Ivan Manzini

Multidrug resistance transporters in the olfactory receptor neurons of Xenopus laevis tadpoles

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.

Cannabinoid action in the olfactory epithelium

The perception of odors is influenced by a variety of neuromodulators, and there is growing evidence that modulation already takes place in the olfactory epithelium. Here we report on cannabinergic actions in the olfactory epithelium of Xenopus laevis tadpoles. First we show that CB1 receptor-specific antagonists AM251, AM281, and LY320135 modulate odor-evoked calcium changes in olfactory receptor neurons. Second, we localize CB1-like immunoreactivity on dendrites of olfactory receptor neurons. Finally, we describe the cannabinergic influence on odor-induced spike-associated currents in individual olfactory receptor neurons. Here we demonstrate that the cannabinergic system has a profound impact on peripheral odor processing and discuss its possible function.

Classes and Narrowing Selectivity of Olfactory Receptor Neurons of Xenopus laevis Tadpoles

In olfactory receptor neurons (ORNs) of aquatic animals amino acids have been shown to be potent stimuli. Here we report on calcium imaging experiments in slices of the olfactory mucosa of Xenopus laevis tadpoles. We were able to determine the response profiles of 283 ORNs to 19 amino acids, where one profile comprises the responses of one ORN to 19 amino acids. 204 out of the 283 response profiles differed from each other. 36 response spectra occurred more than once, i.e., there were 36 classes of ORNs identically responding to the 19 amino acids. The number of ORNs that formed a class ranged from 2 to 13. Shape and duration of amino acid-elicited [Ca2+]i transients showed a high degree of similarity upon repeated stimulation with the same amino acid. Different amino acids, however, in some cases led to clearly distinguishable calcium responses in individual ORNs. Furthermore, ORNs clearly appeared to gain selectivity over time, i.e., ORNs of later developmental stages responded to less amino acids than ORNs of earlier stages. We discuss the narrowing of ORN selectivity over stages in the context of expression of olfactory receptors.

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.

cAMP-independent responses of olfactory neurons in Xenopus laevis tadpoles and their projection onto olfactory bulb neurons

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.

Improved fluorescent (calcium indicator) dye uptake in brain slices by blocking multidrug resistance transporters

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.

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.

ATP activates both receptor and sustentacular supporting cells in the olfactory epithelium of Xenopus laevis tadpoles

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.

The endocannabinoid 2-arachidonoyl-glycerol controls odor sensitivity in larvae of Xenopus laevis.

Cannabinoids modulate the activity of many neuronal cells, among them sensory neurons in the olfactory epithelium. Here we show that the endocannabinoid 2-arachidonoyl-glycerol (2-AG) is synthesized in both olfactory receptor neurons and glia-like sustentacular cells in larval Xenopus laevis. Its production in the latter depends on the hunger state of the animal. The essential effect of 2-AG in olfactory receptor neurons is the control of odorant detection thresholds via cannabinoid CB1 receptor activation. Hunger renders olfactory neurons more sensitive. Endocannabinoid modulation in the nose may therefore substantially influence food-seeking behavior.