1. Odor dilutions
<ol>
<li>Most odorants are soluble in paraffin oil. However, DMSO or ethanol can also be used as alternative solvents for particular odors. Prepare appropriate dilutions (e.g. 1:10 volume : volume; v:v) from pure odorants in glass vials. Most odor dilutions are stable at room temperature, but for highly volatile compounds it is better to make working dilutions on a weekly basis. Each sensillum responds to different odors within a different concentration range. For Drosophila, a useful look-...

<ol>
	<li>Boeckh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elektrophysiologische+Untersuchungen+an+einzelnen+Geruchsrezeptoren+auf+der+Antenne+des+TotengrAbers+(Necrophorus+Coleoptera).">Elektrophysiologische Untersuchungen an einzelnen Geruchsrezeptoren auf der Antenne des TotengrAbers (Necrophorus Coleoptera).</a> Z. Vergl. Physiol. 46, 212-248 (1962).</li><li>Schneider, D., Hecker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Elektrophysiologic+der+Antenne+des+Seidenspinners+Bombyx+mori+bei+Reizung+mit+angereicherten+Extrakten+des+Sexuallockstoffes.">Zur Elektrophysiologic der Antenne des Seidenspinners Bombyx mori bei Reizung mit angereicherten Extrakten des Sexuallockstoffes.</a> Z. Naturforschg. 11b, 121-124 (1956).</li><li>Touhara, K., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+odorants+and+pheromones+with+chemosensory+receptors.">Sensing odorants and pheromones with chemosensory receptors.</a> Annu Rev Physiol. 71, 307-332 (2009).</li><li>Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+olfactory+receptors+are+heteromeric+ligand-gated+ion+channels.">Insect olfactory receptors are heteromeric ligand-gated ion channels.</a> Nature. 452, 1002-1006 (2008).</li><li>Wicher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+odorant+receptors+are+both+ligand-gated+and+cyclic-nucleotide-activated+cation+channels.">Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels.</a> Nature. 452, 1007-1011 (2008).</li><li>Hallem, E. A., Ho, M. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+odor+coding+in+the+Drosophila+antenna.">The molecular basis of odor coding in the Drosophila antenna.</a> Cell. 117, 965-979 (2004).</li><li>Hallem, E. A., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+odors+by+a+receptor+repertoire.">Coding of odors by a receptor repertoire.</a> Cell. 125, 143-160 (2006).</li><li>Boeckh, J., Kaissling, K. E., Schneider, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+olfactory+receptors.">Insect olfactory receptors.</a> Cold Spring Harb Symp Quant Biol. 30, 263-280 (1965).</li><li>de Bruyne, M., Foster, K., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+coding+in+the+Drosophila+antenna.">Odor coding in the Drosophila antenna.</a> Neuron. 30, 537-552 (2001).</li><li>Lu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+coding+in+the+maxillary+palp+of+the+malaria+vector+mosquito+Anopheles+gambiae.">Odor coding in the maxillary palp of the malaria vector mosquito Anopheles gambiae.</a> Curr Biol. 17, 1533-1544 (2007).</li><li>Hallem, E. A., Fox, A. N., Zwiebel, L. J., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction:+mosquito+receptor+for+human-sweat+odorant.">Olfaction: mosquito receptor for human-sweat odorant.</a> Nature. 427, 212-213 (2004).</li><li>Couto, A., Alenius, M., Dickson, B. J. M. o. l. e. c. u. l. a. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=anatomical,+and+functional+organization+of+the+Drosophila+olfactory+system.">anatomical, and functional organization of the Drosophila olfactory system.</a> Curr. Biol. 15, 1535-1547 (2005).</li><li>Fishilevich, E., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+functional+subdivision+of+the+Drosophila+antennal+lobe.">Genetic and functional subdivision of the Drosophila antennal lobe.</a> Curr Biol. 15, 1548-1553 (2005).</li></ol>

Olfactory cues are used by organisms to identify food sources, potential mates, and predators. Olfactory sensory neurons (OSNs) are the first relay center between external stimuli and higher centers of the brain where the information is further processed. In Drosophila melanogaster and Anopheles gambiae, OSNs are easily accessible and their electrical activity can be monitored while stimulated by odor puffs.
The single sensillum recording (SSR) technique explained in this vi...

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<td>Paraffin oil</td>
<td>Odors</td>
<td>Fluka</td>
<td>76235</td>
<td>&nbsp;</td>
<tr>
<td>High purity odors (&gt;98%)</td>
<td>Odors</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>Methyl acetate 
#296996 
1-octen-3-ol 
#74950 </td>
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<tr>
<td>Filter paper strips</td>
<td>Odors</td>
<td>Fisherbrand</td>
<td>05-714-1</td>
<td>Chromatography paper</td>
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<tr>
<td>Connectors</td>
<td>Odors</td>
<td>Cole-Parmer</td>
<td>EW-06365-40</td>
<td>1/16x1/8"</td>
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<tr>
<td>Glass vials</td>
<td>Odors</td>
<td>Agilent Technologies</td>
<td>5182-0556</td>
<td>&nbsp;</td>
<tr>
<td>Air line plastic tubing</td>
<td>Odor Delivery</td>
<td>Python Products</td>
<td>500PAL</td>
<td>&nbsp;</td>
<tr>
<td>1 serological pipette</td>
<td>Odor Delivery</td>
<td>Corning</td>
<td>4101</td>
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<td>Plastic tubing</td>
<td>Odor Delivery</td>
<td>Cole-Parmer</td>
<td>EW-06418-0</td>
<td>0.050"x0.090"OD</td>
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<tr>
<td>Disposable borosilicate glass Pasteur pipettes</td>
<td>Odor Delivery</td>
<td>FisherBrand</td>
<td>13-678-20A</td>
<td>5-3/4 inches</td>
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<tr>
<td>Programmable stimulus controller</td>
<td>Odor Delivery</td>
<td>Syntech</td>
<td>CS-55</td>
<td>&nbsp;</td>
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<td>Anti-vibration table</td>
<td>Electrophysiology Equipment</td>
<td>TMC</td>
<td>63533</td>
<td>36&#x201D;Wx30&#x201D;Dx29&#x201D;H</td>
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<td>Faraday cage</td>
<td>Electrophysiology Equipment</td>
<td>TMC</td>
<td>MI8133303</td>
<td>&nbsp;</td>
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<td>Electrophysiology Equipment</td>
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<td>10x and 100x objectives</td>
<td>Electrophysiology Equipment</td>
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<td>10x Plan Fluor 100x L Plan</td>
<td>&nbsp;</td>
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<td>Dissecting microscope</td>
<td>Electrophysiology Equipment</td>
<td>Nikon</td>
<td>EZ645</td>
<td>electrode sharpening/insect prep microscope</td>
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<tr>
<td>Magnetic stands</td>
<td>Electrophysiology Equipment</td>
<td>Newport</td>
<td>MODEL 150</td>
<td>&nbsp;</td>
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<td>IDAC</td>
<td>Electrophysiology Equipment</td>
<td>Syntech</td>
<td>IDAC-4</td>
<td>&nbsp;</td>
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<td>Crocodile clip</td>
<td>Electrophysiology Equipment</td>
<td>Pomona</td>
<td>AL-B-12-0</td>
<td>&nbsp;</td>
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<td>Pomona</td>
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<td>2 electrode holders</td>
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<td>N/A</td>
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<td>AC probe</td>
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<td>Electrophysiology Equipment</td>
<td>Microprobes</td>
<td>M210</td>
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<td>Electrophysiology Equipment</td>
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<td>&nbsp;</td>
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<td>Microscope slide</td>
<td>Insect prep</td>
<td>FisherBrand</td>
<td>12-550A</td>
<td>&nbsp;</td>
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<td>Cover glass</td>
<td>Insect prep</td>
<td>FisherBrand</td>
<td>12-541A</td>
<td>18X18 #1.5</td>
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<tr>
<td>Polypropylene mesh</td>
<td>Insect prep</td>
<td>Small Parts inc.</td>
<td>CMP-0500-B</td>
<td>&nbsp;</td>
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<td>Glass electrode</td>
<td>Insect prep</td>
<td>Frederick Haer & Co.</td>
<td>27-32-0-075</td>
<td>Capillary tubing borosilicate 1.5mm OD x 1.12mm ID x 75 mm</td>
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<td>Double-sided tape (3M)</td>
<td>Insect prep</td>
<td>3M</td>
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<td>Double-sided tape (3M)</td>
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single sensillum recordings in the insects drosophila melanogaster and anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Watch this Scientific Journal Video about Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae at JoVE.com

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

single-sensillum-recordings-insects-drosophila-melanogaster-anopheles

Research

JoVE Journal

Neuroscience

24.9K 次观看.  Rockefeller University. 在使用单一感器录音昆虫气味的嗅觉感官神经元的电生理反应，可测。在这个视频文章，我们将演示如何执行单一飞醋的触角感器录音（。果蝇）和疟疾的蚊子的上颌触须（按蚊）。

视频: Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles...

single sensillum recordings for locust palp sensilla basiconica

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect...

identification of olfactory volatiles using gas chromatography-multi-unit recordings (gcmr) in the insect antennal lobe

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds.  Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

electrophysiological recording from drosophila labellar taste sensilla

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

measuring and altering mating drive in male drosophila melanogaster

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

electrophysiological recording from drosophila trichoid sensilla in response to odorants of low volatility

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

multi-unit recording methods to characterize neural activity in the locust (schistocerca americana) olfactory circuits

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

microinjection method for anopheles gambiae embryos

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

Microinjection Method for Anopheles gambiae Embryos

using the gal4-uas system for functional genetics in anopheles gambiae

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile tool for modification of gene expression in a controlled spatiotemporal manner which permits functional genetic analysis in Anopheles gambiae. The procedures described for using this system are a semi-standardized cloning strategy, sexing and screening of pupae for fluorescent protein markers and embryo...

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

rnai trigger delivery into anopheles gambiae pupae

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal...

RNAi Trigger Delivery into Anopheles gambiae Pupae

dissection and immunostaining of larval salivary glands from anopheles gambiae mosquitoes

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further...

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune...

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Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of...

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IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx explores insecticide resistance-related transcriptional profiles in the species Anopheles gambiae. Provided here are full instructions for using the application, modifications for exploring multiple transcriptomic datasets, and using the framework to build an interactive database for collections of transcriptomic data from any organism, generated in any...

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labeling of single cells in the central nervous system of drosophila melanogaster

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal...

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using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process...

determination of the spontaneous locomotor activity in drosophila melanogaster

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...

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oral bacterial infection and shedding in drosophila melanogaster

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

electrophysiological recordings from the giant fiber pathway of d. melanogaster

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber...

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

dyeing insects for behavioral assays: the mating behavior of anesthetized drosophila

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

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.

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 ...

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

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 ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

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 ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...