同时电生理记录和抑制剂在啮齿动物大脑微注射
Summary
Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.
Abstract
在这里，我们描述了使用商业上获得和负担得起份的单次使用“injectrode”的结构的方法。甲探测系统的开发，其允许注射药物的同时记录来自受影响的神经元群的电生理信号。此方法提供了一种简单且经济的替代的商业解决方案。玻璃移液管将其与一皮下注射针和银长丝组合改性。该injectrode附着到商业微量注射泵用于药物递送。这导致了一种技术，通过多单元外信号从药物递送位点发端提供实时药效反馈。作为概念证明，我们记录了神经元活动从上丘通过光的闪烁大鼠引起，与之同时通过injectrode递送药物。该injectrode记录容量允许每次注射的功能分析现场挠度利于精确控制药物释放的本地化。这种方法的应用也远远超出这里所展示，作为化学物质装入injectrode的选择是巨大的，包括跟踪标记解剖实验。
Introduction
皮质区和副皮质细胞核失活是在不同脑结构间的2-4个功能关系的研究非常重要。最近的文献采用功能丧失的化学或低温技术来研究大脑结构2,5的作用。关于药理显微注射，小体积的药物可以控制的速率施用到脑区域，同时最小化对周围组织6,7的附带损害。这种技术可以用于递送特异性激动剂，反向激动剂或拮抗剂来研究不同的药理学靶上的神经元活动的影响。这样的效果也可以通过测量变化从遥远地点神经元的反应，使研究人员研究了不同的皮质和皮质下结构之间的关系的研究。
在这里，我们展示了一个设备的装配，injectrode，可博个记录电生理信号，并在目标位置输送少量的药物。我们证明该系统的功能，通过注入的GABA，神经元活性的共同抑制剂，在大鼠上丘。这个地区是视觉刺激，使我们能够利用视觉诱发多组活动确认injectrode本地化敏感。失活的可逆性通过正常神经元活性的GABA注射结束后恢复进行评估。
从注射部位监测多单位活动的能力允许为达到所需药效应答所需的注射速率和体积的微调。因此，这种技术的优点是由微灌组织损伤的限制性的潜力，因为最小的有效量被注入。所提出的协议提供了一种成本有效的方法，用于产生所述一次性硬件necessary代表进行实验，其中药物输送和本地的神经元活动的记录是需要的。
Protocol
Representative Results
Discussion
该协议旨在解决与当前可逆灭活方法所带来的挑战。具体而言，该项目旨在改进用于物质调节神经活动，特别是在脑深部结构的化学显微注射方法。一个技术难题摆脱这种类型的设置是需要两个探头将在共定位在体内同样有限的空间，以便在注射部位，以获得精确的记录。这个问题可以通过使用设备，如一个在这里提出，其能够注与记录在同一部位来克服。替代的方法包括使用根据气体的?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Supported by grants from CIHR (MOP231122) and NSERC (RGPIN-2014-06503). We would like to thank Geneviève Cyr for her help preparing experiments and supervising laboratory work. MAL received a scholarship from The Natural Sciences and Engineering Research Council of Canada (NSERC).
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Injection pump (UltraMicroPump III) | WPI | #UMP3 | |
| Injection console (Micro4 Controller) | WPI | #SYS-MICRO4 | |
| Hamilton syringe | Hamliton | (80301) 701LT 10 µL SYR | Syringes between 5 and 10 μL used |
| Gel cyanoacrylate adhesive | Krazy Glue | KG86648R | The gel form is easier to apply on the shaft of the 30G hypodermic needle |
| Glass pipettes | WPI | #TW100F-4 | Thin wall, 1mm OD, 0.75mm ID with filament pipettes used |
| 720 Needle Pipette Puller | Kopf | 720 | |
| Silver wire | A-M Systems, Inc. | 782500 | Bare 0.010” |
References
- Martin, J. H., Ghez, C. Pharmacological inactivation in the analysis of the central control of movement. Journal Of Neuroscience Methods. 86 (2), 145-159 (1999).
- Ponce, C. R., Hunter, J. N., Pack, C. C., Lomber, S. G., Born, R. T. Contributions of indirect pathways to visual response properties in macaque middle temporal area MT. The Journal Of Neuroscience The Official Journal Of The Society For Neuroscience. 31 (10), 3894-3903 (2011).
- Lomber, S. The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function. Journal Of Neuroscience Methods. 86 (2), 109-117 (1999).
- Malpeli, J., Schiller, P. A method of reversible inactivation of small regions of brain tissue. Journal Of Neuroscience Methods. 1 (2), 143-151 (1979).
- Lomber, S. G., Payne, B. R., Horel, J. A. The cryoloop: an adaptable reversible cooling deactivation method for behavioral or electrophysiological assessment of neural function. Journal Of Neuroscience Methods. 86 (2), 179-194 (1999).
- Gonzalez-Perez, O., Guerrero-Cazares, H., Quiñones-Hinojosa, A. Targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants. Journal Of Visualized Experiments. (46), (2010).
- Hupé, J., Chouvet, G., Bullier, J. Spatial and temporal parameters of cortical inactivation by GABA. Journal Of Neuroscience Methods. 86 (2), 129-143 (1999).
- Casanova, C., McKinley, P., Molotchnikofff, S. Responsiveness of Reorganized Primary Somatosensory (SI) Cortex after Local Inactivation of Normal SI Cortex in Chronic Spinal Cats. Somatosensory & Motor Research. 8 (1), 65-76 (1991).
- Malpeli, J. Reversible inactivation of subcortical sites by drug injection. Journal Of Neuroscience Methods. 86 (2), 119-128 (1999).
- Minville, K., Casanova, C. Spatial frequency processing in posteromedial lateral suprasylvian cortex does not depend on the projections from the striate-recipient zone of the cat’s lateral posterior-pulvinar complex. Neurosciences. 84 (3), 699-711 (1998).
- Diao, Y., Wang, Y., Xiao, Y. Representation of the binocular visual field in the superior colliculus of the albino rat. Experimental Brain Research. 52 (1), 67-72 (1983).
