Visualization of the secondary olfactory pathway in tubb2b promoter-driven transgenic Xenopus laevis larvae

Abstract

The olfactory system of vertebrates consists of an olfactory epithelium containing olfactory sensory neurons that send axons to the olfactory bulb (OB), where they are connected to interneurons and projection neurons (PNs) via structures called glomeruli. PNs are the first relay station of olfactory information processing and are directly connected with their axons to higher brain regions. Among vertebrates the morphology, molecular characteristics, position in the OB, and axonal projections of PNs vary. In the olfactory system of larval Xenopus laevis, two odor-processing streams with different molecular and functional characteristics exist. Those streams terminate in a lateral and medial glomerular cluster within the ventral OB. It is not known whether these streams are continuous within PNs and higher olfactory processing centers. In this work, I analyzed the morphology of lateral and medial PNs and compared their structure as well as axonal projection pattern. For the visualization of neurons in the forebrain I used transgenic tubb2b-Katushka Xenopus laevis tadpoles. In this transgenic line the tubb2b promoter drives the expression of the fluorescent protein Katushka. The tubb2b promoter expresses class II β-tubulin protein, which is part of microtubules in neurons of the frog. Although the tubb2b promoter is used in different transgenic frog lines, a thorough investigation of the expression pattern of tubb2b has not been done. Therefore, I performed a detailed analysis of the expression pattern of tubb2b in the whole forebrain including the olfactory system of larval Xenopus laevis at cellular resolution. I found that tubb2b-dependent fluorescence is not present in all neurons and thus cannot be used as a pan-neuronal marker. tubb2b-dependent fluorescence is present in the OB, parts of the basal ganglia, the amygdaloid complex, pallium, optic nerve, preoptic area, hypothalamus, prethalamus and thalamus. In the OB, tubb2b-dependent fluorescence is present in axons of olfactory sensory neurons from the nose, cells in the mitral cell layer, fibers of the extra bulbar olfactory system, but not in interneurons. I used the tubb2b-dependent expression to target PNs in the mitral cell layer of the OB for sparse cell electroporation. I labelled lateral and medial PNs and 3D reconstructed them for a detailed morphological analysis. I found that both PN populations share many morphological characteristics, but differ in the number of dendritic tufts. I then injected dye into the whole lateral and medial PN population to trace their axons and observed that lateral PNs have a different axonal projection pattern than medial PNs. Finally, I performed functional multiphoton calcium imaging in tubb2b-GCaMP6s transgenic Xenopus larvae and was able to identify the lateral and central amygdala as higher olfactory processing centers. Together, this present work provides the first description of the larval secondary olfactory pathway in Xenopus laevis and a detailed map of the expression pattern of tubb2b in the larval forebrain.