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

Aleisha M Moore,Lique M Coolen,Kathryn A Lucas,Michael N Lehman,Robert L Goodman

doi:10.1038/s41598-018-20563-2

Abstract

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.

Highlights

Neurons expressing the peptides kisspeptin or neurokinin B (NKB) play a critical role in the regulation of fertility
We report here the application of Immunolabelling-enabled 3D imaging of solvent cleared organs (iDISCO), a solvent-based optical tissue clearing protocol commonly used for imaging of intact cellular structures, to study the arcuate Kisspeptin/Neurokinin B/Dynorphin (KNDy) neuron population in the rat and sheep brain
We provide the first 3D visualization of the arcuate KNDy population in any species using a dual-label approach with kisspeptin and NKB antibodies, and, the first use of iDISCO clearing and immunolabeling in sheep tissue

Summary

Introduction

Neurons expressing the peptides kisspeptin or neurokinin B (NKB) play a critical role in the regulation of fertility. Taken together, these observations suggest functionally distinct subpopulations of KNDy neurons may regulate GnRH neuron activity and peptide release according to multiple physiological conditions, but little delineation of these subpopulations has occurred. The last five years have seen an increase in the development and use of optical tissue clearing techniques that permits rapid imaging of fluorescent cell populations in intact organs. Given the size of the rat brain and sheep hypothalamus, it is necessary to adapt immunolabelling and clearing techniques for larger tissue volumes. We optimized the iDISCO technique for labelling of KNDy peptides and clearing of larger tissue sizes using the rat brain before applying the optimized iDISCO to the sheep hypothalamic block

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