这项工作是支持部分由一个国家AI06​​3433卫生研究院（NIH）的资助机构，一个国立卫生研究院（NIH）的中心格兰特（P50GM021700）罗纳德G.汤普金斯（A.咏叹调Tzika，主任劳伦斯G. Rahme核磁共振核心），并为儿童的研究经费（＃8893）A.咏叹调Tzika一个Shriner的医院。我们感谢Dionyssios Mintzopoulos博士在本议定书和奥维迪乌C. Andronesi博士开发的初始阶段的援助TOBSY脉冲序列的援助。

第1部分：准备HRMAS测量果蝇 <ol><li>使用标准flylab程序1，收集新eclosing 3天的苍蝇，他们转移到飞瓶含有新鲜飞食品。孵育5天收集到的苍蝇，苍蝇将成为老实验前5-8天。只有一种性别（通常是男性）是用于实验。 </li><li>使用健康，完整的野生型果蝇与治疗如创伤或转基因苍蝇1。 </li><li>放入含有一块飞食品24小时（〜0.2毫升）实验前2毫升管单苍蝇。这些管子承受使用火焰加热的胰岛素针管杯孔。 </li><li>权衡的管飞采用了高精密天平插入之前和之后，并定义每个飞的体重减去后者的第一个值。一个单一的男性飞的重量一般为0.7-1毫克。 </li></ol>第2部分：HRMAS 转子准备。 <ol><li>不到一分钟麻醉里面飞单管置于冰上。 </li><li>铺设一层铝箔置于冰上飞，飞轻轻推入使用软毛刷，以确保完成飞行的插入，同时避免飞伤害的NMR转子插入中空的半球形空间。 </li><li>将插入到氧化锆（ZrO 2的 ）转筒（4毫米直径，50μL），定位和转子的转子插入底部（见图1）之间的苍蝇。 </li><li>关闭用螺丝插入和封口膜（见图1）覆盖，以防止飞和TSP的标准溶液（TSP之间的接触：三甲基硅烷基丙酸- 2 ,2,3,3 - D4酸，MW = 172，δ = 0.00 ppm时，氘水- D 2 O，这两个共振化学位移和量化的参考功能）50毫米。 </li><li> TSP的转子插入顶部的标准解决方案新增8μL。 （见图1）。 </li><li>安全和收紧了整个设置在顶盖与转子（见图1）。 </li></ol>第3部分：数据采集HRMAS <ol><li> HRMAS探头引入转子和魔角54.7 °（见图1）的位置。 </li><li>在结合的BTO - 2000 MAS气动装置的单位，设置在4 ° C的温度。苍蝇都保持在4 ° C，而在光谱仪，麻醉维持。 </li><li>设置在2 kHz的MAS HRMAS 的1 H MRS纺企。 </li><li> MAS旋转频率稳定在2.0 ± 0.001千赫的MAS速度控制器（旋转频率）。 </li><li>对于磁铁的准备：调整和匹配最佳的性能和垫片质量最优的光谱磁铁线圈。 </li><li>采集一维的1 H谱使用同步卡尔赛尔Meiboom -吉尔（CPMG）自旋回波脉冲序列2，转子[90 ° - （τ- 180 ° - τ）N -收购，作为T 2过滤工程删除，以收购一维数据，对所有样品的光谱展宽的1 H核磁共振单飞光谱。同步脉冲间的延迟（τ=500μS）的MAS旋转频率（2千赫）。设置在256 32,768（32K）数据点的瞬态。采集时间9分钟。 </li><li>采集二维（2D）的H - 1 H HRMAS核磁共振对所有使用3绝热脉冲一个TOBSY序列样本单飞光谱。采集参数：2K数据点直接的尺寸（11 ppm的谱宽），1预饱和的水松弛延迟期间，8％的增量扫描，128个递增，2秒的总重复时间​​，搅拌时间45毫秒，总收购29分钟的时间。 </li></ol>第4部分：数据处理/分析<ol><li>分析使用MestReC的软件（Mestrelab研究，www.mestrec.com标本MRspectra） </li><li>使用一个0.5赫兹扩大切趾的功能，并适用于所有HRMAS的 1 H FID的傅立叶变换之前。 </li><li>参考尊重议员光谱​​TSP的δ= 0.0 PPM（外部标准）。 </li><li>手动相位谱，和应用惠特克基线估计广泛的组件前，以峰面积计算减去基准。 </li><li>估计使用MestReC软件的峰面积。峰高与定额TSP的每个收购频谱（TSP峰高= 1）。使用t检验（双尾，P &lt;0.05），组内比较组之间的比率。 </li><li> 2D TOBSY工艺参数是：在两个维度QSINE = 2的窗口函数，直接维和零填充到1K，在第二个维度，阶段中的第二个维度的尺寸和基线校正校正2K点英尺。 </li><li>使用斯帕克方案（TD戈达德和DG Kneller得利3，USCF， <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/</a>产生的二维光谱首页/得利/） </li></ol>第5部分：从Live 果蝇代表光谱苍蝇上述程序允许收集活果蝇苍蝇重现光谱。图2和图3显示了在野生型（WT）俄勒冈州- R代表致辞光谱收购苍蝇。图2给出了1D 的1 H HRMAS CPMG光谱。主要脂质成分[C H 3（0.89 ppm）的，（2）C H N（1.33 PPM），C H 2架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），甘油（1,3 - C H 4.10 PPM，4.30 PPM; 2 - CH 2 5.24 PPM），和小的代谢物：β-丙氨酸（β-丙氨酸，2.57 PPM），醋酸乙烯酯（AC，1.97 PPM），phosphocholine（PC，3.22 PPM）和phophoetanolamine（PE，3.23ppm）进行检测，并根据与以前的报告4，第5指派。 2.02 PPM信号被分配到的 C H2 - CH = CH基团的单不饱和脂肪酸（如棕榈油）亚甲基质子。不饱和脂肪酸被确定在5.33 PPM - CH = CH -基团的质子产生的信号。无法分配或使用1D的频谱不可见的小代谢产物检测 2D 1 H - 1 H TOBSY HRMAS（见图3）。 <img src="/files/ftp_upload/1710/1710fig1.jpg" alt="图1" /> 图1。实验设在体内 HRMAS 1H MRS 14.1 T.外标三甲基硅丙酸- 2 ,2,3,3 - D4氘水酸（TSP / D 2 O）的活果蝇调查。在广场：在HRMAS探头魔角转子位置。 <img src="/files/ftp_upload/1710/1710fig2.jpg" alt="图2" /> 图2 在体内 1D HRMAS 的1 H CPMG的一个活的果蝇重飞光谱。脂质成分：C H 3（0.89 ppm）的，（2）C H N（1.33 PPM），C H 2 C - CO（1.58ppm），醋酸（AC），C H 2 C = C（2.02 PPM），C H 2 C = O（2.24 PPM），β-丙氨酸（β- ALA），phosphocholine（PC）和phophoetanolamine（PE），甘油（1,3 - C H，4.30每分钟4.10; 2 - C H 2 5.22 PPM ） C H = C H（5.33 PPM）。 <img src="/files/ftp_upload/1710/1710fig3.jpg" alt="图3" /> 图3。 体内 2D 1H - 1H TOBSY HRMAS现场果蝇重 14.1吨小代谢产物和脂质成分飞的光谱进行了鉴定。代谢物：丙氨酸（ALA），β-丙氨酸（β- ALA），精氨酸（Arg），谷氨酰胺（Gln），谷氨酸（Glu），PC phosphocholine（电脑），phophoetanolamine（PE），牛磺酸（头），α-葡萄糖（α- GLC）和甘油。血脂组件：C H 3（0.89 PPM），（2）C H 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>

在马萨诸塞州总医院的机构动物研究审查委员会委员会提出的准则和法规的规定进行动物实验。

随着最近在体内 MRI水果中的可行性报告的异常苍蝇6，在果蝇体内MRS研究尚未报道。在本议定书中，我们描述了在体内HRMAS 1 H - MRS检测生物的重要分子方法的小说实施。具体来说，我们检测血脂和小活果蝇的代谢产物在14.1 T苍蝇在约45分钟，它允许有足够的采集时间，同时实现零飞死亡率。使用转子同步WURST 8绝热脉冲（C9 1月 15日 ）在TOBS...

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		<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>
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magnetic resonance spectroscopy of live drosophila melanogaster using magic angle spinning

高分辨魔角旋转（HRMAS）质子核磁共振光谱（<sup&gt; 1</sup&gt; H - MRS）是一种新型的非破坏性技术，提高了光谱线的宽度和允许提取物，完整的细胞，细胞培养，更重要的是完整的组织获得高分辨率光谱研究代谢产物和细胞过程之间的关系。<em&gt;在体内</em&gt; HRMAS<sup&gt; 1</sup&gt; H - MRS的研究还没有要​​在活的果蝇报告<em&gt;果蝇。</em&gt;<em&gt;果蝇，</em&gt;作为一个简单的基因生物体，允许多种生物学功能和各种进化上保守的信号转导通路在整个生物体水平的检验，它是一个有用的模型研究遗传学和生理学。为此，我们制定并实施了<em&gt;在体内</em&gt; HRMAS<sup&gt; 1</sup&gt; H - MRS方法来调查现场<em&gt;果蝇</em&gt; 14.1 T.这里，我们介绍一个HRMAS<sup&gt; 1</sup&gt; H - MRS协议的分子特性<em&gt;果蝇</em与传统的磁共振光谱仪配备HRMAS探头。这种技术是一种新型的，<em&gt;在体内，</em&gt;非破坏性<em&gt;果蝇</em&gt;代谢产物的测量方法，从而使疾​​病的生物标志物的鉴定，从而可能导致新的治疗发展。

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

这项技术使活的分子特征的高分辨率魔角旋转的质子磁共振波谱（1H - MRS HRMAS）<em&gt;果蝇</em与传统的14.1特斯拉光谱仪配备HRMAS探头。

生活磁共振波谱果蝇</em用魔角旋转

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 次观看.  Massachusetts General Hospital, Harvard Medical School. 这项技术使活的分子特征的高分辨率魔角旋转的质子磁共振波谱（1H - MRS HRMAS）果蝇

视频: 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.

多鼠神经解剖学磁共振成像

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

科研

神经科学

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.

Optogenetic刺激的逃避行为<em&gt;果蝇</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;

在自发活动的测定<em&gt;果蝇</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.

磁共振波谱的用途，用于对初级运动皮层新陈代谢双半球经颅电刺激效果的测量工具一

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.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

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).

大鼠脑组织被提取物的高分辨核磁共振代谢组学分析

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.

在条件影响社会空间<em&gt;果蝇</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.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

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.

觅食路径长度协议<em&gt;果蝇</em&gt;幼虫

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.

果蝇幼虫和成年脑的代谢分析

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