우리는 현미경에 도움이 토론 게리 Laevsky 감사합니다. NIH에 의해 투자 된이 작품은 ERG에 R01GM061107 및 R01GM067758을 부여

주 : 근육을 제거 (도 1)에 대한 절차는 유충 필렛을 제조하는 전술 한 방법의 변형이다. 선행 근육 제거를 수행하는 단계는 간단하게 설명하고 리더 상세한 설명은 이전 연구 10, 11로 지칭된다. 차가운 식염수 1. 해부 애벌레 <ol><li> 차가운 HL3.1 식염수 15 차가운 칼슘 2 + - 무료 HL3.1 식염수 (11) (표 1)의 작동 희석을 준비합니다. 접시의 바닥을 충당하기 위해 충분한 차가운 생리 식염수와 실리콘 엘라스토머 접시에 애벌레를 놓습니다. 참고 : 식염수의 선택에 관한 토론을 참조하십시오. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1 배 HL3.1 식염수 (PH 7.2) </td></tr><tr><td> 5 mM의 HEPES </td></tr><tr><td> 70 mM의 염화나트륨 </td></tr><tr&gt; <td> 5 밀리미터의 KCl </td></tr><tr><td> 1.5 mM의 염화칼슘 2 (칼슘 - 무료 식염수를 위해 생략) </td></tr><tr><td> 4 밀리미터의 MgCl 2 </td></tr><tr><td> 10 밀리미터의 NaHCO3 </td></tr><tr><td> 5mM의 트레 </td></tr><tr><td> 115 MM의 자당 </td></tr><tr><td></td></tr><tr><td> 4 ° C에서 필터 소독 및 저장 </td></tr><tr><td> 참고 : 체액 곤충 구성을 모방 </td></tr></tbody></table> 차가운 식염수 표 1. 구성. <ol start="2"><li> 복부면을 위로 유충을 배치합니다. 유충의 복부 표면은 전방에서 후방으로 실행하는 기본 애벌레 기관 튜브에 의해 복부 dentical 벨트와 지느러미 측에서 식별 할 수 있습니다. 전방 - 후방 방향으로 유충을 스트레칭과에 머리와 꼬리를 핀곤충 핀을 사용 접시 해부 실리콘 엘라스토머. 한쪽 끝에서 시작하여 다른 방향으로 진행, 복부 정중선을 따라 잘라 미세 해부 가위를 사용합니다. 참고 :이 방향은 다 신경 세포의 일반적 연구 등의 클러스터를 유지합니다. </li><li> 유충이 절개 된 후, 책을 여는 것처럼 해부 접시 네 모서리를 고정. 중추 신경계, 창자 및 기관을 포함한 내부 장기를 잡아 제거하는 집게를 사용합니다. 필렛이 팽팽하지만 최대한 뻗어있다 그래서 곤충 핀을 조정합니다. 참고 : 근육은 분절 경계에서 몸 벽에 고정된다. 근육이 몸 벽의 대부분을 커버하지만, 그들은 지느러미 중간 선 부근의 좁은 영역에 존재하지 않는다. </li></ol> 2. 근육 제거 <ol><li> 근육 조직이 존재하지 유충의 지느러미 중간 선을 찾습니다. 이 평평한 가능한 방향으로 근육 조직 아래에 삽입 될 수있는 하나의 셉 단자를 배치합니다. </li><li> 에서 시작지느러미 중간 선은 세그먼트의 전방 경계 근처에 조심스럽게 집게와 표피 사이의 접촉을 최소화하기 위해주의하면서 근육과 표피 사이의 겸자 단자를 밀어 넣습니다. </li><li> 하나의 고정 점에서 몸 벽 근육의 부착을 깰 위로 집게를 잡아 당깁니다. 관심의 나머지 헤미 세그먼트 (들)에 대해이 과정을 반복합니다. 참고 :이 프로토콜은 각각 다 신경 세포의 수상 돌기 필드의 후방의 보존을 최적화합니다. 수지상 필드의 앞쪽 부분을 유지하려면, 각 세그먼트의 후방 끝 부분에 집게의 단자를 삽입하는 것이 가장 좋습니다. </li><li> 애벌레 필렛이 최대로 모든 방향으로 뻗어되도록 곤충 핀을 다시 조정합니다. </li><li> 그것은 아직도 25 분 동안 PBS에서 감기, 새로 제조 한 4 % 포름 알데히드를 사용하여 해부 접시에 고정되어있는 동안 필렛을 수정합니다. </li><li> PBS에 5 번 씻어. </li><li> 접점을 최소화하기 위해주의하면서 조심스럽게 나머지 고정 점에서 근육 조직을 멀리 당겨 집게를 사용하여표피와 CT. </li><li> 고정 해제 및 1.5 ml의 microcentrifuge 관에 해부 접시에서 필렛을 제거합니다. 이전 11 설명 된 바와 같이, 모든 후속 세척, 차단 및 면역 형광 염색 단계를 수행합니다. </li></ol> 3. 마운트 애벌레 필렛 <ol><li> 우선 가능한 샘플이 평평하게하기 위해 미세 해부 가위를 사용하여 머리와 꼬리를 제거합니다. 커버 슬립에 직면 필렛의 내면 antifade mountant에 커버 슬립에 필렛을 탑재합니다. </li><li> 커버 슬립에 현미경 슬라이드를 삽입하고 설치 매체를 분산 부드럽게 누르십시오. 위에 현미경 슬라이드 플립과 매니큐어를 사용하여 슬라이드에 커버 슬립을 밀봉. 프레젠테이션은 적어도 한 달 동안 -20 ℃에서 저장 될 수있다. </li></ol>

<ol>
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저자는 공개 아무것도 없어.

여기 프로토콜 초파리 유충 필렛의 근조직의 수동 제거를 위해 설명된다. 이 프로토콜은 이전에 애벌레 해부 기술 10, 11 설명 수정합니다. 유충은 실리콘 엘라스토머 접시에 해부 한 후, 지느러미 중간 선이 있습니다. 단일 셉 가닥은, 그 평탄한 가능한 방향으로 조심스럽게 지느러미 중간 선 근처의 근육 조직과 표피 사이에 삽입됩니다. 포셉 부드럽게 관심의 각 애벌레 세그먼트에...

초파리 애벌레 수지상 arborization (다) 신경 세포는 그들의 유전자 조작에 복종 할 의무 그들이 이미지화 할 수있는 용이성에 신경 발달을 연구하기위한 귀중한 모델을 제공합니다. 이 감각 뉴런은 수상 돌기의 형태 형성 1-3을 제어하는 다양한 경로의 식별 수단이되고있다. 다 뉴런의 네 가지 클래스 (클래스 I - IV) 애벌레 표피 신경을 분포시키다. 이러한 신경 세포는 수상 돌기는 주로 2 차원 배열을 형성 4,5-으로 기저막와 표피 사이에 놓여. 네 개의 클래스, 클래스 IV 다 신경 세포는 다른 동물의 감각 뉴런과 같은 가장 높은 분기 아버과를 가지고,이 아버의 동화는 개발 6-9에 대한 주변 조직에서 고유 요소뿐만 아니라 큐, 특히 표피가 필요합니다 . 연구 방법 등의 연결 및 추가-신경 사실을 확인하는 방법ORS 면역 형광에 의해 반응계 내에서 단백질 발현을 검출하는 능력에서 수지상 형태 형성 이득을 제어한다. 유충의 외부 표피는 항체에 꿰 뚫을 수없는, 그러나이 장애는 쉽게 잘 확립 된 해부 방법 10, 11을 통해 애벌레 필렛의 준비에 의해 극복된다. 그러나, 기저막 단지 내부 놓여 체벽 근육 조직 DA 신경 세포 및 상피 세포의 가시화를 향해 여러 과제를 제공한다. 우선, 근육 조직, 체벽 대부분 라인은 크게 신경 또는 표피 조직에서 나오는 형광 신호를 가린다. 이것은 실질적으로 샘플 신호 대 잡음비를 감소시킨다. 둘째, 많은 관련 단백질은 근육 조직에서뿐만 아니라 신경 세포 또는 표피에서 발현 될 수있다. 이 근육 유래 형광 신호는 신경 세포 또는 표피에서 형광 신호의 상기 모호한 검출 쉽다. 마지막으로, 아니 현미경 기술의 발전서브 회절 해상도에서 샘플 w 허가 이미징 및 상피 세포 (12, 13)를 신경 세포에서 발현되는 단백질의 현지화 안목과 주변에 특히 도움이 될 수 있습니다. 그러나 잡음비 및 커버 슬립을 시료의 근접에 강한 신호로부터 초해 현미경 이점 통해 촬상. 신호 대 잡음 비를 감소 이외에 유생 체벽 근육 거리시켜 초 해상 현미경 방법으로 달성 될 수있는 개선 된 이미지 해상도를 한정 커버 슬립에서 DA 뉴런. 면역 형광 분석을위한 과제 외에도, 근육 조직은 애벌레 몸 벽에 감각 뉴런에서 전기 생리 기록에 대한 장벽을 제공합니다. 그것의 제거는 따라서 감각 뉴런 (14)의 신경 생리 학적 조작 도움이됩니다. 여기 초파리 유충 근조직의 수동 제거하는 방법이 기재되어있다. 우리는 우리의 프로토콜 페이지 입증그렇지 근조직 의해 가려 단백질 ermits 면역 촬상 클래스 IV 다 뉴런의 시각화를위한 신호 대 노이즈 비율을 개선하고, 더 나은 DA 신경 단백질 및 세포 구조의 공간적 관계를 식별하기 위해 초해 현미경의 사용을 가능하게하며 표피.

<table border="0" cellpadding="0" cellspacing="0" width="1299">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dumont #5 tweezers</td>
			<td style="width:304px;">Electron Microscopy Sciences</td>
			<td style="width:151px;">72701-D</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips</td>
			<td>World Precision Instruments</td>
			<td>14003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>for dissecting plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Austerlitz insect pins, 0.1 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fostec 8375 light source</td>
			<td>Artisan Technology Group</td>
			<td>62792-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss Stemi 2000</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Diamond antifade mountant</td>
			<td>Life Technologies</td>
			<td>P36970</td>
			<td>for structured illumination microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro cover glass, 22x22 mm, No. 1.5</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus microscope slides, 25 x 75 x 1.0 mm</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Coracle antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>C615.16</td>
			<td>supernatant, dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Discs large antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td>supernatant, dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-dsRed antibody</td>
			<td>Clontech</td>
			<td>632496</td>
			<td>dilute 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit antibody, Alexa Fluor 568 conjugated</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11011</td>
			<td>dilute 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse antibody, Alexa Fluor 488 conjugated</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11001</td>
			<td>dilute 1:500</td>
		</tr>
	</tbody>
</table>

removal of drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells

초파리 애벌레 수지상 arborization (다) 신경 세포는 신경 세포의 형태 형성의 메커니즘을 조사하는 인기 모델입니다. 다 신경 따라서 표피가 신경을 분포시키다 세포와 면역에 의해 모두 neuronally 및 epidermally 발현 된 단백질의 현장 시각화에서 자신의 분석 혜택과 통신 개발한다. 면역 실험 애벌레 필렛을 준비하는 전통적인 방법은 신경 세포와 표피 단백질 이미징 몇 가지 과제를 제시, 신체 벽의 대부분을 덮고있는 근육 조직을 그대로 둡니다. 여기에서 우리는 초파리 애벌레 필레에서 근육 조직을 제거하는 방법을 설명합니다. 이 프로토콜은, 달리 근육 조직에 의해 가려 단백질의 촬상을 가능하게 신호 대 잡음 비를 향상시키고, DA 신경 발달을 연구 초해 현미경의 사용을 용이하게한다.

우리는 함께 막 마커로 표지 클래스 IV 다 신경과 중격 접합 단백질 Coracle (코라) 및 디스크 대형 (통해 Dlg)를 공동 시각화 면역 실험에서 신호 대 잡음 비를 향상시키기 위해 근육 제거의 유용성을 입증 CD4- tdTomato. 코라 이전 DA 신경 돌기는 상피 세포에 의해 둘러싼 다 신경 세포의 형태 형성 및 기능과 관련하여 연구를 4,6-9,16 돌기 한 많은 식?...

Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

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

초파리 애벌레 수지상 arborization (다) 신경 세포를 사용하여 신경 세포의 형태 형성의 연구는 면역에 의해 신경 세포와 표피 단백질의 현장 시각화 혜택을 누릴 수 있습니다. 우리는 애벌레 몸 벽에서 근육 조직을 제거하여 다 신경 세포와 주변 표피 세포의 면역 형광 분석을 개선하는 절차를 설명합니다.

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 ...

removal-drosophila-muscle-tissue-from-larval-fillets-for

Research

JoVE Journal

Neuroscience

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

8.9K 조회수.  Princeton University.  초파리 애벌레 수지상 arborization (다) 신경 세포를 사용하여 신경 세포의 형태 형성의 연구는 면역에 의해 신경 세포와 표피 단백질의 현장 시각화 혜택을 누릴 수 있습니다. 우리는 애벌레 몸 벽에서 근육 조직을 제거하여 다 신경 세포와 주변 표피 세포의 면역 형광 분석을 개선하는 절차를 설명합니다. 

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

연구

신경과학

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

의 형태학의 분석 Drosophila</em유전 모자이크 사용하기> 애벌레 주변 감각 신경 세포의 Dendrites 및 Axons

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

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 ...

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 ...

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 ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

생화학 적 분석에 대한 감각 뉴런에서 생산 및 축삭의 분리는 다공성 필터를 사용하여

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

병아리 배아에서 분리 및 해리 감각 뉴런의 문화

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

미국 황소 개구리의 Sacculus (헤어 세포의 생리 준비<em&gt; 모이 catesbeiana</em&gt;)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

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.