概要

普通狨猴层流皮层活动的电生理学

Published: August 04, 2023
doi:

概要

定制的微型驱动器能够通过线性硅阵列实现皮质记录位点的亚毫米级靶向。

Abstract

狨猴因其光滑的皮质表面,为检查层流皮层回路提供了理想的模型,便于使用线性阵列进行记录。狨猴最近越来越受欢迎,因为它的神经功能组织与其他灵长类动物相似,而且在记录和成像方面具有技术优势。然而,由于体积小且缺乏作为解剖标志的脑回,该模型中的神经生理学带来了一些独特的挑战。使用定制的微型驱动器,研究人员可以将线性阵列放置控制到亚毫米级精度,并在记录日内在相同的视网膜定位位置可靠地记录。该协议描述了微驱动定位系统的分步构建和硅线性电极阵列的神经生理记录技术。通过精确控制整个记录会话中的电极位置,研究人员可以轻松地遍历皮层,根据其视网膜组织和记录神经元的调谐特性来识别感兴趣的区域。此外,使用这种层流阵列电极系统,可以应用电流源密度分析(CSD)来确定单个神经元的记录深度。该协议还演示了层流记录的示例,包括在 Kilosort 中分离的尖峰波形,这些波形跨越阵列上的多个通道。

Introduction

近年来,普通狨猴(Callithrix jacchus)作为研究大脑功能的模型迅速流行起来。这种日益普及的原因是狨猴光滑皮层的可及性,与人类和其他灵长类动物的神经功能组织的相似性,以及体积小、繁殖速度快1。随着这种模式生物越来越受欢迎,适用于狨猴大脑的神经生理学技术得到了迅速发展。电生理学方法在神经科学中被广泛用于研究啮齿动物和灵长类动物皮层中单个神经元的活动,从而实现无与伦比的时间分辨率和位置访问。由于狨猴作为视觉神经科学模型的相对新颖性,清醒行为电生理学技术的优化仍在不断发展。先前的研究表明,在麻醉制剂中建立了稳健的电生理学方案2,早期觉醒行为神经生理学研究表明了单通道钨电极3 的可靠性。近年来,研究人员已经确定了硅基微电极阵列在清醒行为神经生理学中的应用4。然而,狨猴由于大脑体积小且缺乏解剖学标志,因此提出了独特的目标挑战。该协议概述了如何构建和使用适用于狨猴的微型驱动器记录系统,该系统允许使用硅线性阵列记录大量神经元,同时产生最小的组织损伤。

与狨猴一起工作是一个挑战,因为与较大的灵长类动物相比,视觉皮层中视网膜图的比例较小。电极稍偏移 1 mm 即可导致图谱发生重大变化。此外,研究人员经常需要在记录会话之间改变电极的位置,以获得视觉皮层中更广泛的视网膜位置。目前的半慢性制剂不允许每天调整电极位置,也无法以足够的精度以亚毫米级5瞄准特定位置。考虑到这一点,所提出的微驱动系统利用X-Y电极级,该电极级将轻量级微驱动安装到记录室中,并允许对皮质部位进行亚毫米级靶向。可移动的 X-Y 载物台组件允许线性阵列的垂直和水平移动,以便系统地遍历皮质区域,这是识别感兴趣区域(通过 视网膜位和调谐特性)所必需的。在录制过程中,研究人员还可以手动调整 X-Y 载物台以移动区域内的目标站点。与使用半慢性记录制剂的替代技术相比,这是一个关键优势,后者没有简单的电极靶向机制。

微型驱动器是一种多功能工具,可以安装各种硅阵列以降低到皮层中。在该协议中,使用具有两个间隔 200 μm 的 32 通道线性阵列的定制探针来研究跨越皮层深度的层流电路。大多数探测神经回路的方法通常对大脑皮层所有层的平均电位或单个单元进行采样。然而,最近的研究揭示了关于皮层层流微电路的有趣发现6.通过利用微型驱动器,研究人员可以使用层流探头并对记录深度进行微调,以确保在所有层中进行全面采样。

该系统可以使用市售组件构建,并且很容易针对不同的实验技术或探针进行修改。这种制备的主要优点是能够以亚毫米级精度改变X-Y记录位置,并控制皮层内记录的深度。该协议提供了构建X-Y阶段微驱动器和神经生理学记录技术的分步说明。

Protocol

实验程序遵循美国国立卫生研究院实验动物护理和使用指南。实验和行为程序的协议已获得罗切斯特大学机构动物护理和使用委员会的批准。 1. 包含用于记录的电极的微型驱动器的结构(图 1) 注意:定制的 X-Y 载物台包含多通道线性硅阵列,允许对记录站点进行亚毫米级目标定位。 收集 图 1</st…

Representative Results

该协议描述了如何构建X-Y电极级(图1),该电极级允许对位点进行亚毫米级定位,并在单独的记录会话中保持可靠的定位。X-Y定位的可靠性如 图6所示,该图表明,相隔一周进行的两次记录会话显示其平均射频位置重叠70.8%(图6A)。此外,对微型驱动器定位的微小调整可以支持视网膜空间的精确运动。通过参考每个阶段中相距 2 …

Discussion

目前有几种方法(例如慢性、半慢性、急性)可用于在非人灵长类动物中进行神经生理学实验。普通狨猴因其体型小且缺乏作为解剖标志的脑回,对神经生理学实验提出了独特的挑战。这要求研究人员使用神经生理学标志,如感兴趣区域的视网膜和调谐特性来识别记录目标。因此,在最初绘制皮质区域时,可能需要每天调整电极位置。目前狨猴神经生理学的制剂通常使用半慢性探针定位,这不允?…

開示

The authors have nothing to disclose.

Acknowledgements

这项工作得到了美国国立卫生研究院 (NIH) 资助 R01 EY030998(J.F.M.、AB 和 SC)的支持。该方法基于 Coop 等人开发的方法(正在审查,2022 年; https://www.biorxiv.org/content/10.1101/2022.10.11.511827v2.abstract)。我们要感谢 Dina Graf 和 Mitchell 实验室的成员在狨猴护理和处理方面的帮助。

Materials

1/4 Hp burr drill bit McMaster & Carr Cat# 43035A32 Carbide Bur with 1/4" Shank Diameter, Rounded Cylinder Head, trade Number SC-1, single Cut(https://www.mcmaster.com/products/bur-bits/burs-7/?s=1%2F4%22+bur+bits)
1x1mm Crist Grid Crist Instruments 1 mm x 1 mm Grid https://www.cristinstrument.com/products/implant-intro/grids
91% isopropyl alcohol Medline N/A https://www.medline.com/product/Medline-Isopropyl-Rubbing-Alcohol/Bulk-Alcohol/Z05-PF03807?question=91%25%20isopropyl%20alcohol
Acquisition Board Open-Ephys N/A https://open-ephys.org/acquisition-system/eux9baf6a5s8tid06hk1mw5aafjdz1
Bacitracin Ointment Medline: Cosette Pharmaceuticals Inc N/A https://www.medline.com/product/Bacitracin-Ointment/Antibiotics/Z05-PF86957?question=bacitr
Blunt straight Forceps Medline N/A https://www.medline.com/category/Central-Sterile/Surgical-Instruments/Forceps/Z05-CA16_02_20/products
Bone wax Medline ETHW31G https://www.medline.com/product/Ethicon-Bone-Wax/Bone-Wax/Z05-PF61528?question=bonewax
C&B Metabond Quick Adhesive Cement System Parkell, Inc. SKU: S380 https://www.parkell.com/C-B-Metabond-Quick-Adhesive-Cement-System
Clavamox MWI Animal Health N/A
Contact lens solution Bausch and lomb Various sources available
Custom Printed 3D printed parts ProtoLab https://marmolab.bcs.rochester.edu/resources.html
DB25-G2 25 Pin Male Plug Port Signal Connector Various Sources DB25-G2 25 DB25-G2 25 Pin Male Plug Port Signal 2 Row Terminal Breakout Board Screw Nut Connector
diamond saw attachement for dremmel Dremmel 545 Diamond Wheel https://www.dremel.com/us/en/p/545-26150545ab
Digitizing Head-stages Intan RHD 32channel (Part #C3314) https://intantech.com/RHD_headstages.html?tabSelect=RHD32ch&yPos=120.80
000305175781
EDOT Sigma Aldrich Product # 483028 https://www.sigmaaldrich.com/US/en/product/aldrich/483028
Helping Hands Harbor Freight N/A https://www.harborfreight.com/helping-hands-60501.html
Hook Electrical Clips Various Sources N/A Hook test Cable wires
Interface Cables (RHD 3-ft (0.9 m) ultra thin SPI cable) Intan  Part #C3213 https://intantech.com/RHD_SPI_cables.html
Lab jack Various Sources N/A https://www.amazon.com/Stainless-Steel-Scissor-Stand-Platform/dp/B07T8FM85H/ref=asc_df_B07T8FM85H/?tag=&linkCode=df0&hvadid=366343
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Meloxicam MWI Animal Health N/A
Micro-drive Crist Instrument 3-NRMD https://www.cristinstrument.com/products/microdrives/miniature-microdrive-3-nrmd
Multi-channel linear silicon arrays with 64 channel connector NeuroNexus A1x32-5mm-25-177 https://www.neuronexus.com/products/electrode-arrays/up-to-10-mm-depth/
NanoZ Omentics Adapter- 32 Channel NeuraLynx ADPT-NZ-N2T-32 https://neuralynx.com/hardware/adpt-nz-n2t-32
NanoZ System Plexon NanoZ Impedence Tester https://plexon.com/products/nanoz-impedance-tester/
Narishige Micromanipulator Narishige Stereotaxic Micromanipulator https://usa.narishige-group.com/
Open-Ephys GUI Open-Ephys https://open-ephys.org/
Polyimide Tubing (OD(in): 0.021 / ID(in) 0.018 ) Various Sources (Chamfr) Chamfr Cat#HPC01895 https://chamfr.com/sellers/teleflex-medical-oem-llc/
Primate Chair Custom made by University of Rochester Machine Shop Designs online https://marmolab.bcs.rochester.edu/resources.html
Poly(sodium 4-styrenesulfonate) (PSS) Sigma Aldrich Product # 243051 https://www.sigmaaldrich.com/US/en/product/aldrich/243051
RHD USB Interface board Intan RHD2000 Evaluation Board Version 1.0 https://intantech.com/RHD_USB_interface_board.html
Silastic gel World Precision Instuments # KWIK-SIL Low Toxicity Silicone Adhesive ((https://www.wpiinc.com/kwik-sil-low-toxicity-silicone-adhesive)
Slow release buprenorphine Compounding Pharmacy
Stainless steel wire 36 gauge McMaster & Carr Cat# 6517K11 Round Bend-and-Stay Multipurpose 304 Stainless Steel Wire, Matte Finish, 1-Foot Long, 0.008" Diameter
Stanley 6-Piece Precision Screwdriver Set Stanley 1.4mm flathead screwdriver https://www.amazon.com/Stanley-Tools-6-Piece-Precision-Screwdriver/dp/B076621ZGC/ref=sr_1_3?crid=237VSK5FNFP9N&keywords=
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Steel Screws McMaster & Carr type 00 stainless steel hex screws and 1/8” in length https://www.mcmaster.com/
Steel Tube McMaster & Carr 28 gauge stainless steel tubing https://www.mcmaster.com/tubing/multipurpose-304-stainless-steel-6/id~0-055/
Superglue Loctite SuperGlue Gel Control https://www.loctiteproducts.com/en/products/fix/super-glue/loctite_super_gluegelcontrol.html

参考文献

  1. Mansfield, K. Marmoset models commonly used in biomedical research. Comparative Medicine. 53 (4), 383-392 (2003).
  2. Solomon, S. G., Rosa, M. G. P. A simpler primate brain: the visual system of the marmoset monkey. Frontiers in Neural Circuits. 8, 96 (2014).
  3. Remington, E. D., Osmanski, M. S., Wang, X. An operant conditioning method for studying auditory behaviors in marmoset monkeys. PLoS One. 7 (10), e47895 (2012).
  4. Walker, J. D., et al. Chronic wireless neural population recordings with common marmosets. Cell Reports. 36 (2), 109379 (2021).
  5. Jendritza, P., Klein, F. J., Fries, P. Multi-area recordings and optogenetics in the awake, behaving marmoset. Nature Communications. 14 (1), 577 (2023).
  6. Pinotsis, D. A., et al. Linking canonical microcircuits and neuronal activity: Dynamic causal modelling of laminar recordings. Neuroimage. 146, 355-366 (2017).
  7. Ludwig, K. A., et al. Poly (3, 4-ethylenedioxythiophene)(PEDOT) polymer coatings facilitate smaller neural recording electrodes. Journal of Neural Engineering. 8 (1), 014001 (2011).
  8. Ludwig, K. A., Uram, J. D., Yang, J., Martin, D. C., Kipke, D. R. Chronic neural recordings using silicon microelectrode arrays electrochemically deposited with a poly (3, 4-ethylenedioxythiophene)(PEDOT) film. Journal of Neural Engineering. 3 (1), 59 (2006).
  9. Lu, T., Liang, L., Wang, X. Neural representations of temporally asymmetric stimuli in the auditory cortex of awake primates. Journal of Neurophysiology. 85 (6), 2364-2380 (2001).
  10. Osmanski, M. S., Song, X., Wang, X. The role of harmonic resolvability in pitch perception in a vocal nonhuman primate, the common marmoset (Callithrix jacchus). Journal of Neuroscience. 33 (21), 9161-9168 (2013).
  11. Nummela, S. U., et al. Psychophysical measurement of marmoset acuity and myopia. Developmental Neurobiology. 77 (3), 300-313 (2017).
  12. Paxinos, G., Watson, C., Petrides, M., Rosa, M., Tokuno, H. . The Marmoset Brain in Stereotaxic Coordinates. , (2012).
  13. Mitchell, J. F., Reynolds, J. H., Miller, C. T. Active vision in marmosets: A model system for visual neuroscience. Journal of Neuroscience. 34 (4), 1183-1194 (2014).
  14. Spitler, K. M., Gothard, K. M. A removable silicone elastomer seal reduces granulation tissue growth and maintains the sterility of recording chambers for primate neurophysiology. Journal of Neuroscience Methods. 169 (1), 23-26 (2008).
  15. Jun, J. J., et al. Fully integrated silicon probes for high-density recording of neural activity. Nature. 551 (7679), 232-236 (2017).
  16. Mitzdorf, U. Current source-density method and application in cat cerebral cortex: investigation of evoked potentials and EEG phenomena. Physiological Reviews. 65 (1), 37-100 (1985).
  17. Coop, S. H., Yates, J. L., Mitchell, J. F. Pre-saccadic neural enhancements in marmoset area MT. bioRxiv. , (2022).
  18. Okun, M., Lak, A., Carandini, M., Harris, K. D. Long term recordings with immobile silicon probes in the mouse cortex. PloS One. 11 (3), e0151180 (2016).

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記事を引用
Bucklaew, A., Coop, S. H., Mitchell, J. F. Electrophysiology of Laminar Cortical Activity in the Common Marmoset. J. Vis. Exp. (198), e65397, doi:10.3791/65397 (2023).

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