Application Of Optical Clearing Methods On The Songbird Brain

Songbirds provide an opportunity to explore the neural mechanism underlying the learning and production of complex sound sequences. Imaging the neural structure at single-cell resolution in large volume tissue helps to disclose the functions of neural circuits involved in the song system. Recent advancements in optical clearing techniques may contribute to successful imaging of large-volume samples of songbird brains. These new clearing methods were originally optimized for rodent nervous tissues and previously have not been tested on avian brains. Here we examined whether SeeDB and ClearT, two of the latest optical clearing methods, are suitable for songbird brains, in addition to rodent brains. These two methods were chosen because of the shortness of time required to complete them and because of their compatibility with common lipophilic dye labeling. We applied these methods to brain sections of Bengalese Finches Lonchura striata var. domestica and laboratory rats Rattus norvegicus, then quantified sample transparency as well as expansion rates. The analyses, using both methods, revealed that finch samples were less transparent than rat brain sections. In samples of both species, ClearT was found to enable higher transparency, but caused more expansion, than SeeDB. It is expected that these clearing methods will be of advantage when investigating the anatomical aspects of the neural basis of birdsong.