저자는 영상 내레이션을 제공하는 기술 지원 및 지원, 제임스 바튼에 대한 알렉산드라 오피을 인정하고자하고, 첨부 된 영상에 나타나는에 대한 라모나 Flatz 및 Joellen 스위니. 이 작품은 MJ 머독 자선 신탁 (JED에 부여 번호 2012205)에 의해 지원되었다.

1 스테레오 현미경 시스템 주 :이 프로토콜은 쉽게 영상을 취득하는 기능과, 디지털 카메라에 결합 된 거의 모든 스테레오 현미경 시스템에 적용 할 수 있지만, 정보는 (재료 / 장비의 도표) 연구실에서 사용되는 시스템에 제공된다. <ol><li> 상업적 디지털 카메라에 결합 안 현미경 스테레오 현미경을 이용하여 디지털 영상을 획득. </li><li> 커플 스테레오 현미경 안 현미경 포트 상업적 디지털 카메라를 위해, 스테레오 현미경 광전관 포트 ½x C-마운트를 제거하고 1X C-마운트로 대체. </li><li> 1X C-마운트에 디지털 카메라 커플러 (43mm 스레드)를 탑재합니다. </li><li> 카메라 커플러에 실장이 스텝 다운 링 48mm까지 58mm, 43mm 및 48mm로는, 디지털 카메라를위한 렌즈 어댑터 키트에 디지털 카메라 커플러에서 연결 다리. </li><li> 렌즈 어댑터 키트에 디지털 카메라를 탑재합니다. </li><li> 약 12 배의 결합 배율 (초당 30 프레임, 640 × 480 픽셀)에 대한 현미경 배율 디지털 카메라 세트의 광학 줌과 비디오를 획득. 참고 : 스테레오 현미경의 배율은 삼안 포트의 새롭게 재구성 1X C-마운트에 따라 보상해야합니다. </li></ol> 2 이미징 초파리 셋째 유충 <ol><li> 테이프 마커 캡의 측면이 대략 차지하도록 디지털 카메라에 결합 스테레오 현미경 검정 스테이지 플레이트 영구 마커 ⅓ 카메라의 LCD 모니터에서 관찰 된 뷰의 수직 필드의 ¼한다. 그들이 색상 코드에 사용 이미지화되는 유충의 유전자형을 구별 할 수있는 색상의 구색에 와서 때문에 애벌레 이미징을 수행 할 수있는 단계로 마커 상판을 사용합니다. </li><li> 미세한 포인트 마커의 상부 표면 상에 디지털 카메라의 LCD 모니터에서 관찰 시야를 구별마커. </li><li> 이미지에 대한 제 령 유충을 선택합니다. 셋째 령 유충을 선택하기위한 기준은 몸체 길이, 생활주기의 애벌레 단계, 전방 및 후방 spiracles의 존재 중에 음식 소스로부터 출현하고, 구강 장치 (11)의 하악 후크의 구조였다. 유충은 물 속에서 철저하게 세척하여 청소합니다. </li><li> 광섬유 조명 시스템으로부터의 광 위에서 영구 마커 위쪽 단을 밝히는. 최적의 조명을 제공하는 입사광의 각도를 조정한다. </li><li> 영구 마커 상단의 가장자리에 현미경 초점을 맞 춥니 다. 디지털 영상을 획득 시작합니다. </li><li> 시야를 향 유충 (그림 1)의 전방으로 바로 시야 밖에서 약 75 ° 떨어진 수직 축에서 마커 캡의 측면에 애벌레를 놓습니다. 주 : 마커 캡의 측면에 배치 유충은 번째의 레코드에 카메라 움직임을 허용측면 관점에서 전자 유충. 그것은 그들이 마커 캡의 측면에서 떨어지지 않도록 물과 습기가 애벌레를 유지하는 데 도움이됩니다. 케어는 들판을 가로 질러 크롤 과도한 양의 유충을 준수하므로 물을 너무 많이 사용하지 않는, 그러나 운동을해야합니다. </li><li> 조심스럽게 찌르고보기의 들판을 가로 질러 크롤링을 강요하는 작은 페인트 브러시와 유충을 자극. 애벌레는 거의 협력하지 않고는 직선 들판을 가로 질러 기어 전에 자주 시작점으로 많은 시간을 반환해야 할 환자로합니다. </li><li> 기록 약 10-15 중단 디지털 비디오 영상과 작물의 분 및 디지털 비디오 편집 소프트웨어와 불필요한 영상의 취득 후 제거합니다. </li></ol> 3 이미징 성인 초파리 <ol><li> 일회용 1.5 ml의 분광 폴리스티렌 큐벳에 하나의 성인 초파리를 놓습니다. 참고 : 성인 초파리의 CO 2 진통제를 즉시 난연성 전에ioral 분석 프로토콜은 결과 (12)을 손상시킬 수 있습니다. 성인 초파리가 행동 검사 (13)에 수행하기 전에 CO 2 진통제를 복구하기 위해 24 시간의 기간을 부여하는 것이이 좋습니다. </li><li> 작은면 공을 큐벳의 끝을 연결합니다. 코튼 볼이 넓은 모자 공간을 차지할 정도로 꽉 포장되어 있는지 확인하고, 베트의 감소 된 구획으로 비행을 제한한다. </li><li> 실체 현미경의 스테이지 흰색 접시에 놓고 큐벳을 적절히 디지털 카메라 LCD 모니터에서 관찰 시야와 큐벳의 감소 된 용적 구획 정렬. </li><li> 광섬유 조명 시스템으로부터의 광 위에서 큐벳 밝히는. 최적의 조명을 제공하는 입사광의 각도를 조정한다. </li><li> 현미경 초점 및 디지털 비디오를 획득 시작합니다. </li><li> 기록 약 30-45 중단 디지털 비디오 영상과 작물의 분, 불필요한 제거디지털 비디오 편집 소프트웨어와 영상의 취득 후. </li></ol>

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저자는 경쟁 이해 관계가 존재하지 않는 것을 선언했다.

신경계의 기능을 연구하기위한 모델 시스템으로 초파리 melanogaster의 강도는 크게 유전 가능한 강력한 도구 수렴 및 개발 강력한 행동 분석의 광범위한 유래한다. 여기서 우리는 측면 관점에서 초파리 성인 및 애벌레 기관차 표현형의 고품질 디지털 영상을 획득하는 간단하고 널리 접근 현미경 기술을 제시한다. 우리는 성공적으로 특성화하고 후방 마비의 정도를 정량화하기 위해이 ...

일반적인 과일 초파리 플라이는 1-3 신경계의 기능을 연구하는 실험 모델 강력한 시스템이다. 구조 진화 및 인간과 신경계의 기능의 보전,뿐만 아니라 유전자 조작의 용이성 및 유전 도구의 광대 한 배열은 초파리보기 인간 신경 퇴행성 질환 (4)를 모델링하는 초연 유기체를 만든다. 신경계의 기능 장애의 원인이 유전자 변이는 종종 장애인 운동으로 가능한 돌연변이 애벌레와 성인 초파리 초래한다. 신경계 결함이있는 돌연변이 체에서 관찰 된 표현형은 운동, 비정상적인 조정하고, 성인의 경련 운동의 속도뿐만 아니라 신체 벽 근육의 연동 수축 적자, 유충의 부분 마비를 감소 등이 있습니다. 이 표현형은 높은 처리량 유전 화면과 돌연변이 유충 5의 운동 분석의 개발에 이용 된, 운동 손상을 정량화하고 신경계의 기능에 필요한 유전자를 식별하기위한 6 성인 7 ~ 10 초파리. 이러한 접근은 유충 및 성인 기관차 동작을 정량화하는 데 매우 유용하지만, 이들은 각각 특정 비정상적인 동작에 대한 정성적인 정보를 전달하지 못한다. 돌연변이 제 령 유충 행동 분석에서 변경된 운동력 파라미터를 나타낼 수있는 반면이 크롤링주기 동안 리듬 연동 수축의 변화, 조정의 일반적인 결여 또는 후방 신체 부분 마비의 결과 인 경우, 예를 들어, 불분명 할 수있다 벽의 근육. 여기서 우리는 측면 관점에서 초파리 성인 및 애벌레 기관차 표현형의 고품질 디지털 영상을 획득하는 간단하고 널리 접근 현미경 기술을 설명합니다. 측면 관점에서 인수 한 디지털 비디오 locomotiv의 미묘한 차이의 직접 관찰 및 분석을 할 수 있습니다더 많은 정보를 사이드 뷰 방향에서 전자의 행동.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Trinocular Stereozoom Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZ6145TR</td>
			<td>&#189; C-mount was removed and replaced with 1x C-mount</td>
		</tr>
		<tr>
			<td>1X C-mount</td>
			<td>Leeds Precision Instruments</td>
			<td>LSZ-1XCMT2</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera Coupler (43mm thread)</td>
			<td>Qioptiq Imaging Solutions</td>
			<td>25-70-10-02</td>
			<td></td>
		</tr>
		<tr>
			<td>58mm to 48mm Step Down Ring</td>
			<td>B&#38;H Video</td>
			<td>GBSDR5848</td>
			<td></td>
		</tr>
		<tr>
			<td>48mm to 43mm Step Down Ring</td>
			<td>B&#38;H Video</td>
			<td>GBSDR4843</td>
			<td></td>
		</tr>
		<tr>
			<td>Lensmate Adapter Kit for Canon G10</td>
			<td>LensMateOnline.com</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Canon PowerShot G10 Digital Camera</td>
			<td>Canon U.S.A., Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Spectroscopic PolystereneCuvette</td>
			<td>Denville Scientific</td>
			<td>U8650-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

acquisition of high-quality digital video of drosophila larval and adult behaviors from a lateral perspective

초파리 melanogaster은 신경계의 기능을 연구하기위한 강력한 실험 모델 시스템이다. 신경계의 기능 장애의 원인이 유전자 변이는 종종 가능한 유충 적절하게 텍스트로 설명하거나 완전히 하나의 사진 이미지로 표현하기 어려운 운동 결함이 표현형이 성인을 생산하고 있습니다. 과학 출판사의 전류 모드는, 그러나, 원고 동행 보조 재료로서 디지털 영상 매체의 제출을​​ 지원한다. 여기에서 우리는 측면 관점에서 모두 초파리 애벌레와 성인 표현형의 고품질 디지털 영상을 획득하는 간단하고 널리 접근 현미경 기술을 설명합니다. 이 탈선 기관차 행동의 관찰과 미묘한 차이와 변화의 분석을 할 수 있기 때문에 사이드 뷰에서 애벌레와 성인 운동의 비디오는 유리하다. 우리는 성공적으로 시각화하고 aberran 정량화하는 기술을 사용했다t는 정리를 포함하여 성인 돌연변이 표현형과 행동뿐만 아니라, 셋째 령 유충의 행동을 크롤링.

우리는 성공적으로 획득하고 stathmin 유전자의 기능의 상실 (그림 2) (14)와 관련된 애벌레 행동 표현형을 정량화하기 위해이 기술을 사용하고 있습니다. stathmin 유전자는 수용성 튜 불린의 풀에서 튜 불린 이량 체를 분할 미세 소관 규제 단백질을 암호화하고, 미세 소관 결합과 해체 (15, 16)을 촉진한다. Stathmin 기능은 말초 신경 축색 돌기 (14)의 미세...

Watch this Scientific Journal Video about Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective at JoVE.com

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

여기서 우리는 측면 관점에서 초파리 성인 및 애벌레 돌연변이 표현형의 고품질 디지털 영상을 획득하는 간단하고 널리 접근 현미경 기술을 설명합니다.

의 고품질의 디지털 비디오 취득<em&gt; 초파리</em&gt; 측면 관점에서 애벌레 및 성인 동작

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

acquisition-high-quality-digital-video-drosophila-larval-adult

Research

JoVE Journal

Neuroscience

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

8.7K 조회수.  Willamette University.  여기서 우리는 측면 관점에서 초파리 성인 및 애벌레 돌연변이 표현형의 고품질 디지털 영상을 획득하는 간단하고 널리 접근 현미경 기술을 설명합니다. 

비디오: Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3 애벌레 Segmental 신경의 시각화 차 Instar Drosophila 애벌레 준비

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

연구

신경과학

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

매핑 및 애벌레와 성인의 확장기 - 트랩 Flippase의 표현의 응용 Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

애벌레의, Pupal 및 성인의 해부 및 Immunohistochemistry<em&gt; Drosophila</em&gt; 망막

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

의 애벌레 먹이 회로의 기능적 분석 초파리</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

의 라이브 영상<em&gt; 초파리</em&gt; 애벌레 Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

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.

의 컴퓨터 추적하여있는 분석 후각 행동<em&gt; 초파리</em&gt; 네 사분면의 후각에

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

제거<em&gt; 초파리</em감각 뉴런과 표피 세포의 면역 형광 분석을위한 애벌레 필렛에서&gt; 근육 조직

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

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.

초파리 애벌레 동작 Optogenetic 후 각 신경의 자극에 대 한 응답 추적

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

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

초파리 melanogaster Larval 및 성인 두뇌의 변화 분석

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