慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激
Summary
成人出生在嗅球的神经元可optogenetically控制使用Channelrhodopsin2表达慢病毒注射在延髓的迁徙流和植入微型慢性photostimulation的LED。
Abstract
当地interneurons不断再生1-3成年啮齿动物的嗅球。在这个过程中，被称为成年神经发生，神经干细胞在侧脑室壁引起神经母细胞迁移沿喙迁徙流（RMS）达到并纳入到嗅球几毫米。为了研究不同的步骤和成人出生的神经元整合到预先存在的嗅觉电路的影响，这是必要的选择性标签和操纵的这种神经元的具体人口的活动。 optogenetic技术最近的发展提供了机遇，利用光，正是这个特定的神经元激活，而不会影响周围神经元 4,5队列。在这里，我们目前在神经母细胞的时间限制在RMS队列一个维拉利表达Channelrhodopsin2系列程序（ChR2）YFP的，在他们到达嗅球之前，成为成人出生NEurons。此外，我们将展示如何植入和校准为慢性LED 在体内刺激的ChR2表达神经元的一个缩影。
Protocol
1。立体定向注射在这些实验中使用的病毒是一个艾滋病毒的复制缺陷型慢病毒载体来表达Channelrhodopsine2 – YFP（黄色荧光蛋白）融合构建一个突触子 6驱动。质粒是慷慨提供K. Deisseroth的研究组（ http://www.stanford.edu/group/dlab/optogenetics/index.html ）。慢病毒可以从商业渠道获得，或在实验室中根据既定协议 7 。平均载体滴度?…
Discussion
Optogenetic工具最近增加了我们对选择性的神经元的人群，包括出生于成人的嗅觉系统神经元的控制。在这里，我们描述了如何执行的慢病毒载体表达ChR2 – YFP的成人出生的神经元的具体人口的立体注射。通过仔细监测期间注射后，我们能够分析和处理出生于一个相对同质化时代的成人神经元群活动。
由于他们的低扩散蔓延，其高转导效率，其相对良好的RMS神经母细胞向性，慢?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
“ERA – NET神经元”的框架，这项工作是由寿险公司“诺瓦利斯Taitbout”，巴黎高等DES神经科学（ENP），法新社国立德拉Recherche“的ANR – 09 – NEUR – 004”的支持由欧洲委员会第七框架计划，“基金会争取RECHERCHE Medicale”，Letten基金会和巴斯德基金会。
Materials
| Material | Company | Catalogue No. |
| Ketamine | Merial | Imalgène 1000 |
| Xylazine | Bayer Health Care | Rompum 0.2% |
| Pipette Puller | Sutter | P-97 |
| Glass Capillaries (3.5″) | Drummond Scientific | 3-000-203-G/X |
| Nanoject II Microinjector | Drummond Scientific | 3-000-204 |
| High speed surgical drill | Fine Science Tools | 18000-17 |
| Micro Drill Steel Burrs 0.7mm tip diameter | Fine Science Tools | 19008-07 |
| LED CMS blue 4.6 W | OSRAM | LBW5SN |
| Female electrical connector (2.54 mm) | Fischer Elektronik | BL5.36Z |
| Current controller for LED (max: 1A) | A1W Electronik | HKO-KL50-1000 |
| Pulse generator | AMPI | Master-8 |
| Optical power meter | Thor Labs | PM100 |
| Cyanoacrylate adhesive | Loctite | 454 |
| Polycarboxylate cement (first layer cement) | Septodont | Selfast (#0068Q) |
| Methacrylate cement (second layer cement) | GC InternationalCorp. | Unifast Trad – Liquid (#339292) Unifast Trad – Powder (#339114) |
| Carprofen | Pfizer | Rimadyl |
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Tags
Selective Viral TransductionAdult-born Olfactory NeuronsChronic In Vivo Optogenetic StimulationLocal InterneuronsAdult NeurogenesisLateral VentricleRostral Migratory Stream (RMS)Olfactory BulbLabel And Manipulate ActivityOptogenetic TechnologiesChannelrhodopsin2 (ChR2)-YFPTemporally Restricted Cohort Of NeuroblastsImplant And Calibrate Miniature LED
Cite This Article
Lepousez, G., Alonso, M., Wagner, S., Gallarda, B. W., Lledo, P. Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation. J. Vis. Exp. (58), e3380, doi:10.3791/3380 (2011).