在在线 MRI 图像的指导下,对流增强将视遗传阿德诺相关病毒载体输送到 Rhesus Macaque 的皮质
Summary
在这里,我们演示了磁共振 (MR) 引导的对流增强病毒载体到皮层 (CED) 到皮层作为一种有效和简化的方法,以实现光遗传学表达在大型皮质区域在猴脑。
Abstract
在非人类灵长类动物(NHP)光遗传学中,用病毒载体感染大型皮质区域通常是一项困难而耗时的任务。在这里,我们演示了使用磁共振 (MR) 引导对流增强交付 (CED) 光遗传学病毒载体到原发性躯体感觉 (S1) 和运动 (M1) 皮质的猴获得高效, 广泛皮质表达光敏性电子通道。在MR引导的CED下,将编码红移蛋白C1V1的Adeno相关病毒(AAV)载体注射到恒河猴皮层中,将这种蛋白注入黄色荧光蛋白(EYFP)。输液后三个月,显微荧光成像在M1和S1中确认两只猴子有大面积的光遗传学表达(>130 mm 2)。此外,我们还能够使用微电皮学阵列记录来自表达区域的可靠光唤起电生理反应。后来对记者进行组织学分析和免疫染色后发现,M1和S1中广泛而密集的光遗传学表达与表显荧光成像所指示的分布相对应。与传统技术相比,这种技术使我们能够在较短的时间内获得皮层大面积的表达,损害最小,并且可能是大型动物(如NHPs)的光遗传学病毒传播的最佳方法。这种方法显示了在动物模型中具有细胞型特异性的神经回路的网络级操作的巨大潜力。
Introduction
光遗传学是一个强大的工具,允许操作神经活动和研究大脑中的网络连接。在非人类灵长类动物(NHPs)中实施这种技术有可能增强我们对灵长类动物大脑中大规模神经计算、认知和行为的理解。虽然光遗传学近年来在NHP中已经成功实施了1、2、3、4、5、6、7,这是一个挑战,研究人员面对的是这些动物在大脑大区域实现高水平的表达。在这里,我们提供一种高效和简化的方法,以实现高浓度的光遗传学表达在大脑皮层的大片区域在猴。这项技术具有巨大的潜力,可以结合最先进的记录8、9和光学刺激10技术,提高这些动物目前的光遗传学研究。
对流增强传递(CED)是一种既定的方法,将药理剂和其他大分子(包括病毒载体)输送到中枢神经系统11、12、13。传统的输液方法涉及分布在大脑小区域的多个低容量输注,而CED可以以更少的输注实现更广泛、更均匀的代理分配。输注过程中压力驱动的散装流体流动(对流)允许在使用CED传播病毒载体时更广泛地均匀地分布目标组织。在最近的研究中,我们使用磁共振(MR)引导的CED演示了大面积原马达(M1)和躯体感觉(S1)皮质9和丘拉丘14的转导和后续光遗传学表达。
在这里,我们概述了使用CED实现光遗传学表达在大型皮质区域,只需几个皮质注射。
Protocol
Representative Results
Discussion
本文概述了一种可行、高效的技术,通过MR引导的CED在NHP原发性躯体感觉和运动皮层中实现大规模光遗传学表达。与NHP大脑中传统的病毒输注方法相比,MR引导的CED的使用具有显著的优势。其中一个优势是能够在需要输注的大面积区域获得表达。例如,在常规方法中,在直径为 2-3 mm 的区域1、2、5、17 中多次注入 1-2 μL 的载体…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国心脏协会博士后奖学金(AY)、国防高级研究计划局(DARPA)重组和可塑性的支持,以加速损伤康复(REPAIR;N66001-10-C-2010),R01。NS073940,由UCSF神经科学成像中心。这项工作还得到了国家卫生研究院尤尼斯·肯尼迪·希弗国家儿童健康与人类发展研究所、华盛顿国家灵长类研究中心(WaNPCR,P51 OD010425)以及神经技术中心(CNT,国家科学基础工程研究中心,由格兰特EEC-1028725)。我们感谢卡米洛·迪亚兹-博蒂亚、蒂姆·汉森、维克托·哈拉齐亚、丹尼尔·西尔弗史密斯、卡伦·麦克劳德、朱莉安娜·米兰尼和布莱克利·安德鲁斯在实验中的帮助,以及南天、何继伟、彼得·莱多克霍维奇、米歇尔·马哈比兹和托尼·豪恩的技术支持。
Materials
| 0.2 mL High Pressure IV Tubing | Smiths Medical Inc., Dublin, OH, USA | 533640 | |
| 0.32 mm ID, 0.43 mm OD Silica Tubing | Polymicro Technologies | 1068150027 | |
| 0.45 mm ID, 0.76 mm OD Silica Tubing | Polymicro Technologies | 1068150625 | |
| AAV2.5-CamKII-C1V1-EYFP | Penn Vector Core, University of Pennsylvania | ||
| ABS plastic | Stratasys, MN, USA | ABSplus-P430 | |
| Antimicrobial incise drape | 3M | 6650EZ | Ioban Drape |
| Dental Acrylic | Henry Schein, Inc. | 1013117 | Acrylic Bonding Agent |
| Elevators | VWR International, LLC. | 10196-564 | Langenbeck Elevator, Wide Tip |
| Fine suture | McKesson Medical-Surgical Inc. | 1034505 | |
| Gadoteridol | Prohance, Bracco Diagnostics, Princeton, NJ | 0270-1111-04 | |
| Laser for light stimulation | Omicron-Laserage, Germany | PhoxX 488-60 | |
| MR compatible 3cc syringe | Harvard apparatus, Holliston, MA, USA | 59-8377 | |
| MR Imaging Software | Pixmeo | OsiriX MD 10.0 | |
| MR-Compatible Pump | Harvard apparatus, Holliston, MA, USA | Harvard PHD 2000 | |
| MR-compatible stereotaxic frame | KOPF | 1430M MRI | |
| Perifix Clamp Style Catheter Connector | B-Braun, Bethlehem, PA, USA | N/A | |
| Plastic Screws | Plastics 1 | 0-80 x 1/8N | Nylon screws |
| Titanium screws | Crist Instrument Co., Inc. | 6-YCX-0312 | Self-tapping bone screws |
| Trephine | GerMedUSA Inc, | SKU:GV70-42 | |
| uPrinter SE 3D printer | Stratasys, MN, USA | N/A | |
| Vitamin E Capsule | Pure Encapsulations, LLC. | DE1 | |
| Wet sterile absorbable gelatin | Pfizer Inc. | AZL0009034201 | Gelfoam |
Riferimenti
- Ruiz, O., et al. Optogenetics through windows on the brain in the nonhuman primate. Journal of Neurophysiology. 110 (6), 1455-1467 (2013).
- Diester, I., et al. An optogenetic toolbox designed for primates. Nature Neuroscience. 14 (3), 387-397 (2011).
- Ohayon, S., Grimaldi, P., Schweers, N., Tsao, D. Y. Saccade modulation by optical and electrical stimulation in the macaque frontal eye field. Journal of Neuroscience. 33 (42), 16684-16697 (2013).
- Gerits, A., et al. Optogenetically induced behavioral and functional network changes in primates. Current Biology. 22 (18), 1722-1726 (2012).
- Jazayeri, M., Lindbloom-Brown, Z., Horwitz, G. D. Saccadic eye movements evoked by optogenetic activation of primate V1. Nature Neuroscience. 15 (10), 1368-1370 (2012).
- Dai, J., Brooks, D. I., Sheinberg, D. L. Optogenetic and electrical microstimulation systematically bias visuospatial choice in primates. Current Biology. 24 (1), 63-69 (2014).
- Afraz, A., Boyden, E. S., DiCarlo, J. J. Optogenetic and pharmacological suppression of spatial clusters of face neurons reveal their causal role in face gender discrimination. Proceedings of the National Academy of Sciences of the United States of America. 112 (21), 6730-6735 (2015).
- Ledochowitsch, P., et al. Strategies for optical control and simultaneous electrical readout of extended cortical circuits. Journal of Neuroscience Methods. 256, 220-231 (2015).
- Yazdan-Shahmorad, A., et al. A Large-Scale Interface for Optogenetic Stimulation and Recording in Nonhuman Primates. Neuron. 89 (5), 927-939 (2016).
- Ju, N., Jiang, R., Macknik, S. L., Martinez-Conde, S., Tang, S. Long-term all-optical interrogation of cortical neurons in awake-behaving nonhuman primates. PLoS Biology. 16 (8), e2005839 (2018).
- Bankiewicz, K. S., et al. Convection-enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach. Experimental Neurology. 164 (1), 2-14 (2000).
- Kells, A. P., et al. Efficient gene therapy-based method for the delivery of therapeutics to primate cortex. Proceedings of the National Academy of Sciences of the United States of America. 106 (7), 2407-2411 (2009).
- Krauze, M. T., et al. Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents. Journal of Neurosurgery. 103 (5), 923-929 (2005).
- Yazdan-Shahmorad, A., et al. Widespread optogenetic expression in macaque cortex obtained with MR-guided, convection enhanced delivery (CED) of AAV vector to the thalamus. Journal of Neuroscience Methods. 293, 347-358 (2018).
- Yazdan-Shahmorad, A., Silversmith, D. B., Kharazia, V., Sabes, P. N. Targeted cortical reorganization using optogenetics in non-human primates. Elife. 7, (2018).
- Yazdan-Shahmorad, A., et al. Demonstration of a setup for chronic optogenetic stimulation and recording across cortical areas in non-human primates. SPIE BiOS. , (2015).
- Lerchner, W., Corgiat, B., Der Minassian, V., Saunders, R. C., Richmond, B. J. Injection parameters and virus dependent choice of promoters to improve neuron targeting in the nonhuman primate brain. Gene Therapy. 21 (3), 233-241 (2014).
- Acker, L., Pino, E. N., Boyden, E. S., Desimone, R. FEF inactivation with improved optogenetic methods. Proceedings of the National Academy of Sciences of the United States of America. 113 (46), (2016).
- Bobo, R. H., et al. Convection-enhanced delivery of macromolecules in the brain. Proceedings of the National Academy of Sciences of the United States of America. 91 (6), 2076-2080 (1994).
- Lieberman, D. M., Laske, D. W., Morrison, P. F., Bankiewicz, K. S., Oldfield, E. H. Convection-enhanced distribution of large molecules in gray matter during interstitial drug infusion. Journal of Neurosurgery. 82 (6), 1021-1029 (1995).
- Lonser, R. R., Gogate, N., Morrison, P. F., Wood, J. D., Oldfield, E. H. Direct convective delivery of macromolecules to the spinal cord. Journal of Neurosurgery. 89 (4), 616-622 (1998).
- Szerlip, N. J., et al. Real-time imaging of convection-enhanced delivery of viruses and virus-sized particles. Journal of Neurosurgery. 107 (3), 560-567 (2007).
