In the African clawed frog Xenopus laevis, class II β-tubulin (tubb2b) is expressed exclusively in neurons, and its promoter is used in different transgenic frog lines. In the frog line NBT-Katushka γ-cry Venus, tubb2b drives the expression of the red fluorescent protein Katushka. In this study, we used tubb2b-dependent fluorescence to visualize the olfactory system from the olfactory epithelium (OE) to the olfactory cortex (OC) and to label olfactory projection neurons (PNs) in the olfactory bulb (OB). We performed immunohistochemical stainings of the whole OE and OB as well as coronal brain sections of premetamorphic NBT-Katushka γ-cry Venus Xenopus laevis. We further injected and electroporated groups and single PNs in whole-mount preparations of the OB with various fluorescent dyes. We found that tubb2b was not active in all neurons of the olfactory system and the forebrain. In the OE, we detected tubb2b-positive cell bodies of olfactory receptor neurons and associated axons in the olfactory nerve. Axon terminals in the OB also show tubb2b-dependent fluorescence. In the OB mitral cell layer, we found tubb2b-positive cells separated into a lateral and medial group. Individual cells of both groups are morphologically similar, but differ in their axonal projection pattern to the OC. We found that axons of medial and lateral PNs divide into two olfactory tracts targeting the subpallium and the ventral pallium. Together, we found that transgenic frog lines, which use the promotor tubb2b, are a great tool to visualize main components of the olfactory system and the forebrain, but notably expression is not panneuronal. Moreover, we found that the secondary olfactory pathway of lateral and medial PNs is segregated into two independent streams that target different areas of the forebrain. Overall, this study builds the foundation for further analysis of odor-processing in larval Xenopus.

This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grant 4113/4-1.