Name: ex vivo culturing of whole, developing drosophila brains
Uploaded: 2012-07-27T13:00:00
Description: Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

우리는 원고에 대한 그들의 조언, 유용한 토론 및 의견을 저희 연구소의 모든 구성원을 감사드립니다. 우리는 또한 특히 우리가 개조 실험에서 culturing 위해 머리를 해부하기 전에 1.5 시간 APF 때까지 기다리는 것이 좋은 제안 마르타 코흐와 Bassem 하산을 감사드립니다. 우리는 또한 그들의 공촛점 현미경의 사용을위한 NHGRI 이미징 시설을 감사드립니다. 이 작품은 NINDS 교내 연구 프로그램, NIH (Z01 NS003106)의 기초 신경 과학 프로그램에 의해 지원되었다.

미디어 A. 준비 <ol><li> 필터 - 살균 슈나이더의 Drosophila 미디어 10 % 태아 소 혈청과 1퍼센트 페니실린 / 스트렙토 마이신과 함께 보완하여 완벽한 미디어를 준비합니다. </li><li> 미디어는 매번 신선한 준비되어야한다. 또는 미리 냉동 미디어의 신선한 나누어지는 각 사용하기 전에 해동 수 있습니다. </li><li> culturing에 앞서, 10 μg / ML의 인슐린과 미디어 2 μg / ML ecdysone를 추가합니다. 약물은 농...

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D., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+pruning+during+Drosophila+metamorphosis:+evidence+for+local+degeneration+and+requirement+of+the+ubiquitin-proteasome+system.">Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system.</a> Neuron. 38 (6), 871-885 (2003).</li><li>Lee, T., Marticke, S., Sung, C., Robinow, S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+requirement+of+the+USP/EcR-B+ecdysone+receptor+for+mushroom+body+neuronal+remodeling+in+Drosophila.">Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila.</a> Neuron. 28 (3), 807-818 (2000).</li><li>Connell-Crowley, L., Le Gall, M., Vo, D. J., Giniger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cyclin-dependent+kinase+Cdk5+controls+multiple+aspects+of+axon+patterning+in+vivo.">The cyclin-dependent kinase Cdk5 controls multiple aspects of axon patterning in vivo.</a> Curr. Biol. 10 (10), 599-602 (2000).</li><li>Truman, J. W., Riddiford, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+insights+into+the+evolution+of+metamorphosis+in+insects.">Endocrine insights into the evolution of metamorphosis in insects.</a> Annu. Rev. Entomol. 47, 467-500 (2002).</li><li>Noguchi, T., Miller, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+actin+dynamics+in+individualization+during+spermatogenesis+in+Drosophila+melanogaster.">A role for actin dynamics in individualization during spermatogenesis in Drosophila melanogaster.</a> Development. 130, 1805-1816 (2003).</li><li>Siller, K. H., Serr, M., Steward, R., Hays, T. S., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+brain+neuroblasts+reveals+a+role+for+Lis1/dynactin+in+spindle+assembly+and+mitotic+checkpoint+control.">Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.</a> Mol. Biol. Cell. 16 (11), 5127-5140 (2005).</li><li>Brown, H. L. D., Cherbas, L., Cherbas, P., Truman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+time-lapse+imaging+and+dominant+negative+receptors+to+dissect+the+steroid+receptor+control+of+neuronal+remodeling+in+Drosophila.">Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling in Drosophila.</a> Development. 133, 275-288 (2006).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Developing+and+Adult+Drosophila+Brains+and+Retinae+for+Live+Imaging.">Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging.</a> J. Vis. Exp. (37), e1936 (2010).</li></ol>

<html> 변이와 transgenes 표현을 포함한 조직 구조와 기능의 만성 유전자 조작은 일반적으로 구분하는 것은 매우 어려울 수 즉각적인 효과와 늦은 간접 결과의 조합을 생산하고 있습니다. 약리학과 유전 방식을 보완하는 것은 표현형 뒤에 시간 코스의 심문을 용이하게하실 수 있습니다. 예를 들어, 이것은 Drosophila 10 spermatogenesis 동안 다른 cytoskeletal 구성 요소의 기여를 해부에 대한 강력...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 최종 농도 </td></tr><tr><td> 슈나이더의 Drosophila 미디어 </td><td> Invitrogen </td><td> 21720-024 </td><td></td></tr><tr><td> 태아 소 혈청 (열-inactivated) </td><td> Invitrogen </td><td> 10082-1​​47 </td><td> 10% </td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> Invitrogen </td><td> 15140-122 </td><td> 1퍼센트 </td></tr><tr><td> 인슐린 </td><td> Invitrogen </td><td> 12585-014 </td><td> 10μg/ml </td></tr><tr><td> Ecdysone </td><td> 시그마 - 올드 리치 </td><td> H5142 </td><td> 2μg/ml </td></tr><tr><td> Roscovitine </td><td> 시그마 - 올드 리치 </td><td> R7772 </td><td> 100μM </td></tr><tr><td> Olomoucine </td><td> 시그마 - 올드 리치 </td><td> O0886 </td><td> 100μM </td></tr><tr><td> 정상 염소 혈청 </td><td> Invitrogen </td><td> 50062Z </td><td> 1퍼센트 </td></tr><tr><td> 소의 알부민 세럼 </td><td> 피셔 과학 </td><td> M9501 </td><td> 1퍼센트 </td></tr><tr><td> 포름 알데히드 </td><td> 시그마 - 올드 리치 </td><td> D4551 </td><td> 4퍼센트 </td></tr><tr><td> 트리톤-X 100 </td><td> 피셔 과학 </td><td> BP151 </td><td> 0.5 % </td></tr><tr><td> 인산염은 염분 버퍼 </td><td> Invitrogen </td><td> 70011-044 </td><td> 1X </td></tr><tr><td> Vectashield </td><td> 벡터 연구소 </td><td> H1000 </TD&gt; <td></td></tr><tr><td> Millicell 셀 문화 삽입물 </td><td> Millipore </td><td> PI8P01250 </td><td></td></tr><tr><td> Multidish 잘 사 </td><td> 써모 과학 </td><td> 176,740 </td><td></td></tr><tr><td> 뒤몽 # 5 포셉 </td><td> 파인 과학 도구 </td><td> 11251-20 </td><td></td></tr><tr><td> 현미경 슬라이드 </td><td> 피셔 과학 </td><td> 12-544-7 </td><td></td></tr><tr><td> 유리 커버 </td><td> 피셔 과학 </td><td> 12-548 - 5M </td><td></td></tr></tbody></table>

ex vivo culturing of whole, developing drosophila brains

우리는 전체 Drosophila의 두뇌의 전직 생체내의 culturing에 대한 방법을 설명합니다. 이것은 급성 pharmacological 개입과 세포 과정의 라이브 영상을 허용하여 세포 생물학 및 중앙 뇌 구조의 개발을 조사하는 만성 유전 조작에 대위법으로 사용할 수 있습니다. 기법의 예를 들어, 이전에 우리 실험실 1부터 작동은 이전에 인식되지 않는 subcellular 구획은 Drosophila 중앙 뇌의 axons의 axonal 및 somatodendritic 구획 사이에 놓여 있다고 나타났습니다. 축삭 초기 세그먼트 (AIS) 2로 언급이 구역의 개발은, 신경 세포 특정 cyclin 의존 키나제, Cdk5에 의존하는 유전자 표시되었다. 우리는 Cdk5 특정 pharmacological 억제제 roscovitine 및 olomoucine 3 야생 형 Drosophila 애벌레 두뇌의 전직 생체내 치료는 actin 조직의 급성 변화를 일으키는, 그리고 여기에 표시유전자 Cdk5 활동이 결여 돌연변이에서 본 변화를 모방 세포 표면 단백질 Fasciclin 2의 국산화 인치 전 생체내 문화 기술의 두 번째 예제는 배아 버섯 본문 (MB) 애벌레에서 어른으로 변태하는 동안 감마 뉴런의 연결을 리모델링을 위해 제공됩니다. 버섯 몸은 후각 학습과 파리 4 메모리의 중심지이며, 이러한 감마 뉴런은 성인 innervation 패턴 5를 확립하기 위해 나중 timepoint에 다시 확장 지점 그리고 pupal 개발 중에 axonal와 돌기 가지를 치다합니다. MB 중 이러한 뉴런의 가지치기는 ecdysone 수용체 지하 1 층 7 시에 연기, ecdysone, 스테로이드 호르몬에 의해 실행되는 메커니즘에 의해, neurite 지점 6 지방 변성을 통해 발생하는 것으로 표시되고, 그의 활동에 따라 좌우되어 ubiquitin-proteasome 시스템 6. 전직 생체내의 culturing 우리의 방법은 B 수e는 더욱 발달 리모델링의 메커니즘을 심문하기 위해 사용. 우리는 전 생체내 문화 환경에서, MB 중 감마 뉴런은 생체내에서 해당과 비슷한 시간 코스 발달 가지치기의 과정을 recapitulated 것으로 나타났습니다. 그것은 세포 변형에 레스터 저지하기 위해 조직을 explanting 전에 1.5 시간 번데기 형성 이후까지 기다려야하지만, 필수입니다; 문화에 거의 없거나 전혀없는 변태로 이끌어 pupariation가 시작될 동물의 해부. 따라서, 적절한 수정으로 전직 생체내 문화 접근법뿐만 아니라 중앙 뇌 생물학의 안정 상태 측면과 같은 역동적인 공부를 적용할 수 있습니다.

Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Ex vivo Culturing of Whole, Developing Drosophila Brains

이 문서 하나가 모방 수있는가하는 방법을 설명합니다<em&gt; 생체내에서</em중&gt; 개발<em&gt; Drosophila</em관련&gt; 버섯 본문<em&gt; 예 생체내</em&gt; 문화 시스템.

전체의 전의 생체내의 Culturing, 개발 Drosophila의 뇌는

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

ex-vivo-culturing-of-whole-developing-drosophila-brains

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Neuroscience

Ex vivo Culturing of Whole, Developing Drosophila Brains

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

개발 마우스 망막의 생체내 Electroporation에

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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신경과학

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

뇌 조각의 공포 회로의 예 생체 Optogenetic 해부

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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 ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

전 Utero Electroporation 및 마이그레이션 GABAergic 수의 라이브 이미징 배아 마우스 두뇌의 Organotypic 조각 문화

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

전 비보 내 분 비 신호 초파리 에 두뇌 응답을 시각화 칼슘 이미징

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

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 ...

마우스 Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

저자 스포트라이트: 녹내장 진단 및 치료 개선을 위한 In Vivo 및 Ex Vivo 망막 영상의 발전 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

Align of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina(가시광선 광간섭 단층 촬영 파이버그램과 동일한 마우스 망막의 컨포칼 이미지 정렬)

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Preparation for Ex Vivo Color Fundus Photography 

To begin the ex vivo color fundus photography, set the high-definition multimedia interface camera to the manual ISO function. Arrange two light sources perpendicular to each other and turn the epi-illumination light sources to full power. At the dissecting microscope, insert the one-millimeter ruby bead into the posterior pole.Next, insert the posterior pole of the eye globe into a 30-milliliter quartz crucible filled with buffer. When the tissue sinks, insert a bracing between the eye and the wall of the crucible. Place the globe in the crucible onto the stereomicroscope stage to observe the interior ocular fundus through the microscope using the lowest magnification.Orient the eye by identifying the tissue mark at the 12 o'clock position, the optic nerve head, and the fovea five degrees below the optic nerve head.