This work was supported in part by a National Institutes of Health (NIH) grant AI063433 to Laurence G. Rahme, a National Institute Institutes of Health (NIH) Center Grant (P50GM021700) to Ronald G. Tompkins (A. Aria Tzika, Director of the NMR core), and a Shriner's Hospital for Children research grant (#8893) to A. Aria Tzika. We thank Dionyssios Mintzopoulos Ph.D. for assistance in the initial phases of developing this protocol and Ovidiu C. Andronesi Ph.D. for assistance with the TOBSY pulse sequence.

Part 1: Preparing Drosophila for HRMAS Measurements
<ol>
<li>Using standard flylab procedures 1, collect newly eclosing flies for 3 days and transfer them to fly vials containing fresh fly food. Incubate the collected flies for 5 days so that the flies will become 5-8 days old just prior to the experiment. Only one sex (usually males) is used for experiments.</li>
<li>Use healthy, intact wild type flies to compare with treated e.g. traumatized or genetically altered flies 1.</li>
<li>Place single flies into 2ml tubes containing a piece (~0.2ml) of fly food 24 hours prior to the experiment. These tubes bear a hole in the tube cup made using a flame-heated insulin needle.</li>
<li>Weigh the tubes immediately before and after the fly insertion using a high precision balance and define each fly s weight by subtracting the first value from the latter. A single male fly weighs usually 0.7-1 mg.</li>
</ol>
Part 2: HRMAS Rotor Preparation.
<ol>
<li>Place a single tube on ice for less than a minute to anesthetize the fly inside.</li>
<li>Lay the fly on a layer of aluminum foil placed on ice and push the fly gently into the semispherical hollow space of the NMR rotor insert using a soft brush to ensure complete fly insertion while avoiding fly injury.</li>
<li>Place the insert into the zirconium oxide (ZrO2) rotor tube (4 mm diameter, 50 &mu;l) to locate the flies between the rotor insert and the bottom of the rotor (see figure1).</li>
<li>Close the insert with a screw and cover it with parafilm (see figure1) to prevent the contact between the fly and the TSP standard solution (TSP: trimethylsilyl- propionic-2,2,3,3-d4 acid, Mw = 172, &delta; = 0.00 ppm, 50 mM in deuterated water-D2O, that function as a reference for both resonance chemical shift and quantification).</li>
<li>Add 8 &mu;l of TSP standard solution on top of the rotor insert. (see figure 1).</li>
<li>Secure and tighten the whole set up in the rotors with a top cap (see figure 1).</li>
</ol>
Part 3: HRMAS Data Acquisition
<ol>
<li>Introduce the rotor into the HRMAS probe and position it at the magic angle 54.7&deg; (see figure 1).</li>
<li>Set temperature at 4&deg;C by a BTO-2000 unit in combination with the MAS pneumatic unit. Flies are kept at 4&deg;C while in the spectrometer to maintain anesthesia.</li>
<li>Set the HRMAS 1H MRS spinning rate at 2 kHz MAS.</li>
<li>Stabilize the MAS rotation frequency at 2.0 &plusmn; 0.001 kHz by a MAS speed controller (Rotation frequency).</li>
<li>For magnet preparation: tune and match the coil for optimum performance and shim the magnet for optimal quality of spectra.</li>
<li>Acquire one dimensional 1H spectra using&nbsp; rotor synchronized Carr-Purcell-Meiboom-Gill (CPMG) spin echo pulse sequence 2, [90&deg;-(&tau;-180&deg;- &tau;)n-acquisition], which works as a T2 filter to remove the spectral broadening 1H NMR single-fly spectra to acquired one-dimensional data on all samples. Synchronize the inter-pulse delay (&tau; = 500&mu;s) to the MAS rotation frequency (2 kHz). Set the number of transients at 256 with 32,768 (32k) data points. Acquisition time 9 min.</li>
<li>Acquire two-dimensional (2D) 1H-1H HRMAS NMR single-fly spectra on all samples using a TOBSY sequence with adiabatic pulses 3. Acquisition parameters are: 2k data points direct dimension (11 ppm spectral width), 1 s water pre-saturation during the relaxation delay, 8 scans per increment, 128 increments, 2 s total repetition time, 45 ms mixing time, and a total acquisition time of 29 min.</li>
</ol>
Part 4: Data Processing/Analysis
<ol>
<li>Analyze the MRspectra of specimens using MestReC software (Mestrelab Research, www.mestrec.com)</li>
<li>Use a line-broadening apodization function of 0.5 Hz and apply to all HRMAS 1H FIDs prior to Fourier transformation.</li>
<li>Reference the MR spectra with respect to TSP at &delta; = 0.0 ppm (external standard).</li>
<li>Phase spectra manually and apply a Whittaker baseline estimator to subtract the broad components of the baseline prior to peak area calculations.</li>
<li>Estimate peak areas using MestReC software. Scale peak heights with respect to TSP for each acquired spectrum (TSP peak height = 1). Use t-tests (two-tailed, p&lt;0.05) to compare within-group to between-groups ratios.</li>
<li>2D TOBSY process parameters are: QSINE = 2 window function in both dimensions, FT with 2k points in the direct dimension and zero-filling to 1k in the second dimension, phase correction in both dimensions and baseline correction in the second dimension.</li>
<li>Produce the 2D spectra using Sparky program (T. D. Goddard and D. G. Kneller, SPARKY 3, USCF, <a href="http://www.cgl.ucsf.edu/home/sparky/">http://www.cgl.ucsf.edu/home/sparky/</a>)</li>
</ol>
Part 5: Representative Spectra from Live Drosophila melanogaster Flies
The procedures described above permit to collect reproducible spectra from live Drosophila melanogaster flies. Figures 2 and 3 show representative MR spectra acquired in wild-type (wt) Oregon-R flies. Figure 2 presents 1D 1H HRMAS CPMG spectra. Principal lipid components [CH3 (0.89 ppm), (CH2)n (1.33 ppm), CH2C-CO (1.58ppm), CH2C=C (2.02 ppm), CH2C=O (2.24 ppm), CH=CH (5.33 ppm)], glycerol (1,3-CH 4.10 ppm and 4.30 ppm; 2-CH2 5.24 ppm), and small metabolites: &beta;-Alanine (&beta;-Ala, 2.57 ppm), acetate (Ac, 1.97 ppm), phosphocholine (PC, 3.22 ppm) and phophoetanolamine (PE, 3.23ppm) were detected and assigned in accordance with prior reports 4, 5. Signals at 2.02 ppm were assigned to methylene protons of the CH2-CH=CH moiety of mono-unsaturated fatty acids (i.e. palmitoleic). The unsaturated acids were identified by a signal at 5.33 ppm produced by protons of the -CH=CH- moiety. Small metabolites that could not be assigned or were not visible using the 1D spectrum were detected using 2D 1H-1H TOBSY HRMAS (see figure 3).
<img src="/files/ftp_upload/1710/1710fig1.jpg" alt="Figure 1" /> Figure 1. Experimental set up of in vivo HRMAS 1H MRS for the investigation of live Drosophila at 14.1 T. External standard trimethylsilyl- propionic-2,2,3,3-d4 acid in deuterated water (TSP/D2O). In the square: rotor position at the magic angle in HRMAS probe.
<img src="/files/ftp_upload/1710/1710fig2.jpg" alt="Figure 2" /> Figure 2. In vivo 1D HRMAS 1H CPMG spectra of a live Drosophila melanogaster wt fly. Lipid components: CH3 (0.89 ppm), (CH2)n (1.33 ppm), CH2C-CO (1.58ppm), Acetate (Ac), CH2C=C (2.02 ppm), CH2C=O (2.24 ppm), &beta;-Alanine (&beta;-Ala), phosphocholine (PC) and phophoetanolamine (PE), Glycerol (1,3-CH 4.10, 4.30 ppm; 2-CH2 5.22 ppm), CH=CH (5.33 ppm).
<img src="/files/ftp_upload/1710/1710fig3.jpg" alt="Figure 3" /> Figure 3. In vivo 2D 1H-1H TOBSY HRMAS spectrum of a live Drosophila melanogaster wt fly at 14.1 T. Small metabolites and lipid components were identified. Metabolites: alanine (Ala), &beta;-Alanine (&beta;-Ala), arginine (Arg), glutamine (Gln), glutamate (Glu), PC phosphocholine (PC), phophoetanolamine (PE), Taurine (Tau), &alpha;-Glucose (&alpha;-Glc) and Glycerol. Lipids components: CH3 (0.89 ppm), (CH2)n (1.33 ppm), CH2C-CO (1.58 ppm), CH2C=C (2.02 ppm), CH2C=O (2.24 ppm), CH=CH (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>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by Massachusetts General Hospital Institutional Animal Research Review Board Committee.

With the exception of the recent report of the feasibility of in vivo MRI in fruit flies 6, in vivo MRS studies in Drosophila have not yet been reported. In the present protocol, we describe the implementation of a novel in vivo HRMAS 1H-MRS approach for detecting biologically important molecules. Specifically, we detected lipids and small metabolites in live Drosophila flies at 14.1 T in approximately 45 min, which allows adequate acquisition time, while...

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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

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS 1H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster. Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS 1H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS 1H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.

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

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

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

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Neuroscience

12.5K visualizações.  Massachusetts General Hospital, Harvard Medical School. Esta técnica permite o uso de alta resolução, ângulo mágico de prótons girando MR espectroscopia (HRMAS 1H-MRS) para a caracterização molecular de viver Drosophila melanogaster Com um espectrômetro convencional 14,1 tesla equipado com uma sonda HRMAS.

Vídeo: 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.

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

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.

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;

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.

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.

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).

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.

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.

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

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

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.

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