Abstract
The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies.
Highlights
Recent innovations in tissue clearing, combined with advances in light sheet microscopy and big data analysis, are bringing the exploration of whole brain three-dimensional space at single-cell resolution within our reach
With the CUBIC protocol, prolonging the immersion in reagent-1 solution to 10 days, instead of the 7 days recommended by the original protocol, helped improve final transparency
We obtained transparencies comparable to those reported for CUBIC and iDISCO+ protocols, enabling single sided light sheet penetration in imaging volumes of least half an adult zebra finch forebrain (Figures 1, 2 and Supplementary Movie S1)
Summary
Recent innovations in tissue clearing, combined with advances in light sheet microscopy and big data analysis, are bringing the exploration of whole brain three-dimensional space at single-cell resolution within our reach. Brain tissue is composed of high-refractive index (RI) molecules, lipids and proteins, embedded in a low RI medium, water. This RI mismatch leads to heterogeneity in light scattering, and turns tissue opaque. Clearing the Song System flourished into several variants, generally try to reduce the RI mismatch by either substituting water by a higher RI medium or by removing or modifying the optical properties of the dry components (for review see Richardson and Lichtman, 2015). Tissue clearing reports are limited to the zebra finch syrinx (Faunes et al, 2017) and the bengalese finch brain (Fujii et al, 2016)
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