Thomas Hassenklöver

Purinergic Signaling Regulates Cell Proliferation of Olfactory Epithelium Progenitors

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. STEM CELLS 2009;27:2022–2031

Nucleotide-induced Ca2+ signaling in sustentacular supporting cells of the olfactory epithelium.

Extracellular purines and pyrimidines are important signaling molecules acting via purinergic cell‐surface receptors in neurons, glia, and glia‐like cells such as sustentacular supporting cells (SCs) of the olfactory epithelium (OE). Here, we thoroughly characterize ATP‐induced responses in SCs of the OE using functional Ca2+ imaging. The initial ATP‐induced increase of the intracellular Ca2+ concentration [Ca2+]i always occurred in the apical part of SCs and subsequently propagated toward the basal lamina, indicating the occurrence of purinergic receptors in the apical part of SCs. The mean propagation velocity of the Ca2+ signal within SCs was 17.10 ± 1.02 μm/s. ATP evoked increases in [Ca2+]i in both the presence and absence of extracellular Ca2+. Depletion of the intracellular Ca2+ stores abolished the responses. This shows that the ATP‐induced [Ca2+]i increases were in large part, if not entirely, due to the activation of G protein‐coupled receptors followed by Ca2+ mobilization from intracellular stores, suggesting an involvement of P2Y receptors. The order of potency of the applied purinergic agonists was UTP > ATP > ATPγS (with all others being only weakly active or inactive). The ATP‐induced [Ca2+]i increases could be reduced by the purinergic antagonists PPADS and RB2, but not by suramin. Our findings suggest that extracellular nucleotides in the OE activate SCs via P2Y2/P2Y4‐like receptors and initiate a characteristic intraepithelial Ca2+ wave.

Bimodal processing of olfactory information in an amphibian nose: odor responses segregate into a medial and a lateral stream

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.

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. J. Comp. Neurol. 524:986–998, 2016.

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 Ca2+ 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.

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.

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 Ca2+. Depletion of intracellular Ca2+ 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 P2Y2/P2Y4-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.

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.

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.

Amino Acid- vs. Peptide-Odorants: Responses of Individual Olfactory Receptor Neurons in an Aquatic Species

Amino acids are widely used waterborne olfactory stimuli proposed to serve as cues in the search for food. In natural waters the main source of amino acids is the decomposition of proteins. But this process also produces a variety of small peptides as intermediate cleavage products. In the present study we tested whether amino acids actually are the natural and adequate stimuli for the olfactory receptors they bind to. Alternatively, these olfactory receptors could be peptide receptors which also bind amino acids though at lower affinity. Employing calcium imaging in acute slices of the main olfactory epithelium of the fully aquatic larvae of Xenopus laevis we show that amino acids, and not peptides, are more effective waterborne odorants.