SG는 버팔로의 뉴욕 주립 대학에서와 존 R. Oishei 재단 기금에 의해 지원됩니다.

<html> 1. 시약의 준비 <ol><li> 100mM 파이프, 2mM EGTA, 1 MM MgSO 4를 사용 PEMFA 버퍼를 준비합니다. 7.0 버퍼의 산도를 조정합니다. PEMFA 버퍼는 필터 소독과 4 ° C.에 저장됩니다 </li><li> 중 1ml를 추가하여 PBT를 준비 트리톤 X - 100 100ml 1X PBS (인산이 살린 버퍼) (산도 7.0). 4 스토어 ° C. </li><li> 1X PBS (산도 7.0)에 0.01 % NaAcide와 5% BSA를 (소 혈청 알부민) 준비합니다. </li><li> 0.7 % NaCl 및 0.03 % 탈이온수에서 트리톤 X - 100을 사용하여 생리 식염수를 준비합니다. </li><li> 한 부분은 37 % 포름 알데히드 4 개 부품 PEMFA 버퍼를 결합하여 정착액을 준비합니다. </li></ol> 2. 태아 수집 <ol><li> 한천 먹이 플레이트와 어댑터를 포함한 플라스틱 병 속에&gt; 100 파리를 배치하여 관심 Drosophila 변형에 케이지를 준비합니다. </li><li> 파리가 케이지에 acclimated하고 그들의 계란을 보유하지 않게, 접시마다 8-12시간을 변경, 72 시간 동안 실온에서 어둠 속에 파리의 새장을 둡니다. </li><li> 실험은 12 시간 동안 배아를 수집하십시오. 컨테이너에서 페트리 접시를 제거하고 교체합니다. 삭제 페트리 접시를 가지고 생리 식염수와 깨끗한 페인트 브러쉬를 사용하여 요리에서 배아를 씻으십시오. </li><li> 훌륭한 체에서 배아를 수집합니다. 초과 누룩을 제거하는 호수와 린스. </li><li> 주걱을 사용하면 부드럽게 배아를 선택하고 모자와 작은 유리관에서 그들을 놓으십시오. 생리 식염수의 1ml에 대해 추가합니다. </li></ol> 3. Chorion 및 Vitelline 점막의 제거 및 고정 <ol><li> 배아의 수집에 따라 50 % 헵탄 50 %의 정착액 결합하여 헵탄 - 수정 솔루션을 준비합니다. 솔루션은 상단과 하단에 정착액에 헵탄와 계층 것입니다. </li><li> 유리관에 5 분 동안 생리 식염수에서 그들을 잠복기로 배아를 씻으십시오. 파스퇴르 피펫을 사용하여 여분의 호수를 제거합니다. </li><li> 탈이온수와 배아를 씻어. </li><li> 3 분 50 % 표백제에서 그들을 잠복기로 배아를 Dechorionate. </li><li> 탈이온수로 철저하게 배아를 씻어. </li><li> 부드러운 흔들림과 헵탄 - 수정 솔루션 30 분 동안 그들을 잠복기로 배아를 수정. </li><li> 파스퇴르 피펫을 사용하면 메탄올이 튜브에서 헵탄를 떠나있는 정착액 (하단 레이어)를 대체합니다. vitelline 막을 제거하기 위해 적극적으로 흔들어. devitellinized 배아는 튜브의 바닥에 싱크 것입니다. </li><li> 파스퇴르 피펫을 사용하여 새 유리관에서 배아를 수집합니다. 태아에게 메탄올이있는 5 번 린스, 각 시간 5 분 동안 태아를 품어. </li></ol> 4. 항체 스테 이닝, 마운트, 그리고 시각화 <ol><li> 50% PBT 솔루션과 5 분 50 % 메탄올의 혼합물에서 배아를 Rehydrate. 그렇다면 5 분 백퍼센트 PBT 솔루션 rehydrate. </li><li> 4 1 시간 0.01 % NaAcide 솔루션 ° C.와 함께 5 % BSA에서 그들을 잠복기로 배아를 차단 </li><li> 배아 지금 항체 염색법에 대한 준비가되어 있습니다. 0.01 % NaAcide 솔루션 5 % BSA를 사용하여 적절한 농도로 diluting하여 기본 항체를 준비합니다. 우리는 시스테인 문자열 단백질 (CSP), 기공을 갖는 시냅스 단백질과 표면 glycoproteins와 라벨 모든 뉴런에 존재의 연결을 특정 - 탄수화물 잔기를 인식 방지 HRP 또는 말 무 퍼옥시데이즈 항체, 대한 항체를 사용합니다. </li><li> 4 밤새 배아 ° C 기본 항체 솔루션 인치를 품어 </li><li> 다음 날, 시간의 과정을 통해 PBT와 배아 10 번 씻으십시오. </li><li> 적절한 농도를 diluting하여 이차 항체 솔루션을 준비합니다. 우리는 1:200의 농도에 알렉사 차 항체를 사용합니다. </li><li> 4 두 시간 동안 태아를 품어 ° C 보조 항체 솔루션 인치 </li><li> 시간의 과정을 통해 태아에게 PBT 10 번 씻으십시오. </li><li> Vectashield 장착 매체 하룻밤 배아를 품어. </li><li> 유리 슬라이드에있는 배아를 탑재하고 매니큐어로 커버 슬립을 봉쇄. 형광 현미경을 사용하여 배아를 시각화. </li></ol> 5. 대표 결과 <img src="/files/ftp_upload/2150/2150fig1a.jpg" alt="그림 1" /> CSP <img src="/files/ftp_upload/2150/2150fig1b.jpg" alt="그림 1" /> HRP - FITC <img src="/files/ftp_upload/2150/2150fig1c.jpg" alt="그림 1" /> 병합 그림 1 : CSP (적색)와 HRP (녹색)에 대한 항체를 사용하여 스테이지 16-17에서 배아 CNS를 보여주는 대표 이미지. 접합면의 별개의 얼룩을 확인합니다. <img src="/files/ftp_upload/2150/2150fig2a.jpg" alt="그림 2" /> CSP <img src="/files/ftp_upload/2150/2150fig2b.jpg" alt="그림 2" /> HRP - FITC &quot;/&gt; 병합 그림 2 : CSP (적색)와 HRP (녹색)에 대한 항체를 사용하여 스테이지 16-17에서 배아 PNS를 보여주는 대표 이미지. 주변 뉴런의 반복 패턴을합니다.

<ol>
	<li>Bodmer, R., Barbel, S., Sheperd, S., Jack, J. W., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+sensory+organs+by+mutations+of+the+cut+locus+of+D.+melanogaster.">Transformation of sensory organs by mutations of the cut locus of D. melanogaster.</a> Cell. 51, 293-307 (1987).</li><li>Bodmer, R., Carretto, R., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+of+the+peripheral+nervous+system+in+Drosophila+embryos:+DNA+replication+patterns+and+cell+lineages.">Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages.</a> Neuron. 3, 21-32 (1989).</li><li>Campos-Ortega, J. A., Hartenstein, V. <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 Embryonic Development of Drosophila melanogaster. , (1985).</li><li>Carroll, S. B., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+fushi+tarazu+protein+during+Drosophila+embryogenesis.">Localization of the fushi tarazu protein during Drosophila embryogenesis.</a> Cell. 43, 47-57 (1985).</li><li>Dambly-Chaudi&#232;re, C., Ghysen, A., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+connections+between+imaginal+discs+in+Drosophila.">Muscle connections between imaginal discs in Drosophila.</a> Developmental Biology. 113, 288-294 (1986).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Drosophila의 배아가의 연결을 개발하는 동안 세포와 분자 변화를 조사위한 강력한 시스템입니다. 이 프로토콜에서는 immunohistochemistry에 대한 전체 마운트 배아를 준비하는 방법을 증명하고있다. 우리는 캐롤과 스콧, 1985 년 설명된 프로토콜에서이 프로토콜을 적응있다. 이 프로토콜은 다른 항체의 연결을 테스트하는 적응 될 수 있지만, 항체의 최적화 할 수 있습니다. 또한, 배아 PNS와 CNS의 발...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>37% formaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP531-25</td>
<td>&nbsp;</td>
<tr>
<td>CSP</td>
<td>&nbsp;</td>
<td>Developmental Studies Hybridoma Bank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HRP-FITC</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch Laboratories</td>
<td>323-095-021</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 568 Goat Anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield Mounting Medium</td>
<td>&nbsp;</td>
<td>Vector Laboratories, Inc.</td>
<td>H-1000</td>
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visualization of the embryonic nervous system in whole-mount drosophila embryos

Drosophila의 배아가의 연결을 발전의 세포 및 분자 기초를 조사하는 매력적인 모델 시스템입니다. 여기의 연결을 단백질에 항체를 사용하여 Drosophila 배아 신경 시스템의 시각화를위한 절차를 설명합니다. 전체 배아 주변 신경 및 중추 신경 시스템이 잘 개별 세포 수준 (..; Bodmer 외, 1987;. Dambly - 쇼디에르 외, 1986 Bodmer 외, 1989)에서 특징 때문에, 이러한 시스템에 어떤 aberrations 수 쉽게 다른 단백질의 연결을하기 위해 항체를 사용하여 식별됩니다. 개발 배아는 배아는 시각화에 대한 적절한 발달 단계에 있는지 확인하기 위해 특정 시간에 수집하고 있습니다. 수집 후, 배 (胚)의 바깥쪽 레이어, chorion 막과 배아를 둘러싼 vitelline 봉투가 고정되기 전에 제거됩니다. 배아는 다음의 연결 항체와 incubated하고 찬란 레이블 차 항체를 사용하여 시각입니다. 단계에서 배아가 12-17 배아 신경 시스템에 액세스할 시각입니다. 12 단계 CNS의 배아 밴드 단축을 시작하고 단계 15 일까지 접합면의 확실한 패턴이 달성되었습니다. 단계 17으로 CNS 계약 및 PNS는 완전히 (캄포스 - 오르테가 외. 1985) 개발되고 있습니다. 따라서 PNS와 CNS의 패턴의 변화는 쉽게 이러한 발달 단계 동안 볼 수 있습니다.

Watch this Scientific Journal Video about Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos at JoVE.com

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

우리는 개최 준비하는 절차를 설명<em&gt; Drosophila</em&gt; embryogenesis 동안 배아 신경 시스템의 시각화를위한 배아.

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 ...

visualization-embryonic-nervous-system-whole-mount-drosophila

Research

JoVE Journal

Neuroscience

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

13.1K 조회수.  SUNY-University at Buffalo. 우리는 개최 준비하는 절차를 설명 Drosophila embryogenesis 동안 배아 신경 시스템의 시각화를위한 배아.

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

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 ...

연구

신경과학

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

의 Proprioceptors의 시각화<em&gt; Drosophila</em&gt; 애벌레와 Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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

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.

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 microscopy.

의 중추 신경계에 단일 세포의 라벨<em&gt; Drosophila melanogaster</em

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

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

에 중추 신경 시스템 복구를 조사하기 위해 상해 패러다임<em&gt; Drosophila</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

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 ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

설치류 중추 신경계의 생체 Optogenetic 자극

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

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 ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

장용 신경계의 제자리 칼슘 이미징에서

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

간단한 한 단계 성인의 전체 마운트 준비에 대한 해부 프로토콜<em&gt; 초파리</em&gt; 두뇌

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...