ABSTRACT
Glomeruli are the functional units of the vertebrate olfactory bulb (OB) connecting olfactory receptor neuron (ORN) axons and mitral/tufted cell (MTC) dendrites. In amphibians, these two circuit elements regularly branch and innervate multiple, spatially distinct glomeruli. Using functional multiphoton-microscopy and single-cell tracing, we investigate the impact of this wiring on glomerular module organization and odor representations on multiple levels of the Xenopus laevis OB network. The glomerular odor map to amino acid odorants is neither stereotypic between animals nor chemotopically organized. Among the morphologically heterogeneous group of uni- and multi-glomerular MTCs, MTCs can selectively innervate glomeruli formed by axonal branches of individual ORNs. We conclude that odor map heterogeneity is caused by the coexistence of different intermingled glomerular modules. This demonstrates that organization of the amphibian main olfactory system is not strictly based on uni-glomerular connectivity.
ABSTRACT
Microtubules are essential components of the cytoskeleton of all eukaryotic cells and consist of α- and ß-tubulin heterodimers. Several tissue-specific isotypes of α- and ß-tubulins, encoded by distinct genes, have been described in vertebrates. In the African clawed frog (Xenopus laevis), class II ß-tubulin (tubb2b) is expressed exclusively in neurons, and its promoter is used to establish different transgenic frog lines. However, a thorough investigation of the expression pattern of tubb2b has not been carried out yet. In this study, we describe the expression of tubb2b-dependent Katushka fluorescence in the forebrain of premetamorphic Xenopus laevis at cellular resolution. To determine the exact location of Katushka-positive neurons in the forebrain nuclei and to verify the extent of neuronal Katushka expression, we used a transgenic frog line and performed several additional antibody stainings. We found tubb2b-dependent fluorescence throughout the Xenopus forebrain, but not in all neurons. In the olfactory bulb, tubb2b-dependent fluorescence is present in axonal projections from the olfactory epithelium, cells in the mitral cell layer, and fibers of the extrabulbar system, but not in interneurons. We also detected tubb2b-dependent fluorescence in parts of the basal ganglia, the amygdaloid complex, the pallium, the optic nerve, the preoptic area, and the hypothalamus. In the diencephalon, tubb2b-dependent fluorescence occurred mainly in the prethalamus and thalamus. As in the olfactory system, not all neurons of these forebrain regions exhibited tubb2b-dependent fluorescence. Together, our results present a detailed overview of the distribution of tubb2b-dependent fluorescence in neurons of the forebrain of larval Xenopus laevis and clearly show that tubb2b-dependent fluorescence cannot be used as a pan-neuronal marker.
ABSTRACT
Sensory systems detect environmental stimuli and transform them into electrical activity patterns interpretable by the central nervous system. En route to higher brain centers, the initial sensory input is successively transformed by interposed secondary processing centers. Mapping the neuronal activity patterns at all of those stages is essential to understand sensory information processing. Larval Xenopus laevis is very well-suited for whole-brain imaging of neuronal activity. This is mainly due to its small size, transparency, and the accessibility of both peripheral and central parts of sensory systems. Here we describe a protocol for calcium imaging at several levels of the olfactory system using focal injection of chemical calcium indicator dyes or a Xenopus transgenic line with neuronal GCaMP6s expression. In combination with fast volumetric multiphoton microscopy, the calcium imaging methods described can provide detailed insight into spatiotemporal activity of entire brain regions at different stages of sensory information processing. Although the methods are broadly applicable to the central nervous system, in this work we focus on protocols for calcium imaging of glomeruli in the olfactory bulb and odor-responsive neurons in the olfactory amygdala.
