JoVE Journal > Neuroscience
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Please note that all translations are automatically generated. Click here for the English version.
Neuroscienze

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

Published: January 19, 2024
doi:
10.3791/65387
, ,
1Institute of Ophthalmology,University College London

Summary

当与头板和与单光子和双光子显微镜兼容的光学设计集成时，微棱镜透镜在测量不同条件下垂直柱中的神经反应方面具有显着优势，包括头部固定状态下的良好控制实验或自由移动动物的自然行为任务。

Abstract

随着多光子显微镜和分子技术的进步，荧光成像正在迅速发展成为研究活体脑组织结构、功能和可塑性的有力方法。与传统的电生理学相比，荧光显微镜可以捕获细胞的神经活动和形态，从而能够以单细胞或亚细胞分辨率长期记录已识别的神经元群。然而，高分辨率成像通常需要一个稳定的、头部固定的设置，以限制动物的运动，并且制备透明玻璃的平面允许在一个或多个水平面上可视化神经元，但在研究跨不同深度的垂直过程方面受到限制。在这里，我们描述了一种将头板固定和微棱镜相结合的程序，该微棱镜可提供多层和多模态成像。这种手术准备不仅可以进入小鼠视觉皮层的整个柱子，而且允许在头部固定位置进行双光子成像，在自由移动的范式中进行单光子成像。使用这种方法，人们可以对不同皮层中已识别的细胞群进行采样，在头部固定和自由移动状态下记录它们的反应，并跟踪几个月的长期变化。因此，该方法提供了对微电路的全面分析，能够直接比较由良好控制的刺激和自然行为范式引起的神经活动。

Introduction

体内双光子荧光成像1,2的出现，结合了光学系统中的新技术和转基因荧光指示剂，已成为神经科学中研究活体大脑中复杂结构、功能和可塑性的强大技术3,4.特别是，这种成像方式通过捕获神经元的形态和动态活动，提供了比传统电生理学无与伦比的优势，从而促进了对已识别神经元的长期跟踪5,6,7,8。

尽管具有显著的优势，但高分辨率荧光成像的应用通常需要静态的头部固定设置，从而限制动物的活动能力9,10,11。此外，使用透明玻璃表面可视化神经元将观察限制在一个或多个水平面上，限制了对跨越不同皮层深度的垂直过程动力学的探索12。

为了解决这些局限性，本研究概述了一种创新的外科手术，该手术集成了头板固定、微棱镜和微型镜，以创建具有多层和多模态功能的成像模式。微棱镜允许观察沿皮层柱13,14,15,16的垂直处理，这对于理解信息在通过皮层的不同层时如何处理和转换以及垂直处理在塑料变化过程中如何改变至关重要。此外，它允许在头部固定范式和自由移动的环境中对相同的神经群体进行成像，包括多功能实验设置17,18,19：例如，头部固定通常需要用于控制良好的范式，如感觉知觉评估和2光子范式下的稳定记录，而自由移动为行为研究提供了更自然、更灵活的环境。因此，在两种模式下进行直接比较的能力对于进一步了解能够实现灵活功能响应的微电路至关重要。

从本质上讲，在荧光成像中集成头板固定、微棱镜和微型镜为探索大脑结构和功能的复杂性提供了一个很有前途的平台。研究人员可以在跨越所有皮质层的不同深度对已识别的细胞群进行采样，直接比较它们在良好控制和自然范式中的反应，并监测它们在20个月内的长期变化。这种方法为这些神经群体在不同实验条件下如何相互作用和随时间变化提供了宝贵的见解，为了解神经回路的动态性质提供了一个窗口。

Protocol

所有实验均根据 1986 年英国动物（科学程序）法案进行，并经英国内政部批准和颁发的个人和项目许可证，并经过适当的伦理审查。成虫转基因品系CaMKII-TTA;培育了GCaMP6S-TRE21 ，并将其后代用于实验。为了实验人员的安全和维持无菌条件，所有程序均在无菌条件下进行，并配备完整的个人防护设备。 1.术前准备 为了尽量减少水肿，手术前1…

Representative Results

已经展示了在自由移动和头部固定条件下，使用单光子和双光子成像模式在数周内对同一神经元群进行慢性多层 体内 钙成像的方法。在这里，当动物在黑暗中探索开放的竞技场时，在单光子成像下识别匹配神经元群的能力已经得到证明（图7A）。从鉴定的神经元中提取钙痕迹并进行z评分以进行比较（图7B）。神经元在相隔 3 周的会话中显示出相…

Discussion

在这里，我们已经展示了在同一神经群体中观察和直接比较头部固定和自由移动条件下神经元的能力。虽然我们演示了在视觉皮层中的应用，但该协议可以适应许多其他大脑区域，包括皮层区域和深核24,25,26,27,28，以及其他数据采集和行为设置29,30<s…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢Charu Reddy女士和Matteo Carandini教授（Cortex实验室）对手术方案和转基因小鼠品系共享的建议。我们感谢 Norbert Hogrefe 医生 （Inscopix） 在手术开发过程中的指导和帮助。我们感谢Andreea Aldea女士（Sun Lab）在手术设置和数据处理方面的协助。这项工作得到了Moorfields Eye Charity的支持。

Materials

0.9% Sodium Chloride solution for infusion (Vetivex 11) 250ml Dechra 20091607 Saline for hydration and drug reconsitution
18004-1 Trephine 1.8mm diameter bur FST 18004-18 Drill bit
1ml syringe Terumo MDSS01SE 1ml syringe
23G x 5/8 inch 6% LUER needle Terumo NN-2316R 23G needle
71000 Automated stereotaxic apparatus w/ built-in software RWD – RWD
Absorbable Haemostatic Gelatin Sponge (10x10x10mm) Surgispon SSP-101010 gel-foam
Alcohol pads 70% isopropyl alcohol Braun 9160612 Alcohol pads
Aluminium foil Any retailer – Foil to cover eyes during surgery
Articifical Cerebrospinal Fluid  Tocris Bioscience a Bio-Techne Brand 3525/25ML ACSF
Automated microinjection pump WPI 8091
Betadine solution (10% iodinated Povidone) 500ml Videne/Ecolab 3030440 Betadine
Bruker Ultime 2Pplus (customised) Bruker – Two-photon imaging system 
Cardiff Aldasorber Vet-Tech AN006 Anaesthesia absorber
CFI S Plan Fluor ELWD ADM 20XC Nikon MRH48230 20x objective lens
Compact Anaesthesia system – single gas – isoflurane K/F, with oxygen concentrator model: ZY-5AC and scavenging unit Vet-Tech AN001 Compact anaesthesia system 
Contec Prochlor  Aston Pharma AP2111L1 Disinfectant (hypochlorous acid)
Dexamethasone Sodium Phosphate Injection, USP, 4mg/ml, NDC: 0641-6145-25 Hikma Covetrus:70789 Dexamethasone
Dissecting Knife, cutting edge 4mm, thickness 0.5mm, stainless steel Fine Science Tools 10055-12 Knife for incisino of cortex
Dual-Sided, Non-Puncture Mouse & Neonatal Rat Ear Bars Stoelting 51649 Ear bar
Dummy microscope Inscopix Dummy microscope To help with implantation
Ethanol (100%)  VWR 40-1712-25 Used to make 70% ethanol 
Fisherbrand Nitrile Indigo Disposable Gloves PPE Cat III FischerScientific 17182182 Gloves
Homeothermic Monitor 50-7222-F Harvard Apparatus 50-7222-F Homeothermic monitoring system/heating pad
Image processing software ImageJ – Image processing software
Inscopix Data Processing Software (IDPS) Inscopix – One-photon calcium imaging processing software
Insight Duals-232, S/N 2043 InSight Insight Spectra X3 Two-photon imaging laser
IsoFlo 250ml 100% w/w inhalation Zoetis WM 42058/4195 Isoflurane
Kwik-Sil Low Toxicity Silicone Adhesive World Precision Intruments (WPI) KWIK-SIL Silicone adhesive
MICROMOT mains adapter NG 2/S, w/ Drill unit 60/E PROXXON NO 28 515 Handheld drill
nVoke Integrated Imaging and Optogenetics System package Inscopix – One-photon Imaging system and software
ProView Implant Kit Inscopix ProView Implant Kit Dummy microscope, stereotaxic arm and attachment 
ProView Prism Probe Inscopix 1050-002203 Microprism lens
Rimadyl (50mg/ml) Zoetis VM 42058/4123 Carprofen 
Stereotaxis Microscope on Articulated arm with table clamp WPI PZMTIII-AAC  Microscope
Super-Bond Universal kit, SUN Medical Prestige-Dental K058E Adhesive cement
Two-photon calcium image software Suite2P – Two-photon calcium imaging processing software
Vapouriser Vet-Tech – Isoflurane vapouriser
Xailin Lubricating Eye Ointment 5g Xailin-Night MLG/28/1551 Ophthalmic ointment 
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Denk, W., Strickler, J. H., Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science. 248 (4951), 73-76 (1990).
  2. Svoboda, K., Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron. 50 (6), 823-839 (2006).
  3. Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., Tank, D. W. Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron. 56 (1), 43-57 (2007).
  4. Vaziri, A., Emiliani, V. Reshaping the optical dimension in optogenetics. Curr Opin Neurobiol. 22 (1), 128-137 (2012).
  5. Holtmaat, A., et al. high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc. 4 (8), 1128-1144 (2009).
  6. Sun, Y. J., Sebastian Espinosa, J., Hoseini, M. S., Stryker, M. P. Experience-dependent structural plasticity at pre- and postsynaptic sites of layer 2/3 cells in developing visual cortex. Proc Natl Acad Sci U S A. 116 (43), 21812-21820 (2019).
  7. Andermann, M. L., Kerlin, A. M., Reid, R. C. Chronic cellular imaging of mouse visual cortex during operant behavior and passive viewing. Front Cell Neurosci. 4, 3 (2010).
  8. Sofroniew, N. J., Flickinger, D., King, J., Svoboda, K. A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. Elife. 5, 14472 (2016).
  9. Puścian, A., Benisty, H., Higley, M. J. NMDAR-dependent emergence of behavioral representation in primary visual cortex. Cell Rep. 32 (4), 107970 (2020).
  10. Trachtenberg, J. T., et al. Long-term in vivo. imaging of experience-dependent synaptic plasticity in adult cortex. Nature. 420 (6917), 788-794 (2002).
  11. Seaton, G., et al. Dual-component structural plasticity mediated by αCaMKII autophosphorylation on basal dendrites of cortical layer 2/3 neurones. J Neurosci. 40 (11), 2228-2245 (2020).
  12. Helmchen, F., Denk, W. Deep tissue two-photon microscopy. Nat Methods. 2 (12), 932-940 (2005).
  13. Andermann, M. L., et al. Chronic cellular imaging of entire cortical columns in awake mice using microprisms. Neuron. 80 (4), 900-913 (2013).
  14. Chia, T. H., Levene, M. J. Microprisms for in vivo multilayer cortical imaging. J Neurophysiol. 102 (2), 1310-1314 (2009).
  15. Low, R. J., Gu, Y., Tank, D. W. Cellular resolution optical access to brain regions in fissures: Imaging medial prefrontal cortex and grid cells in entorhinal cortex. Proc Natl Acad Sci U S A. 111 (52), 18739-18744 (2014).
  16. Buxhoeveden, D. P., Casanova, M. F. The minicolumn hypothesis in neuroscience. Brain. 125, 935-951 (2002).
  17. Chen, S., et al. Miniature fluorescence microscopy for imaging brain activity in freely-behaving animals. Neurosci Bull. 36 (10), 1182-1190 (2020).
  18. Gulati, S., Cao, V. Y., Otte, S. Multi-layer cortical Ca2+ imaging in freely moving mice with prism probes and miniaturized fluorescence microscopy. J Vis Exp. (124), e55579 (2017).
  19. Resendez, S. L., et al. Visualization of cortical, subcortical, and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nat Protoc. 11 (3), 566-597 (2016).
  20. Guo, Z. V., et al. Procedures for behavioral experiments in head-fixed mice. PLoS One. 9 (2), 88678 (2014).
  21. Wekselblatt, J. B., Flister, E. D., Piscopo, D. M., Niell, C. M. Large-scale imaging of cortical dynamics during sensory perception and behavior. J Neurophysiol. 115 (6), 2852-2866 (2016).
  22. Pnevmatikakis, E. A., et al. Simultaneous denoising, deconvolution, and demixing of calcium imaging data. Neuron. 89 (2), 285-299 (2016).
  23. Zhou, P., et al. Efficient and accurate extraction of in vivo calcium signals from microendoscopic video data. Elife. 7, 28728 (2018).
  24. Beckmann, L., et al. Longitudinal deep-brain imaging in mouse using visible-light optical coherence tomography through chronic microprism cranial window. Biomed Opt Express. 10 (10), 5235-5250 (2019).
  25. Wenzel, M., Hamm, J. P., Peterka, D. S., Yuste, R. Reliable and elastic propagation of cortical seizures in. Cell Rep. 19 (13), 2681-2693 (2017).
  26. Heys, J. G., Rangarajan, K. V., Dombeck, D. A. The functional micro-organization of grid cells revealed by cellular-resolution imaging. Neuron. 84 (5), 1079-1090 (2014).
  27. Barson, D., Hamodi, A. S. Simultaneous mesoscopic and two-photon imaging of neuronal activity in cortical circuits. Nat Methods. 17 (1), 107-113 (2020).
  28. Paquelet, G. E., et al. Single-cell activity and network properties of dorsal raphe nucleus serotonin neurons during emotionally salient behaviors. Neuron. 110 (16), 2664-2679 (2022).
  29. Yang, Q., et al. Transparent microelectrode arrays integrated with microprisms for electrophysiology and simultaneous two-photon imaging across cortical layers. bioRxiv. , (2022).
  30. Priestley, J. B., Bowler, J. C., Rolotti, S. V., Fusi, S., Losonczy, A. Signatures of rapid plasticity in hippocampal CA1 representations during novel experiences. Neuron. 110 (12), 1978-1992 (2022).
  31. Zong, W., et al. Miniature two-photon microscopy for enlarged field-of-view, multi-plane and long-term brain imaging. Nat Methods. 18 (1), 46-49 (2021).
  32. Engelbrecht, C. J., et al. Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo. Opt Express. 16 (8), 5556-5564 (2008).
  33. Suzuki, M., Aru, J., Larkum, M. E. Double-μ Periscope, a tool for multilayer optical recordings, optogenetic stimulations or both. Elife. 10, 72894 (2021).
  34. Stibůrek, M., et al. 110 μm thin endo-microscope for deep-brain in vivo observations of neuronal connectivity, activity and blood flow dynamics. Nat Commun. 14 (1), 1897 (2023).

Tags

Keywords: Long-term ImagingNeural PopulationsMicroprismsHead-fixed AnimalsFreely Moving AnimalsSystem NeuroscienceChronic RecordingCell Type-specific TargetingAwake StatesNatural ConditionsSensory Information ProcessingCortical LayersMulti-photon MicroscopyFluorescence ImagingElectrophysiologyNeural ActivityMorphologyHead Plate FixationMicroprismMouse Visual Cortex
check_url/it/65387?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Burrows, R., Ma, C., Sun, Y. J. Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals. J. Vis. Exp. (203), e65387, doi:10.3791/65387 (2024).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image