Three-dimensional imaging of KNDy neurons in the mammalian brain using optical tissue clearing and multiple-label immunocytochemistry.

Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in the regulation of fertility. The ability to detect features of KNDy neurons that are essential for fertility may require three-dimensional (3D) imaging of the complete population. Recently developed protocols for optical tissue clearing permits 3D imaging of neuronal populations in un-sectioned brains. However, these techniques have largely been described in the mouse brain. We report 3D imaging of the KNDy cell population in the whole rat brain and sheep hypothalamus using immunolabelling and modification of a solvent-based clearing protocol, iDISCO. This study expands the use of optical tissue clearing for multiple mammalian models and provides versatile analysis of KNDy neurons across species. Additionally, we detected a small population of previously unreported kisspeptin neurons in the lateral region of the ovine mediobasal hypothalamus, demonstrating the ability of this technique to detect novel features of the kisspeptin system.