Journal Article

SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction

Imai, Takeshi, Ke, Meng-Tsen, Fujimoto, Satoshi

2013

Overview

This technical report describes a method to clear fixed brain tissues while allowing for fluorescent dye tracing and retaining cellular morphology. The authors demonstrate the utility of the technique by obtaining a wiring diagram for sister mitral cells. We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: Animal Genetics and Genomics / Animals / Behavioral Sciences / Benzoates - pharmacology / Benzyl Alcohol - pharmacology / Biological Techniques / Biomedicine / Brain / Brain - drug effects / Brain - embryology / Brain - ultrastructure / Chemical tests and reagents / Dendrites - ultrastructure / Electroporation / Female / Fixatives - pharmacology / Fluorescent Dyes - analysis / Fluorescent Dyes - chemistry / Formaldehyde - pharmacology / Fructose - chemistry / Fructose - pharmacology / Gene Knock-In Techniques / Glycerol - analogs & derivatives / Glycerol - chemistry / Glycerol - pharmacology / Histocytological Preparation Techniques / Light / Mice / Mice, Inbred C57BL / Mice, Inbred ICR / Mice, Transgenic / Microscopy / Microscopy - methods / Morphology / Nerve Net - ultrastructure / Neurobiology / Neurons - ultrastructure / Neurophysiology / Neurosciences / Odorants / Olfactory Bulb - cytology / Olfactory Bulb - drug effects / Olfactory Perception - physiology / Phenyl Ethers - pharmacology / Physiological aspects / Polymers - pharmacology / Pregnancy / Properties / Proteins / Scattering, Radiation / Solutions - chemistry / Solutions - pharmacology / Sucrose / Sucrose - pharmacology / technical-report / Tissue Fixation - methods / Urea - pharmacology