 비교하는 T - 테스트 (2 꼬리, P는 &lt;0.05)를 사용합니다. </li><li> 2D TOBSY 프로세스 매개 변수는 다음과 같습니다 두 차원에 QSINE = 2 창 기능, 직접 치수 두 번째 차원, 두 번째 차원의 두 차원과 기본 수정의 위상 보정에 1K에 제로 채울 2K 점 FT. </li><li> 스파키 프로그램 (TD 고더드 및 DG 넬러, 스파키 3, USCF 사용 2D 스펙트럼 제작 <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/을</a>홈 / 스파키 /) </li></ol> 제 5 부 : 대표 스펙트럼 라이브 Drosophila melanogaster에서 난다 절차는 라이브 Drosophila melanogaster의 파리에서 재현할 수 스펙트럼을 수집하는 것을 허용 위에서 설명한. 야생 형 (WT) 오레곤 - R 인수 그림 2, 3 쇼 대표 MR 스펙트럼 난다. 그림 2 선물 1D 1 H HRMAS CPMG 스펙트럼에게. 주요 지질 구성 요소 [C H 3 (0.89 PPM), (C H 2) N (1.33 PPM), C H 2 C - CO (1.58ppm), C H 2 C = C (2.02 PPM), C H 2 C = O (2.24 PPM), C H = C H (5.33 PPM)], 글리세롤 (1,3 - C H 4.10 PPM과 4.30 PPM, 2 - CH 2 5.24 PPM), 그리고 작은 metabolites : β - 알라닌 (β - 에이러 , 2.57 PPM), 아세테이트 (AC, 1.97 PPM), phosphocholine (PC, 3.22 PPM)와 phophoetanolamine (PE, 3.23ppm)를 감지하고 사전에보고 4, 5에 따라 배정했다. 2.02 PPM에서 신호는 모노 - 불포화 지방산 (즉 palmitoleic)의 C H2 - CH = CH 잔기의 메틸렌의 양자에 할당되었습니다. 불포화 지방산은 - CH = CH - 잔기의 양자에 의해 만들어진 5.33 PPM에서 신호를 식별했습니다. 할당할 수 없습니다거나 1D 스펙트럼을 사용하여 표시되지 않은 수 작은 metabolites은 2D 1 H - 1 H를 사용하여 감지되었습니다 TOBSY HRMAS (그림 3 참조). <img src="/files/ftp_upload/1710/1710fig1.jpg" alt="그림 1" /> 그림 1. 실험은 생체내에의 준비 진정제를 맞았을 물에 14.1 T. 외부 표준 trimethylsilyl - 프로피 - 2 ,2,3,3 - D4의 산성 (TSP / D 2 O)에서 살고 Drosophila의 조사 HRMAS 1H MRS. HRMAS 프로브에있는 마법의 각도에서 로터 위치 : 광장에서. <img src="/files/ftp_upload/1710/1710fig2.jpg" alt="그림 2" /> 그림 2. Drosophila melanogaster 라이브 WT 날아의 생체내 1D HRMAS 1 H CPMG 스펙트럼에서. 지질 구성 요소 : C H 3 (0.89 PPM), (C H 2) N (1.33 PPM), C H 2 C - CO (1.58ppm), 아세테이트 (AC), C H 2 C = C (2.02 PPM), C H 2 C = O (2.24 PPM), (β - 에이러) β - 알라닌, phosphocholine (PC)와 phophoetanolamine (PE), 글리세롤 (1,3 - C H 4.10, 4.30 PPM, 2 - C H 2 5.22 PPM) , C H = C H (5.33 PPM). <img src="/files/ftp_upload/1710/1710fig3.jpg" alt="그림 3" /> 그림 3. 생체내 2D 1H - 1H에 14.1 T. 중소 metabolites 및 지질 구성 요소에 살고 Drosophila melanogaster WT 날아의 TOBSY HRMAS 스펙트럼이 확인되었습니다. Metabolites : 알라닌 (에이러), (β - 에이러) β - 알라닌, 아르기닌 (ARG), 글루타민 (Gln), 글루 탐 산염 (GLU), PC의 phosphocholine (PC), phophoetanolamine (PE), 타우린 (타우), α - 포도당 (α - Glc)과 글리세롤. Lipids 구성 요소 : C H 3 (0.89 PPM), (C H 2) N (1.33 PPM), C H 2 C - CO (1.58 PPM), C H 2 C = C (2.02 PPM), C H 2 C = O (2.24 PPM), C H = C H (5.33 PPM).

<ol>
	<li>Apidianakis, Y., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+host+for+studying+Pseudomonas+aeruginosa+infection.">Drosophila melanogaster as a model host for studying Pseudomonas aeruginosa infection.</a> Nat Protoc. 4, 1285-1294 (2009).</li><li>Meiboom, S., Gill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+spin-echo+method+for+measuring+nuclear+relaxation+time.">Modified spin-echo method for measuring nuclear relaxation time.</a> Rev Sci Instrum. 29, 688-691 (1958).</li><li>Andronesi, O. C., Mintzopoulos, D., Struppe, J., Black, P. M., Tzika, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-state+NMR+adiabatic+TOBSY+sequences+provide+enhanced+sensitivity+for+multidimensional+high-resolution+magic-angle-spinning+1H+MR+spectroscopy.">Solid-state NMR adiabatic TOBSY sequences provide enhanced sensitivity for multidimensional high-resolution magic-angle-spinning 1H MR spectroscopy.</a> J Magn Reson. 193, 251-258 (2008).</li><li>Astrakas, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton+NMR+spectroscopy+shows+lipids+accumulate+in+skeletal+muscle+in+response+to+burn+trauma-induced+apoptosis.">Proton NMR spectroscopy shows lipids accumulate in skeletal muscle in response to burn trauma-induced apoptosis.</a> Faseb J. 19, 1431-1440 (2005).</li><li>Fan, T. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+profiling+by+one-+and+two+dimensional+NMR+analysis+of+complex+mixtures.">Metabolite profiling by one- and two dimensional NMR analysis of complex mixtures.</a> Prog Nuc Magn Reson Spec. 28, 161-219 (1996).</li><li>Null, B., Liu, C. W., Hedehus, M., Conolly, S., Davis, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution,+in+vivo+magnetic+resonance+imaging+of+Drosophila+at+18.8+Tesla.">High-resolution, in vivo magnetic resonance imaging of Drosophila at 18.8 Tesla.</a> PLoS One. 3, e2817-e2817 (2008).</li><li>Zektzer, 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=Improved+signal+to+noise+in+high-resolution+magic+angle+spinning+total+correlation+spectroscopy+studies+of+prostate+tissues+using+rotor-synchronized+adiabatic+pulses.">Improved signal to noise in high-resolution magic angle spinning total correlation spectroscopy studies of prostate tissues using rotor-synchronized adiabatic pulses.</a> Magn Reson Med. 53, 41-48 (2005).</li></ol>

동물 실험은 매사 추세츠 종합 병원 기관 동물 연구 검토위원회위원회에 의해 명시된 지침과 규정에 따라 수행되었다.

과일의 생체내 MRI에의 가능성의 최근 보고서를 제외하고 6 파리, Drosophila의 생체내 MRS 연구 아직보고되지 않았습니다. 현재 프로토콜, 우리는 생물학적으로 중요한 분자를 검출 생체내 HRMAS 1 H - MRS 방식의 소설의 구현을 설명합니다. 제로 파리 사망률을 달성하면서 특히, 우리는 lipids, 라이브 Drosophila의 작은 metabolites 충분한 수집 시간을 허...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Deuterium oxide</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>7789-20-0</td>
<td>&nbsp;</td>
<tr>
<td>3-(trimethylsilyl)propionic-2,2,3,3-d4 acid</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>24493-21-8</td>
<td>&nbsp;</td>
<tr>
<td>agar, sucrose, yeast, cornmeal</td>
<td>Food</td>
<td>Genesee Scientific Corporation</td>
<td>&nbsp;</td>
<td><a href="http://www.flystuff.com/">http://www.flystuff.com/</a></td>
</tr>
<tr>
<td>Oregon RS or Canton-S flies</td>
<td>Adult fly lines</td>
<td>Bloomington Stock Center</td>
<td>&nbsp;</td>
<td><a href="http://flystocks.bio.indiana.edu/">http://flystocks.bio.indiana.edu/</a></td>
</tr>
<tr>
<td>Paintbrush</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>(size 0)</td>
</tr>
<tr>
<td>2ml tubes</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>K749521-1590</td>
<td>&nbsp;</td>
<tr>
<td>Fly incubators</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>high humidity capacity (60-75%), adjustable temperature, and a 12 h:12 h light: dark cycle.</td>
</tr>
<tr>
<td>Bruker Bio-Spin Avance NMR spectrometer (600.13 MHz) 4mm triple resonance (1H, 13C, 2H) HRMAS probe </td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BTO-2000 unit in combination with a MAS pneumatic unit</td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>4mm zirconium oxide rotor (capacity 50 ul)</td>
<td>Equipment</td>
<td>Bruker Bio-Spin</td>
<td>B3829 (Bruker store)</td>
<td>&nbsp;</td>
<tr>
<td>MestReC (Mestrelab Research) </td>
<td>Software</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1D NMR spectra analysis <a href="http://mestrelab.com/">http://mestrelab.com/</a></td>
</tr>
<tr>
<td>SPARKY 3, USCF</td>
<td>Software</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2D NMR spectra
analysis
 <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/home/sparky/</a></td>
</tr>		</tbody>
		</table>
		</div>

magnetic resonance spectroscopy of live drosophila melanogaster using magic angle spinning

고해상도 매직 각도 스피닝 (HRMAS) 프로톤 자기 공명 분광 (<sup&gt; 1</sup&gt; H - MRS) 스펙트럼 라인 폭을 향상시키고 추출, 손상 세포, 세포 배양, 그리고 더 중요한 것은 그대로 조직에서 얻을 수 고해상도 스펙트럼은 metabolites와 셀룰러 프로세스 간의 관계를 조사 수있는 새로운 비파괴 기술입니다.<em&gt; 생체내에서</em&gt; HRMAS<sup&gt; 1</sup&gt; H - MRS 연구 라이브 과일 파리에보고해야 아직<em&gt; Drosophila melanogaster.</em&gt;<em&gt; Drosophila,</em&gt; 간단한 유전자 유기체로, 여러 생물 학적 기능과 다양한 evolutionarily 보존 신호 경로가 전체 유기체 수준에서 검사 수 있으며 그것은 유전자와 생리를 조사하는 유용한 모델입니다. 이를 위해, 우리는 개발 및 구현<em&gt; 생체내에</em&gt; HRMAS<sup&gt; 1</sup라이브 조사&gt; H - MRS 방법<em&gt; Drosophila</em&gt; 여기 14.1 T.에서 우리는 HRMAS을 설명<sup&gt; 1</sup의 분자 특성에 대한&gt; H - MRS 프로토콜<em&gt; Drosophila</em&gt; HRMAS 프로브가 장착된 기존의 MR 분광계와 함께. 이 기법은 소설이다<em&gt; 생체내에</em&gt; 비파괴<em&gt; Drosophila</em따라서 질병 생체 식별을 가능하게하고&gt; 신진 대사 측정 방법은, 소설 치료 발전에 기여 수 있습니다.

Watch this Scientific Journal Video about Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning at JoVE.com

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

이 기술은 생활의 분자 특성에 대한 양성자 MR 분광법 (1H - MRS HRMAS)를 회전 고해상도 마법 각도의 사용을 가능하게<em&gt; Drosophila melanogaster</em&gt; 기존의 14.1 테슬라 분광계와이 HRMAS 프로브가 장착된.

라이브의 자기 공명 분광학 Drosophila melanogaster 매직 앵글 스피닝를 사용하여

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

magnetic-resonance-spectroscopy-live-drosophila-melanogaster-using

Research

JoVE Journal

Neuroscience

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

12.4K 조회수.  Massachusetts General Hospital, Harvard Medical School. 이 기술은 생활의 분자 특성에 대한 양성자 MR 분광법 (1H - MRS HRMAS)를 회전 고해상도 마법 각도의 사용을 가능하게 Drosophila melanogaster 기존의 14.1 테슬라 분광계와이 HRMAS 프로브가 장착된.

비디오: Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

여러 마우스 Neuroanatomical 자기 공명 영상

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

연구

신경과학

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

의 이스케이프 문제의 Optogenetic의 자극<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

의 자발적인 운동 활성의 결정<em&gt; 초파리</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

일차 운동 피질 대사에 양성 반구 경 두개 전기 자극 효과의 측정을위한 도구로 자기 공명 분광학의 사용

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

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 ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

조직 추출물의 고해상도 핵 자기 공명 분광법에 의한 쥐 뇌의 대사 체 분석

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

사회적 공간에 영향을 미치는 조건<em&gt; 초파리</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

에서 냄새 유도 행동의 고해상도 정량화<em&gt; 초파리</em사용&gt;<em&gt; Flywalk</em&gt; 패러다임

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

대한 꼴 경로 길이 프로토콜<em&gt; 노랑 초파리</em&gt; 애벌레

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

초파리 melanogaster Larval 및 성인 두뇌의 변화 분석

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...