对调查信息处理的神经药理学清醒行为猕猴皮质压力喷射系统
Summary
Here, we show the pressure injection of neuropharmacological substances during single-cell recording in an awake, behaving macaque monkey. This procedure allows pharmacological manipulation in the direct vicinity of a cortical recording site.
Abstract
The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation of sensory information processing by attention. However, little is known about which neurotransmitter systems are involved in such modulations. A practical way to address this question is to combine single-cell recording with local and temporary neuropharmacological manipulation in a suitable animal model. Here we demonstrate a technique combining acute single-cell recordings with the injection of neuropharmacological agents in the direct vicinity of the recording electrode. The video shows the preparation of the pressure injection/recording system, including preparation of the substance to be injected. We show a rhesus monkey performing a visual attention task and the procedure of single-unit recording with block-wise pharmacological manipulations.
Introduction
In cortical and subcortical areas, neuronal activity is affected by various neuromodulators, for example acetylcholine1. These modulatory effects on neuronal responses have been reported in in vitro studies2, as well as in electrophysiological recordings from anesthetized animals3 and systemic pharmacological manipulations in humans4. Nevertheless, the exact role of different neuromodulators and the involvement of various receptor subtypes are largely unknown. To measure the effects of specific neuromodulators on the activity of single neurons, it is desirable to induce a temporary neuromodulator change as close as possible to the recording electrode. Furthermore, it is important that those manipulations are done in awake animals, as cognitive functions are only present in the absence of anesthesia. Additionally, anesthesia interacts with cholinergic and GABAergic systems5,6 and can lead to changes in neural activity3.
Within the last decades, two main methods of local drug delivery have been developed and refined: iontophoresis and pressure injection. In both methods drugs are delivered through micropipettes made of either glass or steel. With iontophoresis, an electrical current regulates the release of the drug7. Additionally, there is a significant contribution of electro-osmosis to the total amount of ejected molecules8, correlating with the tip diameter9 of the micropipette as well as with the concentration8 of the substance used. Iontophoresis is a powerful tool to quickly and precisely manipulate small volumes of nervous tissue. For iontophoretic injections, multi-barrel micropipettes are usually used10, with one acting as a recording device while the other positions serve as delivery pipettes. A limitation of this method is that only charged molecules can be used, severely limiting the selection of drugs.
Pressure injection uses either air compression or mechanical pressure to eject a substance from a micropipette. Using this method any soluble substance, charged or uncharged, can be used, including large molecules. The method of pressure injection was first described by Reyniers in 1933 and further refined in the 1950s (see Lalley11 for a review). In the 1980s the method was further refined to allow delivery of amounts in the nanoliter range (mainly lidocain12) to a defined brain area13 while simultaneously performing single-cell recording. The ejected volume was usually monitored by observing the movement of a marker, such as the meniscus in the upper part of the pipette13. Pressure injection was first used in the 1990s in awake animals, both extracellularly14 and intracellularly15,16. Based on the cumulative expertise gained in these studies it is now possible to reliably record from different brain structures in combination with pharmacological manipulation (see17 for a comparison of recent pressure injection systems).
An enduring open issue for both drug delivery methods is the difficulty in determining the precise volume injected. This is an even bigger challenge for experiments with awake, behaving rhesus monkeys where the animal performs the experimental task in a separate room. This can be alleviated by the use of a software-controlled system instead of relying on a visual marker to continuously monitor an injection.
The system described here is an extension of a well-established electrophysiological recording system (Mini Matrix System) and combines an injection pipette with multiple parallel-oriented recording electrodes at defined distances in a customizable arrangement. Pharmacological manipulation of the tissue near the recording electrode is possible using only a small amount of substance, ensuring a fast recovery and allowing multiple blocks of injection and control/recovery within the limited time window offered by the behavioral task of the animal.
Protocol
Representative Results
Discussion
在这里，我们详细说明了如何与一个“关闭的，现成的”压力喷射系统进行可靠和精确的注射和高品质的单细胞记录。而药物递送的此方法先前已在行为猴（在17中综述）使用，这里提出的系统具有的优点，下面综述。
如图4A中所示，这里描述的系统可以提供有和没有在记录现场的直接附近药理注射单一神经元活动的稳定的测量。 如图4B所示，控制物质，盐水的注射，并没有导致在燃烧率的变化。这个控制表明，注射过程本身对记录神经元的放电性能没有可测量的影响。
神经元，记录电极，以及微量的空间配置是关键在这些实验中的重要性。虽然在记录期间组织它们的相对位置的精确测量是不可能的，我们可以考虑和方差的可能来源的控制。第一，体积注射期间有一个危险，即所关注的神经元可从记录电极位移远，影响记录信号的稳定性。出于这个原因，它是谨慎前和注射块来验证信号稳定后，比较燃烧率。第二，该记录系统的引导管配置定义电极和微量之间的距离（ 例如 ，305微米的同心3通道系统在该实验中使用）。由于该系统提供了精确的位置控制为在组织电极和微量的深度，它们之间的距离可以通过仔细校准相对深度的记录（步骤3.5）之前，和录音过程中保持它们在共同的深度被最小化。
ENT“> 潜在的局限性除了在内部质量控制由生产，系统需要在实验室条件下进行验证，因为不同品牌管，注射器等均可使用，并可能导致在喷射量的差异。虽然该系统可被用于注入非常小的体积如这里所示的实验中，这些是可以由于在正常实验室环境进行验证，以实际测量极限的最小体积的下方。然而，更大的注射体积可以用来推断软件定义体积和由硬件喷射的体积之间的关系。如果使用透明管，喷射过程的一个附加的视觉检查可以通过测量一个可视标记的位移。
插入微量进系统比电极插入更为苛刻，因为微量的直径稍大并且材料是更脆弱。此外，加入管到微量的销是具有挑战性，它需要打破微量的上部的高风险。然而，一个成功加载微量的寿命是数月，甚至与日常使用。
在实践中，我们还没有系统的记录后的清洗过程中所遇到的喷射系统的堵塞。尽管如此，没有“在线”检查是可能的，而且是有风险的，一个物理障碍（如在枪头组织）可能会阻止物质注入。因此它可能是可取的保守分析数据，例如仅包括在进一步的分析，显示在烧制该实验的控制和注射块之间率显著变化的那些细胞。
尽管他们的直径小，微电极和吸液管将取代脑组织，并可能导致一些局部组织损伤。这可以通过手动定位日的前端被最小化E导游管正上方的硬脑膜。电极然后穿透硬脑膜及完整性是通过在线测量它们的阻抗推断。然后，将微量被插入。当使用这种方法，定期清除组织硬脑膜以上的建议，以进一步降低电极或吸管断裂的危险。
比较另类的方法
这里使用的系统显示相比其他压力喷射系统明显的优势。一个很强的优势是微量（约100微米），这是其它的探测器17的大小的一半的直径，因此，最大限度地减少神经组织的损伤。与以前的设计中，当前系统使用空间上分离的记录电极和微量。虽然其他系统提供电极和吸管之间的较小的距离，在这里所描述的系统允许电极和移液管，从而permi独立深度变化拟合记录会话中的变量相对距离。重要的是，需要对记录质量没有妥协制成，作为喷射系统是一个既定的记录装置的一个扩展。而只有一个微量，因此一种物质在该协议中使用，它可以在一实验程序内注入几种物质。为了实现这一点，一些微量可以拧入单独的导管和连接到安装在各个喷射泵的注射器。最后，控制该系统是容易的，因为只有一个计算机程序是需要推动电极和微量，并在实验过程中进行压注。
压注入相比离子电渗疗法，有相对的优点和缺点。例如，高压喷射需要被引入到组织中比离子导入，从而增加了神经元的位移的危险性更大的卷。目前原山坳中使用的卷在NL范围内，我们很少经历了一个记录细胞的信号质量显着的变化。该系统还允许被注入更大的体积，这是行为的操作可能有用的，但可能会影响神经元记录的稳定性。注射压力超过离子导入具有明显的优势是规模较大的各种可用物质，因为没有规定使用电荷的物质。然而，pH值下应检查和实验组和对照物质之间进行比较（ 例如 ，盐水）。
这个问题可能出现为什么要使用加压注入，而不是新技术的长期建立的方法，如光遗传学操纵的神经活动。虽然还有在啮齿类动物中建立的，光遗传学尚未确立可靠的恒河猴。尤其是，它还不允许选择性地对特定神经递质类型细胞的本地操纵。从长远来看，我们看到用于治疗与optogentic操作的优点药理操作的优点在阐明的认知功能的神经基础组合巨大潜力。
在这里，我们已经表明压喷射如何可用于药理学上操纵的清醒大脑局部禁区，表现恒河猴。我们希望，这种方法激励其他科学家来研究神经元活动的动态神经调节的贡献。
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由通过合作研究中心889“蜂窝机制感觉处理”，以ST（项目C04）德意志研究联合会的资助。我们感谢新浪普卢默，雷奥诺拉Burchardt，德克Prüsse，克劳斯Heisig和拉尔夫Brockhausen技术和动物相关的支持和我们的合作者在德国灵长类研究中心，卡塔琳娜Debowski博士和安娜Magerhans的干细胞单元，在技术援助过滤过程。
Materials
| (-)-Scopolamine hydrochloride | Sigma-Aldrich | 55-16-3 | Mr 339.81 g/mol |
| NaCl 0.9% | B. Braun Melsungen AG | 3079870 | 5ml |
| Terg-a-zyme | Sigma-Aldrich | Z273287 | enzyme detergen |
| Hydrogen peroxide | Roth | Used in 3% solution with deionized water | |
| Ethanol | Chemie-Vertieb Hannover | 104642 | 70% |
| Deionized water | |||
| Injekt 40 Duo | B. Braun Melsungen AG | 9166432V | Syringe and needle |
| Eppendorf Safe-Lock microcentrifuge tubes, amber | Eppendorf | 0030 120.191 | 1,5ml |
| Quarzglass micropipette | Thomas Recording | ||
| Recording electrode | Thomas Recording | quartz/platinum-tungsten fiber electrode; impedance value 1-2 MΩ and 0.3-0.5 MΩ | |
| PharmedBPT-Schlauch | Saint-Gobain Performance Plastics | 3702003 | Size: 0,25 x 2,05 mm (Wd: 0,9mm) |
| Loctite 401 | Henkel | 233641 | Superglue |
| Silicon oil | Thomas Recording | M-1000 | |
| Minisart RC15 | Sartorius | 17761———-R | Syringe filter |
| Multichannel Micro Injection System | Thomas Recording | multichannel microelectrode manipulator “System Eckhorn” equipped with microelectrodes and micropipettes and a precision multichannel microinjection pump | |
| McLab | custom | internal lab software to control stimulus presentation |
References
- Noudoost, B., Moore, T. The role of neuromodulators in selective attention. Trends Cogn Sci. 15 (12), 585-591 (2011).
- Jochems, A., Reboreda, A., Hasselmo, M., Yoshida, M. Cholinergic receptor activation supports persistent firing in layer III neurons in the medial entorhinal cortex. Behav Brain Res. 254, 108-115 (2013).
- Thiele, A., Herrero, J. L., Distler, C., Hoffmann, K. P. Contribution of cholinergic and GABAergic mechanisms to direction tuning, discriminability, response reliability, and neuronal rate correlations in macaque middle temporal area. J Neurosci. 32 (47), 16602-16615 (2012).
- Thienel, R., et al. Muscarinic antagonist effects on executive control of attention. Int J Neuropsychopharmacol. 12 (10), 1307-1317 (2009).
- Anthony, B. L., Dennison, R. L., Aronstam, R. S. Disruption of muscarinic receptor-G protein coupling is a general property of liquid volatile anesthetics. Neurosci Lett. 99 (1-2), 191-196 (1989).
- Yamakura, T., Bertaccini, E., Trudell, J. R., Harris, R. A. Anesthetics and ion channels: molecular models and sites of action. Annu Rev Pharmacol Toxicol. 41, 23-51 (2001).
- Herr, N. R., Wightman, R. M. Improved techniques for examining rapid dopamine signaling with iontophoresis. Front Biosci. 5, 249-257 (2013).
- Bevan, P., Bradshaw, C. M., Pun, R. Y., Slater, N. T., Szabadi, E. The relative contribution of iontophoresis and electro-osmosis to the electrophoretic release of noradrenaline from multi barrelled micropipettes [proceedings]. Br J Pharmacol. 67 (3), 478-479 (1979).
- Herr, N. R., Kile, B. M., Carelli, R. M., Wightman, R. M. Electroosmotic flow and its contribution to iontophoretic delivery. Anal Chem. 80, 8635-8641 (2008).
- Thiele, A., Delicato, L. S., Roberts, M. J., Gieselmann, M. A. A novel electrode-pipette design for simultaneous recording of extracellular spikes and iontophoretic drug application in awake behaving monkeys. J Neurosci Meth. 158 (2-4), 207-211 (2006).
- Lalley, P. M., Johansson, H., Windhorst, U. . Microiontophoresis and Pressure Ejection: Modern Techniques in Neuroscience. , 193-209 (1999).
- Malpeli, J. G., Schiller, P. H. A method of reversible inactivation of small regions of brain tissue. J Neurosci Meth. 1 (2), 145-159 (1979).
- Malpeli, J. G. Reversible inactivation of subcortical sites by drug injection. J Neurosci Meth. 86 (2), 119-128 (1999).
- Dias, E. C., Segraves, M. A. A pressure system for the microinjection of substances into the brain of awake monkeys. J Neurosci Meth. 72 (1), 43-47 (1997).
- Szente, M. B., Baranyi, A., Woody, C. D. Effects of protein kinase C inhibitor H-7on membrane properties and synaptic responses of neocortical neurons of awake cats. Brain Res. 506 (2), 281-286 (1990).
- Woody, C. D., Bartfai, T., Gruen, E., Nairn, A. lntracellular injection of cGMP-dependent protein kinase results in increased input resistance in neurons of the mammalian motor cortex. Brain Res. 386 (1-2), 379-385 (1986).
- Noudoost, B., Moore, T. A reliable microinjectrode system for use in behaving monkeys. J Neurosci Meth. 194 (2), 218-223 (2011).
- Association of Primate Veterinarians. . Cranial Implant Care Guidelines for Nonhuman Primates in Biomedical Research. , (2015).
- Treue, S., Martinez-Trujillo, J. C. Feature-based attention influences motion processing gain in macaque visual cortex. Nature. 399, 575-579 (1999).
- Martinez-Trujillo, J. C., Treue, S. Feature-based attention increases the selectivity of population responses in primate visual cortex. Curr Biol. 14 (9), 744-751 (2004).
