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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.

Aggregation and spreading of α-Synuclein (αSyn) are hallmarks of several neurodegenerative diseases, thus monitoring human αSyn (hαSyn) in animal models or cell cultures is vital for the field. However, the detection of native hαSyn in such systems is challenging. We show that the nanobody NbSyn87, previously-described to bind hαSyn, also shows cross-reactivity for the proteasomal subunit Rpn10. As such, when the NbSyn87 is expressed in the absence of hαSyn, it is continuously degraded by the proteasome, while it is stabilized when it binds to hαSyn. Here, we exploit this feature to design a new Fluorescent Reporter for hαSyn (FluoReSyn) by fusing NbSyn87 to fluorescent proteins, which results in fluorescence signal fluctuations depending on the presence and amounts of intracellular hαSyn. We characterize this biosensor in cells and tissues to finally reveal the presence of transmittable αSyn in human cerebrospinal fluid, demonstrating the potential of FluoReSyn for clinical research and diagnostics., α-Syn in CSF is a biomarker of neurodegenerative diseases; however, the detection of clinically relevant species is difficult. Here, the authors create a nanobody biosensor that reveals the presence of α-Syn in cells, which allow the detection of transmittable forms of α-Syn present in human CSF.

The olfactory system of anuran tadpoles requires substantial restructuring to adapt to the lifestyle of the adult frogs. Xenopus laevis tadpoles have a single main olfactory epithelium in the principal nasal cavity associated with aquatic olfaction. After metamorphosis, this epithelial surface is transformed into the adult air-nose and a new epithelium, the adult water-nose, is present in the middle cavity. Impacts of this massive remodeling on odor processing, behavior and network structure are still unexplored.In the present study, we used neuronal tracings, calcium imaging and a behavioral assay to examine the functional connectivity between the epithelium and the main olfactory bulb during metamorphosis. In tadpoles, olfactory receptor neurons in the principal cavity epithelium project axons to glomeruli in the ventral main olfactory bulb. During metamorphosis, these projections are gradually replaced by receptor neuron axons emerging from the newly forming middle cavity epithelium. Despite this metamorphotic reorganization in the ventral bulb, two spatially and functionally segregated odor processing streams remain undisrupted. In line with this, metamorphotic rewiring does not alter behavioral responses to waterborne odorants. Contemporaneously, newly formed receptor neurons in the remodeling principal cavity epithelium project their axons to the dorsal part of the bulb. The emerging neuronal networks of the dorsal and ventral main olfactory bulb show substantial differences. Glomeruli around the midline of the dorsal bulb are innervated from the left and right nasal epithelia. In addition, postsynaptic projection neurons in the dorsal bulb predominantly have smaller tufts and connect to multiple glomeruli, while more than half of projection neurons in the ventral bulb have a single, bigger tuft.Our results show that during the metamorphotic reconstruction of the olfactory network the ‘water system’ remains functional. Differences of the neuronal network of the dorsal and ventral olfactory bulb imply that a higher degree of odor integration takes place in the dorsal main olfactory bulb. This is likely connected with the processing of different odorants, airborne vs. waterborne, in these two parts of the olfactory bulb.

The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. The epithelial sodium channel (ENaC) is present in the apical membrane of sodium-absorbing vertebrate epithelia and evolved as part of a machinery for efficient sodium conservation. ENaC belongs to the degenerin/ENaC protein family and is the only member that opens without an external stimulus. We hypothesized that ENaC evolved from a proton-activated sodium channel present in ionocytes of freshwater vertebrates and therefore investigated whether such ancestral traits are present in ENaC isoforms of the aquatic pipid frog Xenopus laevis. Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from αβγ- or δβγ-subunit combinations, we demonstrate that Xenopus δβγ-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges (pH 8.0–6.0). This effect was not observed in Xenopus αβγ-ENaC or human ENaC orthologs. We show that protons interfere with allosteric ENaC inhibition by extracellular sodium ions, thereby increasing the probability of channel opening. Using homology modeling of ENaC structure and site-directed mutagenesis, we identified a cleft region within the extracellular loop of the δ-subunit that contains several acidic amino acid residues that confer proton-sensitivity and enable allosteric inhibition by extracellular sodium ions. We propose that Xenopus δβγ-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutively-active ion channel. Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water–to–land transition.

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.

The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits (α, β, and γ), but a fourth δ-subunit may replace α and form atypical δβγ-ENaCs. Using Xenopus laevis as a model, here we found that mRNAs of the α- and δ-subunits are differentially expressed in different tissues and that δ-ENaC predominantly is present in the urogenital tract. Using whole-cell and single-channel electrophysiology of oocytes expressing Xenopus αβγ- or δβγ-ENaC, we demonstrate that the presence of the δ-subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the α- and γ-subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike α-ENaC, the δ-subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by δβγ-ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the δ-subunit prevented cleavage of γ-ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the Xenopus δ-ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution.

Complete segregation of the main olfactory epithelium (MOE) and the vomeronasal epithelium is first observed in amphibians. In contrast, teleost fishes possess a single olfactory surface, in which genetic components of the main and vomeronasal olfactory systems are intermingled. The transient receptor potential channel TRPC2, a marker of vomeronasal neurons, is present in the single fish sensory surface, but is already restricted to the vomeronasal epithelium in a terrestrial amphibian, the red-legged salamander (Plethodon shermani). Here we examined the localization of TRPC2 in an aquatic amphibian and cloned the Xenopus laevis trpc2 gene. We show that it is expressed in both the MOE and the vomeronasal epithelium. This is the first description of a broad trpc2 expression in the MOE of a tetrapod. The expression pattern of trpc2 in the MOE is virtually undistinguishable from that of MOE-specific v2rs, indicating that they are co-expressed in the same neuronal subpopulation.

Mammalian olfactory receptor families are segregated into different olfactory organs, with type 2 vomeronasal receptor ( v2r ) genes expressed in a basal layer of the vomeronasal epithelium. In contrast, teleost fish v2r genes are intermingled with all other olfactory receptor genes in a single sensory surface. We report here that, strikingly different from both lineages, the v2r gene family of the amphibian Xenopus laevis is expressed in the main olfactory as well as the vomeronasal epithelium. Interestingly, late diverging v2r genes are expressed exclusively in the vomeronasal epithelium, whereas “ancestral” v2r genes, including the single member of v2r family C, are restricted to the main olfactory epithelium. Moreover, within the main olfactory epithelium, v2r genes are expressed in a basal zone, partially overlapping, but clearly distinct from an apical zone of olfactory marker protein and odorant receptor-expressing cells. These zones are also apparent in the spatial distribution of odor responses, enabling a tentative assignment of odor responses to olfactory receptor gene families. Responses to alcohols, aldehydes, and ketones show an apical localization, consistent with being mediated by odorant receptors, whereas amino acid responses overlap extensively with the basal v2r -expressing zone. The unique bimodal v2r expression pattern in main and accessory olfactory system of amphibians presents an excellent opportunity to study the transition of v2r gene expression during evolution of higher vertebrates.

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.

Interleukin (IL)-1β is a potent pro-inflammatory cytokine of innate immunity involved in host defense. High systemic IL-1β levels, however, cause life-threatening inflammatory diseases, including systemic inflammatory response syndrome. In response to various danger signals, the pro-form of IL-1β is synthesized and stays in the cytoplasm unless a second signal, such as extracellular ATP, activates the inflammasome, which enables processing and release of mature IL-1β. As pulmonary surfactant is known for its anti-inflammatory properties, we hypothesize that surfactant inhibits ATP-induced release of IL-1β. Lipopolysaccharide-primed monocytic U937 cells were stimulated with an ATP analog in the presence of natural or synthetic surfactant composed of recombinant surfactant protein (rSP)-C, palmitoylphosphatidylglycerol, and dipalmitoylphosphatidylcholine (DPPC). Both surfactant preparations dose-dependently inhibited IL-1β release from U937 cells. DPPC was the active constituent of surfactant, whereas rSP-C and palmitoylphosphatidylglycerol were inactive. DPPC was also effective in primary mononuclear leukocytes isolated from human blood. Experiments with nicotinic antagonists, siRNA technology, and patch-clamp experiments suggested that stimulation of nicotinic acetylcholine receptors (nAChRs) containing subunit α9 results in a complete inhibition of the ion channel function of ATP receptor, P2X7. In conclusion, the surfactant constituent, DPPC, efficiently inhibits ATP-induced inflammasome activation and maturation of IL-1β in human monocytes by a mechanism involving nAChRs.

In the olfactory epithelium (OE) continuous neurogenesis is maintained throughout life. The OE is in direct contact with the external environment, and its cells are constantly exposed to pathogens and noxious substances. To maintain a functional sense of smell the OE has evolved the ability to permanently replenish olfactory receptor neurons and sustentacular cells lost during natural turnover. A cell population residing in the most basal part of the OE, the so-called basal cells (BCs), keep up this highly regulated genesis of new cells. The population of BCs is thought to include both the stem cells of the OE and various progenitor cells. In recent years a number of regulatory factors that positively and/or negatively regulate the proliferation within the OE have been identified, but a thorough comprehension of the complex interplay of these regulatory factors and the role of the different epithelial cell types is still illusive. Combining labeling techniques, immunohistochemistry, electron microscopy, functional calcium imaging, and a bromo-2′-deoxyuridine incorporation assay, we show for the first time that purinergic receptors are expressed in BCs of the OE of larval Xenopus laevis and that nucleotide-induced Ca2+ signaling in these cells is involved in the regulation of the cell turnover in the OE. Our data contribute to a better understanding of the regulation of the cell turnover in the OE in particular and also of how the proliferation of neuronal progenitor cells is regulated in general. Disclosure of potential conflicts of interest is found at the end of this article.

ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that, also known as multidrug resistance proteins, transport a wide variety of substrates across biological membranes in an energy-dependent manner. Recently it has been shown that members of this protein family interfere with fluorescent (calcium indicator) dye uptake in taste buds of rat and in cells in the olfactory epithelium of larval Xenopus laevis, including olfactory receptor neurons. It has, however, not been resolved whether this effect only serves to extrude xenobiotics in sensory taste and olfactory cells, or alternatively, whether it is a more general feature of many central nervous system neurons. In the latter case blocking these transporters would improve fluorescent dye uptake in general. Here we show, by means of cell imaging, that also neurons of the olfactory bulb express multidrug resistance transporters, whereby a marked inhomogeneity among cells in the main and accessory olfactory bulb was observed. Blocking these transporters improved the net uptake of fluorescent dyes not only in cell somata of the olfactory bulb, but especially in fine neuronal structures such as individual dendrites or olfactory glomeruli, which consist of a tangle of tiny neuronal processes. We therefore suggest that the expression of multidrug resistance proteins may be common in cells of the central nervous system, and that the application of specific transport inhibitors could generally improve fluorescent dye uptake in brain slices, thereby improving calcium imaging conditions. peerReviewed

The sensory input layer in the olfactory bulb (OB) is typically organized into spheroidal aggregates of dense neuropil called glomeruli. This characteristic compartmentalization of the synaptic neuropil is a typical feature of primary olfactory centres in vertebrates and most advanced invertebrates. In the present work we mapped the location of presynaptic sites in glomeruli across the OB using antibodies to presynaptic vesicle proteins and presynaptic membrane proteins in combination with confocal microscopy. In addition the responses of glomeruli upon mucosal application of amino acid-odorants and forskolin were monitored using functional calcium imaging. We first describe the spatial distribution of glomeruli across the main olfactory bulb (MOB) in premetamorphic Xenopus laevis. Second, we show that the heterogeneous organization of glomeruli along the dorsoventral and mediolateral axes of the MOB is associated with a differential distribution of synaptic vesicle proteins. While antibodies to synaptophysin, syntaxin and SNAP-25 uniformly labelled glomeruli in the whole MOB, intense synaptotagmin staining was present only in glomeruli in the lateral, and to a lesser extent in the intermediate, part of the OB. Interestingly, amino acid-responsive glomeruli were always located in the lateral part of the OB, and glomeruli activated by mucosal forskolin application were exclusively located in the medial part of the OB. This correlation between odour mapping and presynaptic protein distribution is an additional hint on the existence of different subsystems within the main olfactory system in larval Xenopus laevis.

Glomeruli in the vertebrate olfactory bulb (OB) appear as anatomically discrete modules receiving direct input from the olfactory epithelium (OE) via axons of olfactory receptor neurons (ORNs). The response profiles with respect to amino acids (AAs) of a large number of ORNs in larval Xenopus laevis have been recently determined and analysed. Here we report on Ca2+ imaging experiments in a nose–brain preparation of the same species at the same developmental stages. We recorded responses to AAs of glomeruli in the OB and determined the response profiles to AAs of individual glomeruli. We describe the general features of AA-responsive glomeruli and compare their response profiles to AAs with those of ORNs obtained in our previous study. A large number of past studies have focused either on odorant responses in the OE or on odorant-induced responses in the OB. However, a thorough comparison of odorant-induced responses of both stages, ORNs and glomeruli of the same species is as yet lacking. The glomerular response profiles reported herein markedly differ from the previously obtained response profiles of ORNs in that glomeruli clearly have narrower selectivity profiles than ORNs. We discuss possible explanations for the different selectivity profiles of glomeruli and ORNs in the context of the development of the olfactory map.

Olfactory perception is mediated by a multitude of olfactory receptors, whose expression in the sensory surface, the olfactory epithelium, is spatially regulated. A common theme is the segregation of different olfactory receptors in different expression domains, which in turn leads to corresponding segregation in the neuronal responses to different odor groups. The amphibian olfactory receptor gene family of trace amine associated receptors, in short TAARs, is exceedingly small and allows a comprehensive analysis of spatial expression patterns, as well as a comparison with neuronal responses to the expected ligands for this receptor family, amines. Here we report that TAAR4b exhibits a spatial expression pattern characteristically different in two dimensions from that of TAAR4a, its close homolog. Together, these two genes result in a bimodal distribution resembling that of amine responses as visualized by calcium imaging. A stringent quantitative analysis suggests the involvement of additional olfactory receptors in amphibian responses to amine odors.

In contrast to the single sensory surface present in teleost fishes, several spatially segregated subsystems with distinct molecular and functional characteristics define the mammalian olfactory system. However, the evolutionary steps of that transition remain unknown. Here we analyzed the olfactory system of an early diverging tetrapod, the amphibian Xenopus laevis, and report for the first time the existence of two odor-processing streams, sharply segregated in the main olfactory bulb and partially segregated in the olfactory epithelium of pre-metamorphic larvae. A lateral odor-processing stream is formed by microvillous receptor neurons and is characterized by amino acid responses and Gαo/Gαi as probable signal transducers, whereas a medial stream formed by ciliated receptor neurons is characterized by responses to alcohols, aldehydes, and ketones, and Gαolf/cAMP as probable signal transducers. To reveal candidates for the olfactory receptors underlying these two streams, the spatial distribution of 12 genes from four olfactory receptor gene families was determined. Several class II and some class I odorant receptors (ORs) mimic the spatial distribution observed for the medial stream, whereas a trace amine-associated receptor closely parallels the spatial pattern of the lateral odor-processing stream. Other olfactory receptors (some class I odorant receptors and vomeronasal type 1 receptors) and odor responses (to bile acids, amines) were not lateralized, the latter not even in the olfactory bulb, suggesting an incomplete segregation. Thus, the olfactory system of X. laevis exhibits an intermediate stage of segregation and as such appears well suited to investigate the molecular driving forces behind olfactory regionalization. peerReviewed

Transforming growth factor β (TGFβ) signalling plays important roles in a variety of tissues and cell types. Impaired TGFβ signalling contributes to several pathologies, including cancer, fibrosis as well as neurodegenerative diseases. TGFβ receptor type I-mediated phosphorylation of Smad2, the formation of the Smad2-Smad4 complex and translocation to the nucleus are critical steps of the TGFβ signalling pathway. Here, we demonstrate that thapsigargin-mediated increase of intracellular Ca(2+) concentrations inhibited TGFβ1-induced Smad2 transcriptional activity in the oligodendroglial cell line OLI-neu. We provide evidence that thapsigargin treatment dramatically reduced the nuclear translocation of Smad2 after TGFβ1 treatment but had no effect on its phosphorylation at Ser465/467. Moreover, using Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitors and a constitutively active CaMKII mutant, we provide evidence that the observed inhibition of TGFβ signalling in OLI-neu cells was strongly dependent on Ca(2+)-mediated CaMKII activation. In summary, this study clearly shows that the TGFβ1-induced Smad2 nuclear translocation is negatively regulated by intracellular Ca(2+) in OLI-neu cells and that increased intracellular Ca(2+) concentrations block Smad2-mediated transcription of TGFβ target genes. These results underline the importance of intracellular Ca(2+) for the regulation of TGFβ signalling.

Nucleotides and amino acids are acknowledged categories of water-borne olfactory stimuli. In previous studies it has been shown that larvae of Xenopus laevis are able to sense amino acids. Here we report on the effect of ATP in the olfactory epithelium (OE) of Xenopus laevis tadpoles. First, ATP activates a subpopulation of cells in the OE. The ATP-sensitive subset of cells is almost perfectly disjoint from the subset of amino acid-activated cells. Both responses are not mediated by the well-described cAMP transduction pathway as the two subpopulations of cells do not overlap with a third, forskolin-activated subpopulation. We further show that, in contrast to amino acids, which act exclusively as olfactory stimuli, ATP appears to feature a second role. Surprisingly it activated a large number of sustentacular supporting cells (SCs) and, to a much lower extent, olfactory receptor neurons. The cells of the amino acid- and ATP-responding subsets featured differences in shape, size and position in the OE. The latencies to activation upon stimulus application differed markedly in these subsets. To obtain these results two technical points were important. We used a novel dextran-tetramethylrhodamine-backfilled slice preparation of the OE and we found out that an antibody to calnexin, a known molecular chaperone, also labels SCs. Our findings thus show a strong effect of ATP in the OE and we discuss some of the possible physiological functions of nucleotides in the OE. peerReviewed

Olfactory receptor neurons (ORNs) are the only class of neurons that is directly exposed to the environment. Therefore, they need to deal with xenobiotic and potentially cytotoxic substances. Here we show for the first time that ORNs possess transporter systems that expel xenobiotics across the plasma membrane. Using calcein and calcium-indicator dyes as xenobiotics, we demonstrate that ORNs appear to express the multidrug resistance P-glycoprotein (MDR1) and multidrug resistance-associated proteins (MRP). This endows ORNs with the ability to transport a large number of substrates including calcium-indicator dyes and calcein across their plasma membranes. Conversely, blocking P-glycoprotein and MRP increases the net uptake of these dyes.

We used a slice preparation of the olfactory epithelium of Xenopus laevis tadpoles to record odorant responses of olfactory receptor neurons (ORNs) and compared these to odorant responses recorded in isolated ORNs. The maximum recording time in the slice was considerably longer than in isolated ORNs, which is essential when many odorants are to be tested. No odorant-induced responses could be obtained from isolated ORNs recorded in the on-cell mode, while recordings in the slice (on-cell and whole-cell) as well as previously reported perforated-patch recordings in isolated ORNs of the same species () were successful, though qualitatively different. In the slice preparation, amino acids as well as an extract from Spirulina algae always induced excitatory responses, while, in a previous study on isolated ORNs, responses were either excitatory or inhibitory. The results of this study show that ORNs obtained using different preparation techniques can give markedly different responses upon the application of odorants. Our experiments indicate that the slice preparation combined with the on-cell configuration of the patch-clamp technique is the method of choice for testing many odorants on individual ORNs. peerReviewed

We report on responses of olfactory receptor neurons (ORNs) upon application of amino acids and forskolin using a novel slice preparation of the olfactory epithelium of Xenopus laevis tadpoles. Responses were measured using the patch-clamp technique. Both amino acids and forskolin proved to be potent stimuli. Interestingly, a number of ORNs that responded to amino acids did not respond to forskolin. This suggests that some amino acids activate transduction pathways other than the well-known cAMP-mediated one. The differential processing of cAMP-mediated stimuli on the one hand and amino acid stimuli on the other was further elucidated by calcium-imaging of olfactory bulb neurons using a novel nose-olfactory bulb preparation of Xenopus laevis tadpoles. The projection pattern of amino acid-sensitive ORNs to olfactory bulb neurons differed markedly from the projection pattern of forskolin-sensitive ORNs. Olfactory bulb neurons activated by amino acids were located laterally compared to those activated by forskolin, and only a small proportion responded to both stimuli. The ensemble of neurons activated by forskolin was also activated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the membrane-permeant cAMP analogue 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (pCPT-cAMP). We therefore conclude that sensory transduction of a number of amino acids is cAMP independent, and amino acid- and cAMP-mediated responses are processed differentially at the level of the olfactory bulb.