This work was supported by grants from the U.S. National Institutes of Health (DA017279, AG033954, DA037743), the Michael J. Fox Foundation, and the Caltech Innovation Initiative funding the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology. We thank Brian Williams for optimising the RNA-Seq library protocol and for providing assistance. We thank Henry Amrhein for computational training. We thank Barbara J. Wold for the use of her equipment and laboratory space. We thank Igor Antoshechkin for library sequencing, and for facility management.

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The authors have nothing to disclose.

여기에서 우리는 단일 셀 RNA-서열과 각 셀을 연구, 형광 태그를 사용하여 이기종 인구에서 단일 세포를 분리. 우리는 수확 서열 라이브 TH-eGFP는 세포 100 % 다음 세 DA 관련된 유전자 전 사체, TH, DDC 및 slc6a3의 존재에 기초하여, 실제로 도파민 뉴런 있다고보고한다. RNA-서열에 의해 평가로 TH-eGFP는 양성 세포는 모두이 세 가지 유전자를 표명했다. 이는 각각의 TH-eGFP는 세포는 도파민 뉴런 ...

파킨슨 병 (PD)는 불치, 노화와 관련된 퇴행성 신경 질환이다. 이 비교적 흔한 질환의 원인은 제대로 이해 남아 있습니다. 운동 완만, 강성 및 진동의 진단 임상 양상에 이르는 Substantia 그라 (SNC)에서 도파민 뉴런의 뚜렷한 신경 세포의 변성과 뇌 전반에 걸쳐 광범위하게 신경 세포의 손실은있다. SNC의 도파민 신경 세포를 포함하는 차 혼합 문화는 파킨슨 병에 특히 적합하다. 복부 피개 영역 (VTA) 도파민 뉴런은 보상과 중독에 연루되어있다. 복부 중뇌 (VM) 차 혼합 배아 문화 SNC와 VTA의 도파민 (DA) 신경 세포와 GABA 성 신경 세포 모두를 포함한다. VM 일차 배양 신경 분석에 유용 할 수 있고, 도파민 성 신경 세포의 선택적 취약점 해명. 형태에 따라 문화에서 도파민 세포를 식별 할 신뢰할 수있는 방법은 없습니다. 그우리는 단일 셀, 고 수율 RNA 시퀀싱 라이브러리를 식별하고 하나의 도파민 신경 세포를 수확하고 구성하는 기술을 개발 재. 우리는 하나의 도파민과 중뇌 문화에서 고립 된 GABA 성 신경 세포에 대한 대표의 RNA 전 사체 데이터를보고합니다. 이 프로토콜은 DA / GABA 사체의 다양한 치료법의 효과를 연구하기 위해 신경 보호, 신경 퇴화, 및 약리학 적 분석에 이용 될 수있다. 도파민 뉴런 VM 차 배양에서 발현 뉴런의 소수를 표현하기 때문에, 안정적으로 생활 문화 뉴런을 식별하는 능력은 단일 셀 연구 향상된 범위를 가능하게 할 것이다. 이 새로운 기술은 세포 수준에서 발생하는 메커니즘을 이해하는 발전을 촉진하고 다른 신경 및 분자 생물학 분야에서의 응용을 가질 수있다.

<table><tbody><tr><td>Papain</td><td>Worthington Biochemical Corporation&amp;nbsp;</td><td>LS003126</td><td></td></tr><tr><td>Hanks-balanced Salt Solution (HBSS), 1x</td><td>Gibco&amp;nbsp;</td><td>14175-095</td><td></td></tr><tr><td>Donor Equine Serum</td><td>Thermo Scientific&amp;nbsp;</td><td>SH30074.03</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma&amp;nbsp;</td><td>A7030-50G</td><td></td></tr><tr><td>Medium: Neurobasal medium</td><td>Gibco&amp;nbsp;</td><td>21103-049</td><td></td></tr><tr><td>Culture media that contains a stabilized form of L-glutamine, L-alanyl-L-glutamine: GlutaMAX</td><td>Gibco&amp;nbsp;</td><td>35050-061</td><td>0.5 mM final concentration.</td></tr><tr><td>L-Ascorbic acid</td><td>Sigma&amp;nbsp;</td><td>A7506</td><td></td></tr><tr><td>B27</td><td>Gibco&amp;nbsp;</td><td>17504-044</td><td>50x stock solution, 1x final concentration.</td></tr><tr><td>35 mm glass bottom dishes</td><td>MatTek&amp;nbsp;&amp;nbsp;</td><td>P35GC-1.5-10-C</td><td></td></tr><tr><td>Poly-L-Ornithine</td><td>Sigma&amp;nbsp;</td><td>P4957</td><td></td></tr><tr><td>Laminin</td><td>Sigma&amp;nbsp;</td><td>L2020</td><td>Stored at -20 &amp;deg;C in 20 &amp;micro;L aliquots</td></tr><tr><td>Deoxyribonuclease I (DNase)</td><td>Sigma&amp;nbsp;</td><td>DN25</td><td></td></tr><tr><td>Blue pipette tips P1000</td><td>Sorenson Bioscience</td><td>&amp;nbsp;Inc. 10130</td><td></td></tr><tr><td>10 mm shallow Petri dish</td><td>VWR&amp;nbsp;</td><td>25384-324</td><td></td></tr><tr><td>37 &amp;deg;C Water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C, 5% humidity incubator</td><td></td><td></td><td></td></tr><tr><td>16% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>&amp;nbsp;X100-500ML</td><td></td></tr><tr><td>Donkey serum</td><td>Equitech&amp;nbsp;</td><td>SD-0100HI</td><td>100% stock solution, 1% final concentration.</td></tr><tr><td>Standard Pattern surgical scissors</td><td>Fine Science Tools</td><td>14000-14</td><td></td></tr><tr><td>Forceps - 2x3 teeth</td><td>Fine Science Tools</td><td>11022-14</td><td></td></tr><tr><td>Forceps - Dumont #55</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Forceps - Dumont #5</td><td>Fine Science Tools</td><td>11252-23</td><td></td></tr><tr><td>10x PBS, pH 7.4</td><td>Gibco&amp;nbsp;</td><td>70011-044</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-buffered solution (DPBS) 1x</td><td>Gibco</td><td>14190-144</td><td>500 mL</td></tr><tr><td>21 G needle</td><td>BD PrecisionGlide Needle</td><td>305167</td><td>100 needles</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>model P-87</td><td></td></tr><tr><td>Micromanipulator&amp;nbsp;</td><td>Sutter Instrument</td><td>&amp;nbsp;MP-285</td><td></td></tr><tr><td>Sequencing Kit: SMARTer Ultra Low RNA Kit for Illumina Sequencing</td><td>Clontech</td><td>634936</td><td>100 rnxs,incl Advantage2PCR Kit</td></tr><tr><td>oligonucleotide (12 &amp;micro;M): SMARTer IIA oligonucleotide (12 &amp;micro;M)</td><td></td><td></td><td>From the SMARTer Kit&amp;nbsp;</td></tr><tr><td>Reverse transcriptase (100 units)</td><td>SMARTcribe reverse transcriptase</td><td></td><td>From the SMARTer Kit&amp;nbsp;</td></tr><tr><td>Primer 1</td><td>3&amp;rsquo; CDSIIa primer&amp;nbsp;</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>Primer 2</td><td>IS PCR primer</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>50x 2 polymerase mix</td><td>50x Advantage 2 polymerase mix</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>10x PCR buffer</td><td>10x Advantage 2 PCR buffer</td><td></td><td>From the SMARTer Kit</td></tr><tr><td>RNA Spikes</td><td>ThermoFisher</td><td>4456740</td><td></td></tr><tr><td>PCR cooler rack</td><td>IsoFreeze PCR cooler rack</td><td></td><td></td></tr><tr><td>DNA Sample Preparation Kit&amp;nbsp;</td><td>(Illumina/Nextera) Nexter</td><td>FC-121-1030</td><td>24 Samples</td></tr><tr><td>PCR master mix</td><td>Nextera PCR master mix (NPM)</td><td></td><td>From the Nextera Kit&amp;nbsp;</td></tr><tr><td>PCR primer cocktail (PPC)&amp;nbsp;</td><td>Nextera PCR primer cocktail (PPC)&amp;nbsp;</td><td></td><td>From the Nextera Kit</td></tr><tr><td>Fluorometer</td><td>The Qubit ThermoFisher</td><td>Q33216</td><td></td></tr><tr><td>HS DNA BioAnalyzer kit</td><td>Agilent</td><td>5067-4626</td><td>110 rxns</td></tr><tr><td>Electrophoresis system&amp;nbsp;</td><td>Agilent 2100 Bioanalyzer.</td><td>G2938C</td><td></td></tr><tr><td>Magnetic beads: Agencourt AMPure&amp;nbsp;</td><td>Beckman Coulter</td><td>A63880</td><td>5 mL</td></tr><tr><td>RNAse wipe: RNAse Zap wipes</td><td>Ambion</td><td>AM9786</td><td>Size 100 Sheets</td></tr><tr><td>Qubit ds HS Assay Kit</td><td>Molecular probes&amp;nbsp;</td><td>Q32854</td><td>500 assays</td></tr><tr><td>Gel extraction kit: Qiaquick Gel Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>28704</td><td></td></tr><tr><td>Glass capillary tubing</td><td>(Kimax-51, 1.5 - 1.8 mm o.d.)</td><td></td><td></td></tr><tr><td>Microforge</td><td>Narishige (or equivalent)&amp;nbsp;</td><td></td><td></td></tr><tr><td>Sequencer</td><td>Illumina HiSeq instrument</td><td></td><td></td></tr><tr><td>GENSAT tyrosine hydroxylase-eGFP mouse strain&amp;nbsp;</td><td></td><td>MMRRC stock number: 000292-UNC</td><td>Stock Tg(Th-EGFP)DJ76Gsat/Mutant Mouse Regional Research Center</td></tr></tbody></table>

reliable identification of living dopaminergic neurons in midbrain cultures using rna sequencing and th-promoter-driven egfp expression

<html> 파킨슨 병 (PD)에서 운동 완만, 강성 및 진동에지도하는 SNC에서 도파민 뉴런의 발음 변성 뇌 전반에 걸쳐 광범위하게 신경 세포의 손실이있다. 형광 마커를 사용하여 기본 복부 중뇌 (VM) 문화 생활 도파민 신경 세포의 식별은 고정 된 세포의 면역 염색에 의존하지 않고 이러한 신경 세포의 선택적 취약점을 연구하는 다른 방법을 제공합니다. 여기, 우리는 분리, 해리, 문화 마우스 VM 신경 삼주합니다. 우리는 그 다음의 (a 티로신 하이드 록 (TH) 프로모터에 의해 구동) eGFP는 형광을 이용하여 문화에서 도파민 신경 세포를 식별합니다. 개별 뉴런 유리 가능한 Micropipette를 사용하여 마이크로 원심 튜브에 수확된다. 다음으로, 우리는 수확 된 세포 용균 및 단일 세포 RNA-SEQ 라이브러리 (1, 2)를 생성하는 cDNA 합성 및 트랜스포존 매개 &quot;tagmentation를&quot;행위엉덩이 = &quot;외부 참조&quot;&gt; 3, 4, 5. 품질 제어 검사를 통과 한 후, 단일 셀 라이브러리는 서열화되고 후속 분석은 유전자 발현을 측정하기 위해 수행된다. 우리는 개인의 도파민과 중뇌 문화에서 고립 된 GABA 성 신경 세포에 대한 사체 결과를보고한다. 우리는 수확 서열 된 라이브 TH-eGFP는 세포의 100 % 도파민 신경 세포라고보고한다. 이러한 기술은 신경 과학 및 분자 생물학에서 광범위하게 응용 프로그램을 제공합니다.

여기에서 우리는, 티로신 수산화 프로모터 서열에 의해 구동 eGFP는 표현 마우스에서 복부 중뇌 신경 세포를 배양하는 문화에서 개별 형광 신경 세포를 수확하고, RNA-SEQ (그림 1)를 사용하여 자신의 RNA 전 사체를 측정하기위한 프로토콜을보고합니다. 데이터 수확 서열화 된 TH-eGFP는 양성 세포 100 % 다음 세 DA 관련된 유전자 전 사체의 존재 TH, DOPA 데카 복실 라제 ...

Watch this Scientific Journal Video about Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression at JoVE.com

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

파킨슨 병 (PD)에서 Substantia 그라 (SNC) 도파민 신경 세포는 운동 기능 장애로 이어지는, 퇴화. 여기에서 우리는 티로신 하이드 록 (TH) 프로모터 서열에 의해 구동 eGFP는 표현 마우스에서 복부 중뇌 신경 세포를 배양하는 문화에서 개별 형광 신경 세포를 수확하고, RNA-서열을 사용하여 사체를 측정하기위한 프로토콜을보고합니다.

RNA 시퀀싱 및 TH-프로모터 중심 eGFP는 표현을 사용하여 중간 뇌 문화에서 생활 도파민 뉴런의 신뢰성 확인

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

reliable-identification-living-dopaminergic-neurons-midbrain-cultures

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.3K 조회수.  California Institute of Technology (Caltech).  파킨슨 병 (PD)에서 Substantia 그라 (SNC) 도파민 신경 세포는 운동 기능 장애로 이어지는, 퇴화. 여기에서 우리는 티로신 하이드 록 (TH) 프로모터 서열에 의해 구동 eGFP는 표현 마우스에서 복부 중뇌 신경 세포를 배양하는 문화에서 개별 형광 신경 세포를 수확하고, RNA-서열을 사용하여 사체를 측정하기위한 프로토콜을보고합니다. 

비디오: Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

쥐 Neonates에서 기본 Dissociated Midbrain의 도파민 세포 문화

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

연구

생물학

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

생체 내 신경 아키텍처 연구를 위한 엔지니어링된 신경 전구체의 내 측 통 이식

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

유전학

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

배아 복부 Midbrain의 Organotypic 슬라이스 문화 : Dopaminergic의 연결을 개발 연구하는 시스템 체외에서</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

신경과학

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic 신경 세포 개발에서 유전자 기능을 유학 칙의 Midbrain에서 비켜 Electroporation에

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

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

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

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

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

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

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

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

마우스 도파민 뉴런의 차 문화

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

배아 쥐의 뇌에서 차 중뇌 도파민 신경 세포의 분리, 문화 및 장기 유지 보수

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

탐지 기술 현장에서 혁신적인 RNA에 의해 설치류 핵 Accumbens에서 도파민 D1-알파 수용 체의 식별

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

표현형 프로파일링을 위한 인간 중뇌 도파민 뉴런 배양

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro PD cellular models lack complexity and do not take multiple phenotypes into account. Phenotypic profiling in human induced pluripotent stem cell (iPSC)-derived mDA neurons can address these shortcomings by simultaneously measuring a range of neuronal phenotypes in a PD-relevant cell type in parallel. Here, we describe a protocol to obtain and analyze phenotypic profiles from commercially available human mDA neurons. A neuron-specific fluorescent staining panel is used to visualize the nuclear, &#945;-synuclein, Tyrosine hydroxylase (TH), and Microtubule-associated protein 2 (MAP2) related phenotypes. The described phenotypic profiling protocol is scalable as it uses 384-well plates, automatic liquid handling and high-throughput microscopy. The utility of the protocol is exemplified using healthy donor mDA neurons and mDA neurons carrying the PD-linked G2019S mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene. Both cell lines were treated with the LRRK2 kinase inhibitor PFE-360 and phenotypic changes were measured. Additionally, we demonstrate how multidimensional phenotypic profiles can be analyzed using clustering or machine learning-driven supervised classification methods. The described protocol will particularly interest researchers working on neuronal disease modeling or studying chemical compound effects in human neurons.

저자 스포트라이트: Generating Neuronal Phenotypic Profiles - A Protocol to Culture and Image Human Midbrain Dopaminergic Neurons (저자 스포트라이트: 신경 세포 표현형 프로파일 생성 - 인간 중뇌 도파민 뉴런을 배양하고 이미지화하는 프로토콜) 

This protocol describes the cell culturing of human midbrain dopaminergic neurons, followed by immunological staining and the generation of neuronal phenotypic profiles from acquired microscopic high-content images allowing the identification of phenotypic variations due to genetic or chemical modulations.

인간 줄기세포 유래 중뇌 도파민 뉴런의 표현형 프로파일링

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro ...

RNA 시퀀싱 및 TH-프로모터 중심 eGFP는 표현을 사용하여 중간 뇌 문화에서 생활 도파민 뉴런의 신뢰성 확인

요약

더 많은 비디오 탐색

이 비디오의 챕터

쥐 Neonates에서 기본 Dissociated Midbrain의 도파민 세포 문화

생체 내 신경 아키텍처 연구를 위한 엔지니어링된 신경 전구체의 내 측 통 이식

배아 복부 Midbrain의 Organotypic 슬라이스 문화 : Dopaminergic의 연결을 개발 연구하는 시스템 체외에서

Dopaminergic 신경 세포 개발에서 유전자 기능을 유학 칙의 Midbrain에서 비켜 Electroporation에

에 신약 표현형을 평가

마우스 도파민 뉴런의 차 문화

배아 쥐의 뇌에서 차 중뇌 도파민 신경 세포의 분리, 문화 및 장기 유지 보수

탐지 기술 현장에서 혁신적인 RNA에 의해 설치류 핵 Accumbens에서 도파민 D1-알파 수용 체의 식별

인간 줄기세포 유래 중뇌 도파민 뉴런의 표현형 프로파일링

인간 줄기세포 유래 중뇌 도파민 뉴런의 표현형 프로파일링