Name: a single-fly assay for foraging behavior in drosophila
Uploaded: 2013-11-04T21:30:00
Description: Watch this Scientific Journal Video about A Single-fly Assay for Foraging Behavior in Drosophila at JoVE.com

इस काम के स्वास्थ्य के राष्ट्रीय संस्थान (R01DK092640) और राष्ट्रीय विज्ञान फाउंडेशन (0920668) से JWW के लिए अनुसंधान अनुदान द्वारा समर्थित किया गया.

1. फ्लाई संग्रह और भुखमरी <ol><li> रियर एक 12 घंटा / प्रकाश अंधेरे चक्र पर नियंत्रित तापमान और नमी की स्थिति (जैसे 21 डिग्री सेल्सियस, 50-60% सापेक्ष आर्द्रता) के तहत प्रयोगात्मक मक्खियों. </li><li> महिला eclosion के दिन पर ?...

<ol>
	<li>Dethier, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The hungry fly : a physiological study of the behavior associated with feeding. , (1976).</li><li>Root, C. M., Ko, K. I., Jafari, A., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+facilitation+by+neuropeptide+signaling+mediates+odor-driven+food+search.">Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search.</a> Cell. 145, 133-144 (2011).</li><li>Root, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+presynaptic+gain+control+mechanism+fine-tunes+olfactory+behavior.">A presynaptic gain control mechanism fine-tunes olfactory behavior.</a> Neuron. 59, 311-321 (2008).</li><li>Semmelhack, J. L., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Select+Drosophila+glomeruli+mediate+innate+olfactory+attraction+and+aversion.">Select Drosophila glomeruli mediate innate olfactory attraction and aversion.</a> Nature. 459, 218-223 (2009).</li><li>Faucher, C., Forstreuter, M., Hilker, M., de Bruyne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+responses+of+Drosophila+to+biogenic+levels+of+carbon+dioxide+depend+on+life-stage,+sex+and+olfactory+context.">Behavioral responses of Drosophila to biogenic levels of carbon dioxide depend on life-stage, sex and olfactory context.</a> J. Exp. Biol. 209, 2739-2748 (2006).</li><li>Quinn, W. G., Harris, W. A., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+behavior+in+Drosophila+melanogaster.">Conditioned behavior in Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. U.S.A. 71, 708-712 (1974).</li><li>Fishilevich, E., Domingos, A. I., Asahina, K., Naef, F., Vosshall, L. B., Louis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+behavior+mediated+by+single+larval+olfactory+neurons+in+Drosophila.">Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila.</a> Curr. Biol. 15, 2086-2096 (2005).</li><li>Venken, K. J., Simpson, J. H., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+genes+and+cells+in+the+nervous+system+of+the+fruit+fly.">Genetic manipulation of genes and cells in the nervous system of the fruit fly.</a> Neuron. 72, 202-230 (2011).</li><li>Hamada, F. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internal+thermal+sensor+controlling+temperature+preference+in+Drosophila.">An internal thermal sensor controlling temperature preference in Drosophila.</a> Nature. 454, 217-220 (2008).</li><li>Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+modification+of+behavior+in+Drosophila+by+targeted+expression+of+a+temperature-sensitive+shibire+allele+in+defined+neurons.">Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.</a> J. Neurobiol. 47, 81-92 (2001).</li><li>Sweeney, S. T., Broadie, K., Keane, J., Niemann, H., O'Kane, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+expression+of+tetanus+toxin+light+chain+in+Drosophila+specifically+eliminates+synaptic+transmission+and+causes+behavioral+defects.">Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.</a> Neuron. 14, 341-351 (1995).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li></ol>

इस प्रोटोकॉल में, हम खाद्य खोज व्यवहार परख के लिए एक कदम दर कदम प्रक्रिया का वर्णन. भोजन संबंधी odors के अलावा, यह भी अन्य गंध वस्तुओं का पता लगाने के लिए उड़ान भरने की क्षमता के अध्ययन के लिए अनुकूलित किया ज?...

खाद्य खोज और भोजन की खपत 1: भूख की अमेरिका भूख बढ़ाने वाला व्यवहार के दो प्रकार को बढ़ावा देने. इस सरल व्यवहार परख 2,3 foraging साथ जुड़े chemotactic व्यवहार के अध्ययन के लिए उपयोगी है. विशेष रूप से, यह एक खाना गंध लक्ष्य लगाने के लिए उड़ान भरने की स्थिति, चलने की गति और विलंबता पटरियों. भोजन खोजने के विलंबता अपने आंतरिक भूख बढ़ाने वाला राज्य में परिवर्तन के बहाव मक्खी की गंध पहचान प्रणाली की संवेदनशीलता में बदलाव को मापने के लिए के रूप में मैट्रिक में कार्य करता है. इस परख की एक पुस्तिका संस्करण पहले से GABA बी रिसेप्टर संकेतन 3 मक्खियों वयस्क में गंध स्थानीयकरण व्यवहार के लिए महत्वपूर्ण है दिखाने के लिए इस्तेमाल किया गया था. परख की वर्तमान स्वचालित संस्करण neuropeptide एफ (sNPF) संकेतन ड्रोसोफिला में घ्राण मानचित्र को नया स्वरूप दे और प्रभावों व्यवहार 2 भूख बढ़ाने वाला कैसे कम के अध्ययन में महत्वपूर्ण भूमिका निभाई थी. परीक्षण एक काले, तापमान और आर्द्रता नियंत्रित कमरे में किया जाता है. डिजिटलस्पष्ट ऐक्रेलिक परीक्षण प्लेटों के ऊपर सेट वीडियो कैमरों 660 एनएम एलईडी रोशनी से backlit मक्खियों ट्रैक. कैमरे से जानकारी अगले परीक्षण क्षेत्र के लिए तैनात एक कंप्यूटर से वास्तविक समय में कार्रवाई की है. हम परीक्षण अवधि के दौरान उड़ान पदों के निर्देशांक रिकॉर्ड और बचाने के लिए डाटा अधिग्रहण सॉफ्टवेयर का उपयोग करें. इस प्रतिमान में, विषय सेंटर में एक खाद्य गंध होता है कि एक क्षेत्र में जारी है, गंध वस्तु मक्खी में खाद्य खोज व्यवहार को प्रेरित करता है कि क्षेत्र के भीतर एक खाद्य गंध ढाल बनाता है. ऐसा ही एक गंध खोज प्रोटोकॉल एकल ड्रोसोफिला लार्वा 7 में chemosensation के अध्ययन की ओर लागू किया गया है. इस तरह के चार क्षेत्र olfactometer 4,5 या टी भूलभुलैया के रूप में 6 अन्य व्यवहार assays गंध घृणा या आकर्षण व्यवहार का मूल्यांकन करते हैं, इस प्रतिमान सबसे अच्छा घ्राण संवेदनशीलता और chemotaxis व्यवहार का मूल्यांकन करने के लिए अनुकूल है. कई महत्वपूर्ण लाभ यह Assa के साथवाई. डेटा संग्रह और विश्लेषण ज्यादातर स्वचालित रहे हैं क्योंकि सबसे पहले, यह, बड़े डेटा सेट के तेजी से अधिग्रहण परमिट. दूसरा, इस परख आइसोलेट्स और इस प्रकार उनके व्यवहार को प्रभावित कर सकते हैं कि सामाजिक घ्राण cues को नष्ट करने, एकल मक्खियों के व्यवहार के उपाय. तीसरा, प्रोटोकॉल और सरल प्रयोगात्मक डिजाइन की सादगी परख कुशल और दूसरों को सिखाने के लिए आसान बनाते हैं. इसके अलावा, इस परख आगे ड्रोसोफिला मेलानोगास्टर 8 के लिए उपलब्ध व्यापक आनुवंशिक टूलकिट के साथ संयोजन से खाद्य खोज व्यवहार अंतर्निहित तंत्रिका सर्किट की जांच के लिए इस्तेमाल किया जा सकता है. मौन या उत्तेजित न्यूरॉन्स ऐसे GAL4-यूएएस प्रणाली के साथ ही यूएएस shibire TS1, यूएएस टिटनेस विष, और यूएएस TRPA1 (बी) के रूप में उपकरणों के साथ प्राप्त किया जा सकता है कि transgenes की लक्षित अभिव्यक्ति 9-12 transgenes.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Apple Cider Vinegar</td>
			<td>Spectrum</td>
			<td></td>
			<td>commercially available</td>
		</tr>
		<tr>
			<td>Agarose, Type VII</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td>low gelling temperature agarose</td>
		</tr>
		<tr>
			<td>Acrylic Testing Plate</td>
			<td></td>
			<td>custom</td>
			<td>Plate contains 6 arenas. Each arena is 60 mm in diameter 6 mm in height. See testing plate diagrams for specific measurements.</td>
		</tr>
		<tr>
			<td>LabVIEW V.8.5</td>
			<td>National Instruments</td>
			<td>776670-09</td>
			<td>platform for programs: PositioningTool.vi, FlyTracking--Six Zones.vi NOTE: &#34;elapsed time.vi&#34;, &#34;time into file.vi&#34;, and &#34;two object detect.vi&#34; are included subroutines that must be available in order for the main data acquisition program &#34;FlyTracking--Six zones.vi&#34; to run.</td>
		</tr>
		<tr>
			<td>LabVIEW Vision 8.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Vision Acquisition Software 8.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Vision Builder AI 3.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Igor Pro V.6</td>
			<td>Wavemetric, Inc.</td>
			<td></td>
			<td>platform for macro: Data Analysis for Fly Tracking--Six Zones</td>
		</tr>
		<tr>
			<td>Basler scA1390-17fm</td>
			<td>National Instruments</td>
			<td>779980-01</td>
			<td>Digital Camera NOTE: driver for camera available at Baslerweb.com</td>
		</tr>
		<tr>
			<td>8 mm lens</td>
			<td>National Instruments</td>
			<td>780024-01</td>
			<td>Lens for Basler Digital Camera</td>
		</tr>
		<tr>
			<td>Ground Glass Diffuser Plate</td>
			<td>Edmund Optics</td>
			<td>custom</td>
			<td>Diffuses light, 25 cm x 30 cm</td>
		</tr>
		<tr>
			<td>US Std. No. 100</td>
			<td>Fischer Scientific</td>
			<td>04-881X</td>
			<td>Sieve with nominal opening of 150 &#956;m</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Lighting Option 1</td>
		</tr>
		<tr>
			<td>LED backlight 660 nm (20 cm x 20 cm)</td>
			<td>Spectra West</td>
			<td>BL47192</td>
			<td>a simpler but more expensive lighting option.</td>
		</tr>
		<tr>
			<td>Power Supply for LED Backlight</td>
			<td>Spectra West</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Lighting Option 2</td>
		</tr>
		<tr>
			<td>660 nm LEDs</td>
			<td>Superbrightleds</td>
			<td>RL5R1330</td>
			<td>Wavelength 660 nm (approximately 7 x 7 LED array for a 14.7 inch x 9.75 inch panel)</td>
		</tr>
		<tr>
			<td>Linear DC Power Supply</td>
			<td>GW Instek</td>
			<td>GPS-1830D</td>
			<td>Power supply for LED Panel</td>
		</tr>
		<tr>
			<td>Solderless Breadboard</td>
			<td>Digikey</td>
			<td>922354-ND</td>
			<td>Breadboard for LEDs</td>
		</tr>
	</tbody>
</table>

a single-fly assay for foraging behavior in drosophila

कई जानवरों के लिए, भूख उपयुक्त खाद्य स्रोतों के लिए खोज की सुविधा है एक तरह से घ्राण प्रणाली में परिवर्तन को बढ़ावा देता है. इस वीडियो लेख में, हम ड्रोसोफिला मेलानोगास्टर मक्खी वयस्क फल में घ्राण निर्भर खाद्य खोज व्यवहार पर भूख या तृप्ति के प्रभाव को मापने के लिए एक स्वचालित परख का वर्णन. फल मक्खियों के लिए अदृश्य है कि लाल बत्ती से प्रकाशित एक हलका सा बॉक्स में, कस्टम डाटा अधिग्रहण सॉफ्टवेयर से जुड़ा हुआ एक कैमरा एक साथ छह मक्खियों की स्थिति पर नजर रखता है. प्रत्येक मक्खी सेंटर में एक भोजन की गंध से युक्त व्यक्ति एरेनास में चलना तक ही सीमित है. परीक्षण एरेनास कार्यों गंध संचय को रोकने के लिए है कि एक झरझरा मंजिल पर आराम. गंध के स्रोत, विभिन्न शारीरिक राज्यों तहत घ्राण संवेदनशीलता को दर्शाता है कि एक मीट्रिक लगाने के लिए विलंबता, सॉफ्टवेयर विश्लेषण द्वारा निर्धारित किया जाता है. यहाँ, हम इस व्यवहार प्रतिमान चलाने का महत्वपूर्ण यांत्रिकी चर्चा और मक्खी Loadi के बारे में विशिष्ट मुद्दों को कवरएनजी, गंध संदूषण, परख तापमान, डेटा की गुणवत्ता, और सांख्यिकीय विश्लेषण.

डेटा विश्लेषण सॉफ्टवेयर और लेआउट, चित्रा 1 में देखा जा सकता है एक उदाहरण है, जिनमें से विश्लेषण मापदंड का एक सेट के अनुसार अपने 10 मिनट की सुनवाई के दौरान प्रत्येक मक्खी के प्रदर्शन का मूल्यांकन कर?...

Watch this Scientific Journal Video about A Single-fly Assay for Foraging Behavior in Drosophila at JoVE.com

A Single-fly Assay for Foraging Behavior in Drosophila

इस वीडियो लेख में, हम ड्रोसोफिला मेलानोगास्टर मक्खी वयस्क फल में घ्राण निर्भर खाद्य खोज व्यवहार पर भूख या तृप्ति के प्रभाव को मापने के लिए एक स्वचालित परख का वर्णन.

Foraging व्यवहार के लिए एक एकल मक्खी परख में<em&gt; ड्रोसोफिला</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

a-single-fly-assay-for-foraging-behavior-in-drosophila

Research

JoVE Journal

Neuroscience

A Single-fly Assay for Foraging Behavior in Drosophila

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.

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).

कीड़े में एकल Sensillum रिकॉर्डिंग ड्रोसोफिला मेलानोगास्टर और एनोफ़ेलीज़ gambiae</em

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

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

परख के लिए तरीके ड्रोसोफिला व्यवहार

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

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 भागने व्यवहार की उत्तेजना<em&gt; ड्रोसोफिला मेलानोगास्टर</em

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

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

अनुदैर्ध्य के लिए एक रीढ़ की हड्डी में चैंबर दाखिल के लिए एक प्रक्रिया<em&gt; Vivo</em&gt; माउस स्पाइनल कॉर्ड की इमेजिंग

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

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

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.

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 anaesthesia.

व्यवहार assays के लिए डाइंग कीड़े: Anesthetized के संभोग व्यवहार<em&gt; ड्रोसोफिला</em

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

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.

चारा के लिए पथ-लंबाई प्रोटोकॉल<em&gt; ड्रोसोफिला मेलानोगास्टर</em&gt; लार्वा

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

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

ड्रोसोफिला ग्रूमिंग व्यवहार की मात्रा

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Drosophila द्वीप परख में हरकत व्यवहार के उच्च प्रवाह विश्लेषण

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

घ्राण न्यूरॉन्स की Optogenetic उत्तेजना के जवाब में ट्रैकिंग Drosophila लार्वा व्यवहार

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.