The olfactory network of larval Xenopus laevis regenerates accurately after olfactory nerve transection.

Across vertebrate species, the olfactory epithelium (OE) exhibits the uncommon feature of lifelong neuronal turnover. Epithelial stem cells give rise to new neurons that can adequately replace dying olfactory receptor neurons (ORNs) during developmental and adult phases and after lesions. To relay olfactory information from the environment to the brain, the axons of the renewed ORNs must reconnect with the olfactory bulb (OB). In Xenopus laevis larvae, we have previously shown that this process occurs between 3 and 7 weeks after olfactory nerve (ON) transection. In the present study, we show that after 7 weeks of recovery from ON transection, two functionally and spatially distinct glomerular clusters are reformed in the OB, akin to those found in non-transected larvae. We also show that the same odourant response tuning profiles observed in the OB of non-transected larvae are again present after 7 weeks of recovery. Next, we show that characteristic odour-guided behaviour disappears after ON transection but recovers after 7-9 weeks of recovery. Together, our findings demonstrate that the olfactory system of larval X. laevis regenerates with high accuracy after ON transection, leading to the recovery of odour-guided behaviour.

S100Z is expressed in a lateral subpopulation of olfactory receptor neurons in the main olfactory system of Xenopus laevis.

In contrast to other S100 protein members, the function of S100 calcium-binding protein Z (S100Z) remains largely uncharacterized. It is expressed in the olfactory epithelium of fish, and it is closely associated with the vomeronasal organ (VNO) in mammals. In this study, we analyzed the expression pattern of S100Z in the olfactory system of the anuran amphibian Xenopus laevis. Using immunohistochemistry in whole mount and slice preparations of the larval olfactory system, we found exclusive S100Z expression in a subpopulation of olfactory receptor neurons (ORNs) of the main olfactory epithelium (MOE). S100Z expression was not co-localized with TP63 and cytokeratin type II, ruling out basal cell and supporting cell identity. The distribution of S100Z-expressing ORNs was laterally biased, and their average number was significantly increased in the lateral half of the olfactory epithelium. The axons of S100Z-positive neurons projected exclusively into the lateral and intermediate glomerular clusters of the main olfactory bulb (OB). Even after metamorphic restructuring of the olfactory system, S100Z expression was restricted to a neuronal subpopulation of the MOE, which was then located in the newly formed middle cavity. An axonal projection into the ventro-lateral OB persisted also in postmetamorphic frogs. In summary, S100Z is exclusively associated with the main olfactory system in the amphibian Xenopus and not with the VNO as in mammals, despite the presence of a separate accessory olfactory system in both classes.

Functional odor map heterogeneity is based on multifaceted glomerular connectivity in larval Xenopus olfactory bulb.

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.

Patterns of tubb2b Promoter-Driven Fluorescence in the Forebrain of Larval Xenopus laevis.

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.

Distinct interhemispheric connectivity at the level of the olfactory bulb emerges during Xenopus laevis metamorphosis.

During metamorphosis, the olfactory system of anuran tadpoles undergoes substantial restructuring. The main olfactory epithelium in the principal nasal cavity of Xenopus laevis tadpoles is associated with aquatic olfaction and transformed into the adult air-nose, while a new adult water-nose emerges in the middle cavity. Impacts of this metamorphic remodeling on odor processing, behavior, and network structure are still unexplored. Here, we used neuronal tracings, calcium imaging, and behavioral experiments to examine the functional connectivity between the epithelium and the main olfactory bulb during metamorphosis. In tadpoles, olfactory receptor neurons in the principal cavity project axons to glomeruli in the ventral main olfactory bulb. These projections are gradually replaced by receptor neuron axons from the newly forming middle cavity epithelium. Despite this reorganization in the ventral bulb, two spatially segregated odor processing streams remain undisrupted and behavioral responses to waterborne odorants are unchanged. Contemporaneously, new receptor neurons in the remodeling principal cavity innervate the emerging dorsal part of the bulb, which displays distinct wiring features. Glomeruli around its midline are innervated from the left and right nasal epithelia. Additionally, postsynaptic projection neurons in the dorsal bulb predominantly connect to multiple glomeruli, while half of projection neurons in the ventral bulb are uni-glomerular. Our results show that the "water system" remains functional despite metamorphic reconstruction. The network differences between the dorsal and ventral olfactory bulb imply a higher degree of odor integration in the dorsal main olfactory bulb. This is possibly connected with the processing of different odorants, airborne vs. waterborne.

Olfaction across the water-air interface in anuran amphibians.

Extant anuran amphibians originate from an evolutionary intersection eventually leading to fully terrestrial tetrapods. In many ways, they have to deal with exposure to both terrestrial and aquatic environments: (i) phylogenetically, as derivatives of the first tetrapod group that conquered the terrestrial environment in evolution; (ii) ontogenetically, with a development that includes aquatic and terrestrial stages connected via metamorphic remodeling; and (iii) individually, with common changes in habitat during the life cycle. Our knowledge about the structural organization and function of the amphibian olfactory system and its relevance still lags behind findings on mammals. It is a formidable challenge to reveal underlying general principles of circuity-related, cellular, and molecular properties that are beneficial for an optimized sense of smell in water and air. Recent findings in structural organization coupled with behavioral observations could help to understand the importance of the sense of smell in this evolutionarily important animal group. We describe the structure of the peripheral olfactory organ, the olfactory bulb, and higher olfactory centers on a tissue, cellular, and molecular levels. Differences and similarities between the olfactory systems of anurans and other vertebrates are reviewed. Special emphasis lies on adaptations that are connected to the distinct demands of olfaction in water and air environment. These particular adaptations are discussed in light of evolutionary trends, ontogenetic development, and ecological demands.

Whole-Brain Calcium Imaging in Larval Xenopus.

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.

Conservation of Glomerular Organization in the Main Olfactory Bulb of Anuran Larvae.

The glomerular array in the olfactory bulb of many vertebrates is segregated into molecularly and anatomically distinct clusters linked to different olfactory functions. In anurans, glomerular clustering is so far only described in Xenopus laevis. We traced olfactory projections to the bulb in tadpoles belonging to six distantly related anuran species in four families (Pipidae, Hylidae, Bufonidae, Dendrobatidae) and found that glomerular clustering is remarkably conserved. The general bauplan consists of four unequally sized glomerular clusters with minor inter-species variation. During metamorphosis, the olfactory system undergoes extensive remodeling. Tracings in metamorphotic and juvenile Dendrobates tinctorius and Xenopus tropicalis suggest a higher degree of variation in the glomerular organization after metamorphosis is complete. Our study highlights, that the anatomical organization of glomeruli in the main olfactory bulb (MOB) is highly conserved, despite an extensive ecomorphological diversification among anuran tadpoles, which suggests underlying developmental constraints.

Multi-glomerular projection of single olfactory receptor neurons is conserved among amphibians.

Individual receptor neurons in the peripheral olfactory organ extend long axons into the olfactory bulb forming synapses with projection neurons in spherical neuropil regions, called glomeruli. Generally, odor map formation and odor processing in all vertebrates is based on the assumption that receptor neuron axons exclusively connect to a single glomerulus without any axonal branching. We comparatively tested this hypothesis in multiple fish and amphibian species (both sexes) by applying sparse cell electroporation to trace single olfactory receptor neuron axons. Sea lamprey (jawless fish) and zebrafish (bony fish) support the unbranched axon concept, with 94% of axons terminating in single glomeruli. Contrastingly, axonal projections of the axolotl (salamander) branch extensively before entering up to six distinct glomeruli. Receptor neuron axons labeled in frog species (Pipidae, Bufonidae, Hylidae, and Dendrobatidae) predominantly bifurcate before entering a glomerulus and 59 and 50% connect to multiple glomeruli in larval and postmetamorphotic animals, respectively. Independent of developmental stage, lifestyle, and adaptations to specific habitats, it seems to be a common feature of amphibian olfactory receptor neuron axons to frequently bifurcate and connect to multiple glomeruli. Our study challenges the unbranched axon concept as a universal vertebrate feature and it is conceivable that also later diverging vertebrates deviate from it. We propose that this unusual wiring logic evolved around the divergence of the terrestrial tetrapod lineage from its aquatic ancestors and could be the basis of an alternative way of odor processing.

Purinergic signalling selectively modulates maintenance but not repair neurogenesis in the zebrafish olfactory epithelium.

Olfactory sensory neurons (OSNs) of the vertebrate olfactory epithelium (OE) undergo continuous turnover but also regenerate efficiently when the OE is acutely damaged by traumatic injury. Two distinct pools of neuronal stem/progenitor cells, the globose (GBCs), and horizontal basal cells (HBCs) have been shown to selectively contribute to intrinsic OSN turnover and damage-induced OE regeneration, respectively. For both types of progenitors, their rate of cell divisions and OSN production must match the actual loss of cells to maintain or to re-establish sensory function. However, signals that communicate between neurons or glia cells of the OE and resident neurogenic progenitors remain largely elusive. Here, we investigate the effect of purinergic signaling on cell proliferation and OSN neurogenesis in the zebrafish OE. Purine stimulation elicits transient Ca2+ signals in OSNs and distinct non-neuronal cell populations, which are located exclusively in the basal OE and stain positive for the neuronal stem cell marker Sox2. The more apical population of Sox2-positive cells comprises evenly distributed glia-like sustentacular cells (SCs) and spatially restricted GBC-like cells, whereas the more basal population expresses the HBC markers keratin 5 and tumor protein 63 and lines the entire sensory OE. Importantly, exogenous purine stimulation promotes P2 receptor-dependent mitotic activity and OSN generation from sites where GBCs are located but not from HBCs. We hypothesize that purine compounds released from dying OSNs modulate GBC progenitor cell cycling in a dose-dependent manner that is proportional to the number of dying OSNs and, thereby, ensures a constant pool of sensory neurons over time.

Dye Electroporation and Imaging of Calcium Signaling in Xenopus Nervous System.

Electroporation is an efficient method of transferring charged macromolecules into living cells in order to study their morphology, function, and connectivity within neuronal networks. Labeling cells with fluorophore-coupled macromolecules can be used to trace projections of whole neuronal ensembles, as well as the fine morphology of single cells. Here, we present a protocol to visualize pre- and postsynaptic components of a sensory relay synapse in the brain, using the olfactory system of Xenopus laevis tadpoles as a model. We apply bulk electroporation to trace projections of receptor neurons from the nose to the brain, and single cell electroporation to visualize the morphology of their synaptic target cells, the mitral-tufted cells. Labeling the receptor neurons with a calcium-sensitive dye allows us to record stimulus-induced presynaptic input to the dendrites of the postsynaptic cells via functional calcium imaging.

Functional Reintegration of Sensory Neurons and Transitional Dendritic Reduction of Mitral/Tufted Cells during Injury-Induced Recovery of the Larval Xenopus Olfactory Circuit.

Understanding the mechanisms involved in maintaining lifelong neurogenesis has a clear biological and clinical interest. In the present study, we performed olfactory nerve transection on larval Xenopus to induce severe damage to the olfactory circuitry. We surveyed the timing of the degeneration, subsequent rewiring and functional regeneration of the olfactory system following injury. A range of structural labeling techniques and functional calcium imaging were performed on both tissue slices and whole brain preparations. Cell death of olfactory receptor neurons and proliferation of stem cells in the olfactory epithelium were immediately increased following lesion. New olfactory receptor neurons repopulated the olfactory epithelium and once again showed functional responses to natural odorants within 1 week after transection. Reinnervation of the olfactory bulb (OB) by newly formed olfactory receptor neuron axons also began at this time. Additionally, we observed a temporary increase in cell death in the OB and a subsequent loss in OB volume. Mitral/tufted cells, the second order neurons of the olfactory system, largely survived, but transiently lost dendritic tuft complexity. The first odorant-induced responses in the OB were observed 3 weeks after nerve transection and the olfactory network showed signs of major recovery, both structurally and functionally, after 7 weeks.

Coordinated shift of olfactory amino acid responses and V2R expression to an amphibian water nose during metamorphosis.

All olfactory receptors identified in teleost fish are expressed in a single sensory surface, whereas mammalian olfactory receptor gene families segregate into different olfactory organs, chief among them the main olfactory epithelium expressing ORs and TAARs, and the vomeronasal organ expressing V1Rs and V2Rs. A transitional stage is embodied by amphibians, with their vomeronasal organ expressing more 'modern', later diverging V2Rs, whereas more 'ancient', earlier diverging V2Rs are expressed in the main olfactory epithelium. During metamorphosis, the main olfactory epithelium of Xenopus tadpoles transforms into an air-filled cavity (principal cavity, air nose), whereas a newly formed cavity (middle cavity) takes over the function of a water nose. We report here that larval expression of ancient V2Rs is gradually lost from the main olfactory epithelium as it transforms into the air nose. Concomitantly, ancient v2r gene expression begins to appear in the basal layers of the newly forming water nose. We observe the same transition for responses to amino acid odorants, consistent with the hypothesis that amino acid responses may be mediated by V2R receptors.

Metamorphic remodeling of the olfactory organ of the African clawed frog, Xenopus laevis.

The amphibian olfactory system undergoes massive remodeling during metamorphosis. The transition from aquatic olfaction in larvae to semiaquatic or airborne olfaction in adults requires anatomical, cellular, and molecular modifications. These changes are particularly pronounced in Pipidae, whose adults have secondarily adapted to an aquatic life style. In the fully aquatic larvae of Xenopus laevis, the main olfactory epithelium specialized for sensing water-borne odorous substances lines the principal olfactory cavity (PC), whereas a separate olfactory epithelium lies in the vomeronasal organ (VNO). During metamorphosis, the epithelium of the PC is rearranged into the adult "air nose," whereas a new olfactory epithelium, the adult "water nose," forms in the emerging middle cavity (MC). Here we performed a stage-by-stage investigation of the anatomical changes of the Xenopus olfactory organ during metamorphosis. We quantified cell death in all olfactory epithelia and found massive cell death in the PC and the VNO, suggesting that the majority of larval sensory neurons is replaced during metamorphosis in both sensory epithelia. The moderate cell death in the MC shows that during the formation of this epithelium some cells are sorted out. Our results show that during MC formation some supporting cells, but not sensory neurons, are relocated from the PC to the MC and that they are eventually eliminated during metamorphosis. Together our findings illustrate the structural and cellular changes of the Xenopus olfactory organ during metamorphosis.

Dual processing of sulfated steroids in the olfactory system of an anuran amphibian.

Chemical communication is widespread in amphibians, but if compared to later diverging tetrapods the available functional data is limited. The existing information on the vomeronasal system of anurans is particularly sparse. Amphibians represent a transitional stage in the evolution of the olfactory system. Most species have anatomically separated main and vomeronasal systems, but recent studies have shown that in anurans their molecular separation is still underway. Sulfated steroids function as migratory pheromones in lamprey and have recently been identified as natural vomeronasal stimuli in rodents. Here we identified sulfated steroids as the first known class of vomeronasal stimuli in the amphibian Xenopus laevis. We show that sulfated steroids are detected and concurrently processed by the two distinct olfactory subsystems of larval Xenopus laevis, the main olfactory system and the vomeronasal system. Our data revealed a similar but partially different processing of steroid-induced responses in the two systems. Differences of detection thresholds suggest that the two information channels are not just redundant, but rather signal different information. Furthermore, we found that larval and adult animals excrete multiple sulfated compounds with physical properties consistent with sulfated steroids. Breeding tadpole and frog water including these compounds activated a large subset of sensory neurons that also responded to synthetic steroids, showing that sulfated steroids are likely to convey intraspecific information. Our findings indicate that sulfated steroids are conserved vomeronasal stimuli functioning in phylogenetically distant classes of tetrapods living in aquatic and terrestrial habitats.

The olfactory system as a model to study axonal growth patterns and morphology in vivo.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

Phospholipase C and diacylglycerol mediate olfactory responses to amino acids in the main olfactory epithelium of an amphibian.

The semi-aquatic lifestyle of amphibians represents a unique opportunity to study the molecular driving forces involved in the transition of aquatic to terrestrial olfaction in vertebrates. Most amphibians have anatomically segregated main and vomeronasal olfactory systems, but at the cellular and molecular level the segregation differs from that found in mammals. We have recently shown that amino acid responses in the main olfactory epithelium (MOE) of larval Xenopus laevis segregate into a lateral and a medial processing stream, and that the former is part of a vomeronasal type 2 receptor expression zone in the MOE. We hypothesized that the lateral amino acid responses might be mediated via a vomeronasal-like transduction machinery. Here we report that amino acid-responsive receptor neurons in the lateral MOE employ a phospholipase C (PLC) and diacylglycerol-mediated transduction cascade that is independent of Ca(2+) store depletion. Furthermore, we found that putative transient receptor potential (TRP) channel blockers inhibit most amino acid-evoked responses in the lateral MOE, suggesting that ion channels belonging to the TRP family may be involved in the signaling pathway. Our data show, for the first time, a widespread PLC- and diacylglycerol-dependent transduction cascade in the MOE of a vertebrate already possessing a vomeronasal organ.

Purinergic receptor-induced Ca2+ signaling in the neuroepithelium of the vomeronasal organ of larval Xenopus laevis.

Purinergic signaling has considerable impact on the functioning of the nervous system, including the special senses. Purinergic receptors are expressed in various cell types in the retina, cochlea, taste buds, and the olfactory epithelium. The activation of these receptors by nucleotides, particularly adenosine-5'-triphosphate (ATP) and its breakdown products, has been shown to tune sensory information coding to control the homeostasis and to regulate the cell turnover in these organs. While the purinergic system of the retina, cochlea, and taste buds has been investigated in numerous studies, the available information about purinergic signaling in the olfactory system is rather limited. Using functional calcium imaging, we identified and characterized the purinergic receptors expressed in the vomeronasal organ of larval Xenopus laevis. ATP-evoked activity in supporting and basal cells was not dependent on extracellular Ca(2+). Depletion of intracellular Ca(2+) stores disrupted the responses in both cell types. In addition to ATP, supporting cells responded also to uridine-5'-triphosphate (UTP) and adenosine-5'-O-(3-thiotriphosphate) (ATPγS). The response profile of basal cells was considerably broader. In addition to ATP, they were activated by ADP, 2-MeSATP, 2-MeSADP, ATPγS, UTP, and UDP. Together, our findings suggest that supporting cells express P2Y(2)/P2Y(4)-like purinergic receptors and that basal cells express multiple P2Y receptors. In contrast, vomeronasal receptor neurons were not sensitive to nucleotides, suggesting that they do not express purinergic receptors. Our data provide the basis for further investigations of the physiological role of purinergic signaling in the vomeronasal organ and the olfactory system in general.

Olfactory wiring logic in amphibians challenges the basic assumptions of the unbranched axon concept.

Olfactory receptor neurons extend axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. The consensus view is that in vertebrates individual receptor neurons project unbranched axons into one specific glomerulus of the olfactory bulb. We report here that, strikingly different from the generally assumed wiring principle in vertebrate olfactory systems, axons of single receptor neurons of Xenopus laevis regularly bifurcate and project into more than one glomerulus. Specifically, the innervation of multiple glomeruli is present in all ontogenetic stages of this species, from the larva to the postmetamorphic frog. Also, we show that this unexpected wiring pattern is not restricted to axons of immature receptor neurons, but that it is also a feature of mature neurons of both the main and accessory olfactory system. This glomerular innervation pattern is unique among vertebrates investigated so far and represents a new olfactory wiring strategy.