Combining confocal laser scanning microscopy with serial section reconstruction in the study of adult mouse GnRH system

Recent advances in imaging techniques have extended the possibility of visualizing and following small structures in large volumes of both fixed and live specimens without sectioning. These techniques have contributed valuable information to study neuronal anatomy in the mouse brain. However, these approaches usually implies the use of expensive advanced optical instruments, not always available in research laboratories. Here, we present an easy and affordable method that combines confocal laser scanning fluorescence microscopy (CLSM) and serial section reconstruction in order to reconstruct a whole mouse brain at cellular resolution. In this method a series of thick sections (50 µm) are imaged by CLSM and the resulting stacks of images are registered and 3D reconstructed. This approach is based on existing free software (Fiji ImageJ) and can be performed on ordinary laboratory personal computers. As a proof of principle, by using this technique we reconstructed the anatomical organization of the GnRH system in the adult mouse brain. GnRH system is the head system of reproductive functions in mammals, and our study confirmed the well-known presence of GnRH-positive structures in hypothalamic and hypothalamic-related regions. Moreover, we describe the presence of GnRH-positive cells and processes at the level of olfactory bulbs (OBs), where they form a ring-like structure that surrounds the posterior OBs, sending processes to the olfactory glomerular layer. In the glomerular layer part of these GnRH-positive processes contact the dopaminergic olfactory interneurons. In conclusion, the method we developed allows high-resolution semi-automatic reconstruction of the organization of labelled anatomical structures using open-source software at reduced costs. Future perspectives include the complete automation of the method and its application to the study of the connectivity between specific brain regions, using retrograde/anterograde fluorescence tracers or viral vectors.