JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Published: April 20th, 2021

DOI:

10.3791/62532

1Department of Genetics and Genomics, Baylor College of Medicine, 2Medical Scientist Training Program, Baylor College of Medicine, 3Department of Neuroscience, Baylor College of Medicine

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Understanding the spatial organization, patterns of connectivity, and morphology of complex three-dimensional biological structures is essential for delineating the functions of specific cells and tissues. This is especially true in neuroscience, in which tremendous effort has been dedicated to building high-resolution neuroanatomical maps of the central nervous system1,2. Close examination of the neurons that comprise these maps yields varied morphologies, with connections and locations that reflect the function of these diverse sets of neurons3,4. Mo....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The following protocol follows all animal care guidelines for Baylor College of Medicine.

1. Dissection and tissue preparation

  1. Euthanize the mouse with an overdose of isoflurane by placing the mouse in a closed container with a towel soaked in isoflurane (or by other IUCAC approved means).
  2. Perfuse the animal transcardially using a 25 G needle with 10 mL of ice cold PBS, followed by 10 mL of 4% PFA.
  3. Dissect the brain region (or tissue) of interest.
  4. Plac.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

After image acquisition, the representative cell morphology was analyzed using embedded statistics and classifying scripts within the analysis software. The collected data (Figure 6A) reflects that neuron 2 has a larger dendritic structure with a higher density of spines. As a whole, the data suggests that neuron 2 has a more complex dendritic structure compared to neuron 1. To substantiate this result, standard Sholl analysis was performed, which affirms that neuron 2 is more dendritically .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Before the advent of contemporary tissue clearing techniques, studying neuronal morphology consisted of time-intensive sectioning, imaging, and reconstruction of adjacent very thin sections. Using electrophoretic tissue clearing in combination with confocal imaging provides an unobstructed view of complete neuronal morphology. From intact dendritic trees, down to the smallest synaptic bouton, imaging and quantifying neuronal morphology has never been more feasible.

The preparation of cleared b.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We would like to thank the viral core NRDDC at the Jan and Dan Duncan Neurological Institute for producing the AAVs and lentiviruses used in these experiments. Additionally, we would like to thank the Baylor College of Medicine Center for Comparative Medicine for mouse husbandry and general maintenance of the mice used. We would like to thank the American Heart Association for their support under award number 20PRE35040011, and BRASS: Baylor Research Advocates for Student Scientists for their support (PJH). Finally, we would like to thank Logos for providing our lab with the Logos X-Clarity electrophoretic tissue clearing system.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
15 mL Conical Tube Thermo Scientific 339650
25 G x 1" Needle BD 305127
30% Acrylamide (No-Bis) National Diagnostics EC-810
50 mL Conical Tube Thermo Scientific 339653
Electrophoretic Tissue Clearing Solution Logos C13001
Histodenz Sigma D2158-100G
Hydrogel Solution Kit Logos C1310X
Imaris Oxford Instruments N/A
Paraformaldehyde 16% EMS 15710
PBS, 1x, 500 mL, 6 bottles/case fisher MT21040CV
VA-044 Wako 925-41020
X-CLARITY Polymerization System Logos C20001
X-CLARITY Tissue Clearing System II Logos C30001

  1. Abbott, L. F., et al. The Mind of a mouse. Cell. 182 (6), 1372-1376 (2020).
  2. White, J. G., Southgate, E., Thomson, J. N., Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Philosophical transactions of the Royal Society of London. Series B, Biological Sciences. 314 (1165), 1 (1986).
  3. Jiang, X., et al. Principles of connectivity among morphologically defined cell types in adult neocortex. Science. 350 (6264), (2015).
  4. Winnubst, J., et al. Reconstruction of 1,000 projection neurons reveals new cell types and organization of long-range connectivity in the mouse brain. Cell. 179 (1), 268-281 (2019).
  5. Araya, R., Vogels, T. P., Yuste, R. Activity-dependent dendritic spine neck changes are correlated with synaptic strength. Proceedings of the National Academy of Sciences of the United States of America. 111 (28), (2014).
  6. Bosch, M., Hayashi, Y. Structural plasticity of dendritic spines. Current Opinion in Neurobiology. 22 (3), 383-388 (2012).
  7. Martínez-Cerdeño, V. Dendrite and spine modifications in autism and related neurodevelopmental disorders in patients and animal models. Developmental Neurobiology. 77 (4), 393-404 (2017).
  8. Arenkiel, B. R., Ehlers, M. D. Molecular genetics and imaging technologies for circuit-based neuroanatomy. Nature. 461 (7266), 900-907 (2009).
  9. Kim, E. H., Chin, G., Rong, G., Poskanzer, K. E., Clark, H. A. Optical probes for neurobiological sensing and imaging. Accounts of Chemical Research. 51 (5), 1023-1032 (2018).
  10. Weissman, T. A., Pan, Y. A. Brainbow: New resources and emerging biological applications for multicolor genetic labeling and analysis. Genetics. 199 (2), 293-306 (2014).
  11. Haggerty, D. L., Grecco, G. G., Reeves, K. C., Atwood, B. Adeno-associated viral vectors in neuroscience research. Molecular Therapy - Methods and Clinical Development. 17, 69-82 (2020).
  12. Chung, K., et al. Structural and molecular interrogation of intact biological systems. Nature. 497 (7449), 332-337 (2013).
  13. Kanning, K. C., Kaplan, A., Henderson, C. E. Motor neuron diversity in development and disease. Annual Review of Neuroscience. 33, 409-440 (2010).
  14. Ledda, F., Paratcha, G. Mechanisms regulating dendritic arbor patterning. Cellular and Molecular Life Sciences. 74 (24), 4511-4537 (2017).
  15. Falougy, H. E., Filova, B., Ostatnikova, D., Bacova, Z., Bakos, J. Neuronal morphology alterations in autism and possible role of oxytocin. Endocrine Regulations. 53 (1), 46-54 (2019).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved