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, ...

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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

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 ...

연구

유전학

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

손상 망막의 특성 Electrophysiological GFP - 표현 세포 집단

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

신경과학

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 ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

1 차적인 뉴런에 있는 Neurite 파생을 공부를 위한 향상된 녹색 형광 단백질 기지를 둔 분석

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

발생학

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

친유성 형광 염료를 사용하여 Drosophila 배아 모터 뉴런의 역행 추적

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

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 ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

해마의 생체 내 해부학 및 기능적 추적

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

유전자 표적화된 망막 세포 집단의 희소 아데노-연관 바이러스 표지를 이용한 뉴런 수목화의 타임랩스 이미징

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

저자 스포트라이트: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

배아 마우스 뇌 절편에서 이동하는 뉴런 및 신경교세포 전구세포의 타임랩스 이미징

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

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

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

손상 망막의 특성 Electrophysiological GFP - 표현 세포 집단

에 신약 표현형을 평가

1 차적인 뉴런에 있는 Neurite 파생을 공부를 위한 향상된 녹색 형광 단백질 기지를 둔 분석

친유성 형광 염료를 사용하여 Drosophila 배아 모터 뉴런의 역행 추적

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

해마의 생체 내 해부학 및 기능적 추적

유전자 표적화된 망막 세포 집단의 희소 아데노-연관 바이러스 표지를 이용한 뉴런 수목화의 타임랩스 이미징

배아 마우스 뇌 절편에서 이동하는 뉴런 및 신경교세포 전구세포의 타임랩스 이미징

배아 마우스 뇌 절편에서 이동하는 뉴런 및 신경교세포 전구세포의 타임랩스 이미징