본 연구는 사립 대학의 전략적 연구 재단과 우리는 비디오 클립을 편집 할 수 오타케 Norihito에게 감사의 말씀을 전합니다 HT에 대한 JSPS 젊은 과학자 B 보조금에 대한 문부 과학성 - 지원 프로그램에 의해 지원되었다.

1. 사과 접시를 준비합니다 <ol><li> 12.5 g 한천을 혼합, 125 ㎖의 100 % 시판 사과 주스, 12.5 g 포도당, 375 ML의 H 2 O 1-2분의 혼합물을 전자 레인지와 3cm 세포 배양 접시에 붓는다. 4 ℃에서 보관 </li></ol> 2. 유전 크로스 <ol><li> 다음과 같은 대표적인 유전 십자가를 사용 </li></ol> Or49a-mCD8 :: GFP / CYO X 1,118 (W) 3. 해부 및 염색 프로토콜 <ol><li> 파리를 마취 한 다음 집게를 사용하여 개최하여 수직으로 비행 머리를 잘라. </li><li> 조심스럽게 사과 접시에 안테나를 포함하는 부분을 놓습니다. </li><li> 미세 해부 가위를 사용하여 안테나의 세 번째 세그먼트를 잘라. </li><li> 유리 바닥 배양 접시의 중간에 () 0.1 % 트리톤 X-100 0.1 % PBST (PBS에 4 % 파라 포름 알데히드)의 고정 솔루션의 9​​0 μl를 놓습니다. </li><li> 조심스럽게 해부 antenn 전송직접 고정 솔루션에 날카로운 바늘 AE. 필요한 경우, 물리적으로 바늘을 사용하여 용액에 안테나를 물속. </li><li> 실온 (RT)에서 40 분 알을 품다. 같은 접시에 그들을 유지 (0.4 % 트리톤 X-100과 PBS) 0.4 % PBST에 각각 3 배 10 분 안테나를 씻으십시오. PBST 솔루션을 제거하고 추가하는 노란색 팁을 사용합니다. 때마다 세척 용액 90 μl를 사용합니다. 참고 : 면역 조직 화학 염색 동안 진탕에 접시를 올려 놓지 마십시오. 접시에 솔루션을 제거하거나 추가하기 전에 바늘을 사용하여 요리의 중심으로 모든 안테나를 가지고 조심스럽게 접시의 가장자리에서 솔루션을 추가하거나 제거 할 수 있습니다. </li><li> 실온에서 20 분 동안 0.1 % PBST 5 % 정상 말 혈청 90 μL와 안테나를 차단합니다. </li><li> 차단 솔루션을 제거한 후, 전술 한 바와 같이 축축한 용기에 4 ° C에서 48 시간 동안 5 % 말 혈청을 포함하는 0.1 % PBST에 차 항체의 90μl와 안테나를 품다 <s업&gt; 8. </li><li> 0.4 % PBST -10 분 6 배 안테나를 씻으십시오. </li><li> 4 ℃에서 48 시간 동안 5 % 말 혈청을 포함하는 0.1 % PBST 이차 항체의 90 μL와 안테나를 품어 0.4 % PBST를 사용하여 6 배 10 분 씻으십시오. </li><li> , 안테나를 탑재 가능한 한 배양 접시에서 PBST를 제거하고 점차적으로 안테나에 글리세롤의 두 개의 서로 다른 농도를 소개합니다. 처음 1 2 분 동안 요리에 40 % 글리세롤을 추가; 다음이를 제거하고 80 % 글리세롤을 추가합니다. </li><li> 조심스럽게 노란색 팁을 사용하여 배양 접시에서 (80 % 글리세롤을 포함한다) 안테나를 추출하고 슬라이드에 배치합니다. 조심스럽게 상단에 커버 슬립을 배치하고 매니큐어로 커버 슬립의 가장자리를 밀봉. 안테나는 지금 형광 현미경으로 몇 군데 준비가​​ 된 것입니다. </li></ol>

<ol>
	<li>Christensen, T. A., White, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Representation of olfactory information in the brain In The Neurobiology of Taste and Smell. , 201-232 (2000).</li><li>Ache, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+common+strategy+for+transducing+olfactory+information.">Towards a common strategy for transducing olfactory information.</a> Sem. Cell Biol. 5, 55-63 (1994).</li><li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction.">Odorant-selective genes and neurons mediate olfaction.</a> C. elegans. Cell. 13, 515-527 (1993).</li><li>Barth, A. L., Justice, N. J., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asynchronous+onset+of+odorant+receptor+expression+in+the+developing+zebrafish+olfactory+system.">Asynchronous onset of odorant receptor expression in the developing zebrafish olfactory system.</a> Neuron. 16, 23-34 (1996).</li><li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+olfactory+system+makes+sense+of+scents.">How the olfactory system makes sense of scents.</a> Nature. 413, 211-218 (2001).</li><li>Stockinger, P., 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=Neural+circuitry+that+governs+Drosophila+male+courtship+behavior.">Neural circuitry that governs Drosophila male courtship behavior.</a> Cell. 121, 795-807 (2005).</li><li>Endo, K., 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=Chromatin+modification+of+Notch+targets+in+olfactory+receptor+neuron+diversification.">Chromatin modification of Notch targets in olfactory receptor neuron diversification.</a> Nat Neurosci. 15, 224-233 (2011).</li><li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics.">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> J Vis Exp. , (2011).</li><li>Suh, G. S., 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+single+population+of+olfactory+sensory+neurons+mediates+an+innate+avoidance+behaviour+in+Drosophila.">A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila.</a> Nature. 431, 854-859 (2004).</li><li>Sachse, S., Galizia, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+inhibition+for+temporal+and+spatial+odor+representation+in+olfactory+output+neurons:+A+calcium+imaging+study.">Role of inhibition for temporal and spatial odor representation in olfactory output neurons: A calcium imaging study.</a> J Neurophysiol. 87, 1106-1117 (2002).</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>Vosshall, L. B., Wong, A. M., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+olfactory+sensory+map+in+the+fly+brain.">An olfactory sensory map in the fly brain.</a> Cell. 102, 147-159 (2000).</li><li>Couto, A., Alenius, M., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular,+anatomical+and+functional+organization+of+the+Drosophila+olfactory+system.">Molecular, anatomical and functional organization of the Drosophila olfactory system.</a> Curr Biol. 15, 1535-1547 (2005).</li><li>Clyne, P., 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=Odorant+response+of+individual+sensilla+on+the+Drosophila+antenna.">Odorant response of individual sensilla on the Drosophila antenna.</a> Invert Neurosci. 3, 127-135 (1997).</li><li>Vosshall, L. B., 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+spatial+map+of+olfactory+receptor+expression+in+the+Drosophila+antenna.">A spatial map of olfactory receptor expression in the Drosophila antenna.</a> Cell. 96, 725-736 (1999).</li><li>Wang, J. W., 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=Two-photon+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain.">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112, 271-282 (2003).</li></ol>

저자가 공개하는 게 없다.

우리가 설명하는 초파리 안테나의 해부는 단순하고 실험실 환경에서 수행하기 쉽습니다. 성공적인 절개를 보장하기 위해, 그것을 잘 양날 가위를 사용하는 것이 필수적입니다. 해부 안테나를 면역 염색 동안, 항체 용액의 증발을 방지하기 위해 습기가 채워진 용기들을 배양하는 것이 중요하다. 해부 안테나 솔루션에 떠있는 경향이있다. 고정 동안 PBS에서 0.1 % 트리톤을 사용하고 단계를 차...

후각 시스템은 악취 분자의 거대한 다양한 구분할이어서 높은 뇌 센터에 생성 된 정보를 전송하기 위해 사용된다. 이 입력은 정확히 같은 먹이와 짝짓기 1-6로 기본적인 동물의 동작을 제어하는 데 사용됩니다. 각각의 후각 신경 세포의 종류가 냄새의 특정 세트, 후각 수용체 뉴런의 다양 화와 연관 될 때 (ORN)의 적절한 후각 시스템 기능 7 필수적이다. 초파리 유전학 ORN 개발 및 생리 기능 8-16과 관련된 분자 메커니즘을 포함하는 단일 세포 수준의 연구를 수행하는 가능하게한다. 초파리 안테나의 전체 마운트 면역 염색은 더 자세히 후각 수용체 뉴런의 다양 화에 관련된 분자 메커니즘 (ORN)의 7을 이해할 수있게되었습니다. 여기에서 우리는 교류에 간단한 방법의 포괄적 인 설명을 제공이 hieve.

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Stemi DV4 dissection microscope</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:113px;">Stemi DV4</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Glass bottom culture dishes&#160;</td>
			<td style="width:143px;">MatTek corporation</td>
			<td style="width:113px;">P35G-0-10-C</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Dissection scissor</td>
			<td style="width:143px;">Fine Science Tools</td>
			<td style="width:113px;">15000-08</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Rat anti-ELAV</td>
			<td style="width:143px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:113px;">7E8A10</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Mouse anti-GFP</td>
			<td style="width:143px;">Invitrogen</td>
			<td style="width:113px;">A11122</td>
			<td style="width:116px;">Dilution 1:400</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Donkey Anti-Rabbit IgG</td>
			<td style="width:143px;">Jackson ImmunoResearch Laboratories</td>
			<td style="width:113px;">711-225-152</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Donkey Anti-Rat IgG</td>
			<td style="width:143px;">Jackson ImmunoResearch Laboratories</td>
			<td style="width:113px;">712-165-150</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
	</tbody>
</table>

whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna

악취 물질 분자는 정확하고 조정 된 방법으로 자신의 목표 수용체에 결합한다. 각 수용체는 특정 신호를 인식하고 뇌에 정보를 중계한다. 이와 같이, 후각 정보 인식과 행동, 장점 조사를 모두 수정, 뇌에 전달하는 방법을 결정하는 단계를 포함한다. 흥미롭게도, 세포 전달 및 전사 인자는 후각 수용체 신경 세포의 다양 화에 관여하는 것을 새로운 증거가있다. 여기에서 우리는 강력한 전체가 생체 내 후각 수용체 신경 세포 조직을 분석 실험하는 면역 표시 방법을 마운트 제공합니다. 이 방법을 사용하여, 우리는 안티 ELAV 항체 알려져 팬 신경 마커 Or49a-mCD8 :: GFP, 특히 안티 GFP 항체를 사용하여 NBA의 신경 세포에서 발현 후각 수용체 신경 세포의 모든 후각 수용체 뉴런을 확인했다.

해부 및 고정 모두가 신속하게 수행하는 것을 보장하는 것은이 프로토콜을 사용하여 성공을 달성하는 핵심 요소입니다. 미세 가위와 집게를 사용하면 매우 중요합니다. 면역 염색 한 후, 형광 표지 된 안테나는 공 초점 현미경으로 관찰 하였다. 우리는 일반적으로 20 배 렌즈를 사용하여 1 ㎛ 섹션을. 우리는 NBA에게 Or49a-mCD8 :: GFP를 사용하여 7 ORNs을 표시하고 야생 형 안테나의 NBA ORNs의 수?...

Watch this Scientific Journal Video about Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna at JoVE.com

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

에있는 후각 수용체 뉴런의 전체 마운트 Immunolabeling<em&gt; 초파리</em&gt; 안테나

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

whole-mount-immunolabeling-olfactory-receptor-neurons-drosophila

Research

JoVE Journal

Neuroscience

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

8.9K 조회수.  Doshisha University. Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

비디오: Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

전체 마운트의 배아 신경 계통의 시각화 Drosophila 태아

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

연구

신경과학

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

성인 전체 마운트 준비 Drosophila</em장 섬유 염료 주입을위한> 복부 신경 코드

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

강한 감각 뉴런의 속눈썹에 갇힌 화합물의 플래시 광분해

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

흡입 피펫 기법을 사용하여 강한 수용체 뉴런에서 기록 Odorant 유발 응답

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

에 Appetitive 연관 강한 학습<em&gt; Drosophila</em&gt; 애벌레

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

에 신약 표현형을 평가<em&gt; 초파리</em&gt; 등산 분석 실험에 의한 도파민 뉴런과 뇌 전체의 면역 염색

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

마우스의 메인 후각 시스템의 섬모의 전체 마운트 라벨링

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

본래 Neuroepithelium에서 마우스 강한 감각 뉴런의 천공 패치 클램프 녹음 : 확인 된 부 취제 수용체를 발현 뉴런의 기능 분석

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

전체 마운트 해부 및 성인 마우스 달팽이관의 면역 형광

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.