同时双光子<em>在体内</em>突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex
Summary
This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.
Abstract
This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex.
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.
Introduction
双光子显微镜彻底改变了大脑活动的观察生活和行为的动物。自1990年推出它迅速得到普及，现在实现为对体内 1,2大脑活动的许多方面考核最有趣和创新的方法之一。这些应用包括血流测量，神经元激活（ 例如 ，使用钙离子浓度指标或立即早期基因表达）和神经细胞的形态。越来越多的实验室使用双光子显微镜，实现在整个科学界的技术，因为体内脑成像的新标准。
标准方法涉及颅窗口植入在桶或小鼠脑3的视觉皮层（在覆盖有盖玻璃的头盖骨的圆孔）。接着，根据不同的实验方案，在谋本身经历一系列可视化和行为训练会话，允许在一段时间4,5-监视在大脑活动和神经元形态的变化。在两种情况下，开颅仅影响顶骨，而不穿过缝线。它主要是认为该技术的主要缺点是其应用受到限制，以方便皮质诸如桶或视觉皮层。在其他地区颅窗口植入带来了很大的困难，因失血过多和/或空间阻碍。
在本文中，我们提出了retrosplenial皮层（RSC）作为用于体内显微镜6双光子兴趣另一可能的区域上方颅窗口的注入。 RSC负责空间记忆形成脑电路的一个重要因素。解剖学上，RSC是连接皮质，海马，丘脑和区域7神经元网络的一部分。它是积极参与各种行为，如空间学习和灭绝以及空间导航6。
为了可视化神经元的形态学变化，我们使用表达该THY1启动子下的绿色荧光蛋白（GFP）的转基因小鼠品系。在这些小鼠中，绿色荧光蛋白是在用双光子显微镜8在大脑中的神经元，允许皮质轴突和树突的清晰的可视化的大约10％来表示。我们提出另一个创新是重组腺相关病毒血清型2/1（的rAAV2 / 1）特定的神经元的CaMKII子9下编码红色荧光蛋白（mCherry）进入脑的突出到RSC更深结构的注射，如海马。的rAAV2 / 1 mCherry的Thy1肾炎-GFP小鼠海马的表达允许hippocampo-cortica的前和突触后元素的同时进行可视化升突触10。 mCherry的重组腺相关病毒驱动的表达需要两到三周的蛋白质在终末达到足够的水平。这一时期是从开颅手术恢复所需的通常时间是一致的。
Protocol
Representative Results
Discussion
在当前的论文中，我们提出在在RSC突触输入和突触后的目标体内成像同时双光子的协议通过颅窗口。植入程序包括几个关键步骤。首先，将动物深度麻醉并固定在立体定位框架，然后经RSC颅骨变薄沿标出圆形线的钻头和圆形骨除去。出血停止之后，所述的rAAV2 / 1 mCherry注入海马，及玻璃盖被固定到颅骨上钻孔区。最后的固定杆被固定在头部和动物放置在回收室48小时。后所需?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢M. Steczkowski语音录音，M. Borczyk图纸，ATrąbczyńska病毒的产生，M.Ziókowska用于基因分型和A. Mirgos与拍摄援助。 KR承认钙调蛋白激酶子从K.戴瑟罗特控制下表达荧光蛋白mCherry重组腺相关病毒（腺相关病毒）的一种恩赐。该项目是在实验室的组织结构和功能，神经生物学，实验生物学Nencki研究所中心的动物模型和实验室的核心功能进行，利用概念的基础设施由欧洲联盟资助 – 在欧洲区域发展基金业务方案“创新经济”2007 – 2013年。这项工作是由国家科学中心的资金支持：索纳塔双2012/05 / E / NZ4 / 02996，异色2013/08 / M / NZ3 / 00861，Symfonia 2013/08 / W / NZ24 / 00691为KR和索纳塔双2014 / 14 / E / NZ4 / 00172 RC
Materials
| Drug | |||
| Isoflurane | Baxter | AErrane 8DG9623 | 5-2% pre-operative |
| Isoflurane | Baxter | AErrane 8DG9623 | 1.5-2% during surgery |
| Dexametasone | Scan Vet | Dexasone 2mg/ml | 0.2 mg/kg intramuscular |
| Baytril | Bayer | 2.50% | 5 mg/kg subcutaneously |
| Tolfedine | Vetoquinol | 4% | 4 mg/kg subcutaneously |
| Butomidor | Richter Pharma | 10 mg/ml | 2 mg/kg subcutaneously |
| Carprofen | KRKA-Polska | Rycarfa 50mg/ml | 10 mg/kg subcutaneously |
| Lidocaine | Jelfa | Lignocainum | topically |
| Lidocaine | Jelfa | 20 mg/g | topically |
| Surgery | |||
| Gelfoam | Ethicon | Spongostan dental; REF MS0005 | |
| Eye ointment | Dedra | Lubrithal | topically |
| CA glue | Pelikan Daniel | 20G Huste | |
| Dental acrylic | SpofaDental | Duracryl Plus | |
| Stereotaxic frame | Stoelting | 51500D | |
| Tool | |||
| Coverglass | Harvard Apparatus | HSE-64-0720 | 3 mm diameter |
| Dental drill | Sigmed | Keystone KVet | |
| Fixation bar | Custom made | N/A | M2 or M3 screw nuts could be used |
| Forceps | Renex | PN-7B-SA | |
| Micro scissors | Falcon | BM.183.180 | |
| Dissection microscope | KOZO | XTL6445T | |
| Imaging | |||
| Holder frame | Custom made | N/A | |
| Two-photon microscope | Zeiss | Upright Axio Examiner Z1 | Laser unit: Coherent Chameleon 690-1040nm with Optical Parametric Oscillator 1050-1300nm. Objectives: EC-PLAN-NEUFLUAR 10x/0.1 and LD Plan-APOCHROMAT 20x/1.0. Detection: Zeiss bandpass filters BP 500-550 (GFP) and BP 570-610 (mCherry) separated by beam splitter at 560nm and coupled to two GaAsP photodetectors. |
| Reagent | |||
| Virus | gift from K. Deisseroth | Recombinant adeno-associated virus (rAAV) expressing fluorescent protein mCherry under the control of CaMK promoter |
Riferimenti
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