Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Summary

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.

Abstract

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.

Introduction

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 system^1,2. 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 neurons^3,4. Mo....

Protocol

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.......

Discussion

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.......

Materials

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