A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System

Abstract

Clearing techniques have been developed to transparentize mouse brains, thereby preserving 3D structure, but their complexity has limited their use. Here, we show that immunolabeling of axonal tracts followed by optical clearing with solvents (3DISCO) and light-sheet microscopy reveals brain connectivity in mouse embryos and postnatal brains. We show that the Robo3 receptor is selectively expressed by medial habenula axons forming the fasciculus retroflexus (FR) and analyzed the development of this commissural tract in mutants of the Slit/Robo and DCC/Netrin pathways. Netrin-1 and DCC are required to attract FR axons to the midline, but the two mutants exhibit specific and heterogeneous axon guidance defects. Moreover, floor-plate-specific deletion of Slit ligands with a conditional Slit2 allele perturbs not only midline crossing by FR axons but also their anteroposterior distribution. In conclusion, this method represents a unique and powerful imaging tool to study axonal connectivity in mutant mice.

Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion

“…Several important new clearing methods have been developed and used: ScaleA2 (Hama et al, 2011), 3DISCO (Belle et al, 2014;Erturk et al, 2012;Erturk and Bradke, 2013;Erturk et al, 2014), SeeDB (Ke et al, 2013;Ke and Imai, 2014), CLARITY Tomer et al, 2014), PACT/PARS and CUBIC Tainaka et al, 2014).…”

Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains

“…The 3DISCO optical clearing procedure has been described previously (Belle et al, 2014;Ertürk et al, 2012) and was employed here for human embryos aged 7-8 GW. Pre-methanol treatment was performed to clear the samples of blood clots, as described in the iDISCO protocol (Renier et al, 2014) on 9 GW or older samples, and in adult mouse brains.…”

“…This method has been combined with an immunolabeling protocol followed by light-sheet laserscanning microscopy (LSM) to study neuronal connectivity in mouse embryos and postnatal brains (Belle et al, 2014;Ertürk et al, 2012). Here, we adapted this technique for the first time to intact human embryos and fetuses and gained excellent clearing of the specimens (Figs 3, 4, Movies 1-3).…”

“…3D imaging was performed as previously described (Belle et al, 2014). An ultramicroscope (LaVision BioTec) using InspectorPro software (LaVision BioTec) was used for imaging.…”

“…However, while the sequence and coordination of the events governing the differentiation and migration of GnRH neurons from the nose to the brain have been studied extensively in several species (Abraham et al, 2009;Bruneau et al, 2003;Murakami and Arai, 1994;Murakami et al, 1992;Palevitch et al, 2007;Quanbeck et al, 1997;Ronnekleiv and Resko, 1990;Witkin et al, 2003;Wray et al, 1989), data in humans are scarce, relying mainly on scattered embryonic/fetal stages (Kim et al, 1999;Quinton et al, 1997;, 1996. To better understand the prenatal development and spatial organization of these neurons in humans, we combined immunohistochemistry with threedimensional (3D) analysis of either reconstructed histological sections (Luzzati et al, 2011) or of optically cleared whole-mount embryos/fetuses (Belle et al, 2014;Ertürk et al, 2012;Renier et al, 2014).…”