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Odor perception begins with the detection of odorant molecules by the main olfactory epithelium located in the nasal cavity. Odorant molecules bind to and activate a large family of G-protein-coupled odorant receptors and trigger a cAMP-mediated transduction cascade that converts the chemical stimulus into an electrical signal transmitted to the brain. Morever, odorant receptors and cAMP signaling plays a relevant role in olfactory sensory neuron development and axonal targeting to the olfactory bulb. This review will first explore the physiological response of olfactory sensory neurons to odorants and then analyze the different components of cAMP signaling and their different roles in odorant detection and olfactory sensory neuron development.

Key points Taste transduction occurs in taste buds in the tongue epithelium.The Ca2+‐activated Cl– channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems.Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds.Large Ca2+‐activated Cl− currents blocked by Ani‐9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells.ATP indirectly activates Ca2+‐activated Cl– currents in type I cells through TMEM16A channels.These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells. Abstract The Ca2+‐activated Cl– channels TMEM16A and TMEM16B have relevant roles in many physiological processes including neuronal excitability and regulation of Cl– homeostasis. Here, we examined the presence of Ca2+‐activated Cl– channels in taste cells of mouse vallate papillae by using immunohistochemistry and electrophysiological recordings. By using immunohistochemistry we showed that only TMEM16A, and not TMEM16B, was expressed in taste bud cells where it largely co‐localized with the inwardly rectifying K+ channel KNCJ1 in the apical part of type I cells. By using whole‐cell patch‐clamp recordings in isolated cells from taste buds, we measured an average current of −1083 pA at −100 mV in 1.5 μm Ca2+ and symmetrical Cl– in type I cells. Ion substitution experiments and blockage by Ani‐9, a specific TMEM16A channel blocker, indicated that Ca2+ activated anionic currents through TMEM16A channels. We did not detect any Ca2+‐activated Cl– currents in type II or III taste cells. ATP is released by type II cells in response to various tastants and reaches type I cells where it is hydrolysed by ecto‐ATPases. Type I cells also express P2Y purinergic receptors and stimulation of type I cells with extracellular ATP produced large Ca2+‐activated Cl− currents blocked by Ani‐9, indicating a possible role of TMEM16A in ATP‐mediated signalling. These results provide a definitive demonstration that TMEM16A‐mediated currents are functional in type I taste cells and provide a foundation for future studies investigating physiological roles for these often‐neglected taste cells., Key points Taste transduction occurs in taste buds in the tongue epithelium.The Ca2+‐activated Cl– channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems.Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds.Large Ca2+‐activated Cl− currents blocked by Ani‐9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells.ATP indirectly activates Ca2+‐activated Cl– currents in type I cells through TMEM16A channels.These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells.

eNeuro / Society for Neuroscience 8(5), ENEURO.0179-21.2021 (2021). doi:10.1523/ENEURO.0179-21.2021, Published by Society for Neuroscience, Washington, DC

Microglia are highly plastic cells that change their properties in response to their microenvironment. By using immunofluorescence, live-cell imaging, electrophysiological recordings and RNA sequencing, we investigated the regulation of modified bacterial cellulose (mBC) nanofibril substrates on microglial properties. We demonstrate that mBC substrates induce ramified microglia with constantly extending and retracting processes, reminiscent of what is observed in vivo. Patch-clamp recordings show that microglia acquire a more negative resting membrane potential and have increased inward rectifier K+ currents, caused by an upregulation of Kir2.1 channels. Transcriptome analysis shows upregulation of genes involved in the immune response and downregulation of genes linked to cell adhesion and cell motion. Furthermore, Arp2/3 complex activation and integrin-mediated signaling modulate microglial morphology and motility. Our studies demonstrate that mBC nanofibril substrates modulate microglial phenotype, paving the way for a microglia-material interface that may be very valuable for anti-neuroinflammatory drug screening.

The sense of smell has been shown to deteriorate in patients with some neurodegenerative disorders. In Parkinson's disease (PD) and Alzheimer's disease (AD), decreased ability to smell is associated with early disease stages. Thus, olfactory neurons in the nose and olfactory bulb (OB) may provide a window into brain physiology and pathophysiology to address the pathogenesis of neurodegenerative diseases. Because nasal olfactory receptor neurons regenerate throughout life, the olfactory system offers a broad variety of cellular mechanisms that could be altered in AD, including odorant receptor expression, neurogenesis and neurodegeneration in the olfactory epithelium, axonal targeting to the OB, and synaptogenesis and neurogenesis in the OB. This review focuses on pathophysiological changes in the periphery of the olfactory system during the progression of AD in mice, highlighting how the olfactory epithelium and the OB are particularly sensitive to changes in proteins and enzymes involved in AD pathogenesis. Evidence reviewed here in the context of the emergence of other typical pathological changes in AD suggests that olfactory impairments could be used to understand the molecular mechanisms involved in the early phases of the pathology.

Glial-like supporting cells of the olfactory epithelium are involved in a variety of processes important to the function of this tissue. Henriques et al. find that supporting cells exhibit an ATP-induced, Ca2+-sensitive Cl− current produced by TMEM16A Cl− channels., Glial-like supporting (or sustentacular) cells are important constituents of the olfactory epithelium that are involved in several physiological processes such as production of endocannabinoids, insulin, and ATP and regulation of the ionic composition of the mucus layer that covers the apical surface of the olfactory epithelium. Supporting cells express metabotropic P2Y purinergic receptors that generate ATP-induced Ca2+ signaling through the activation of a PLC-mediated cascade. Recently, we reported that a subpopulation of supporting cells expresses also the Ca2+-activated Cl− channel TMEM16A. Here, we sought to extend our understanding of a possible physiological role of this channel in the olfactory system by asking whether Ca2+ can activate Cl− currents mediated by TMEM16A. We use whole-cell patch-clamp analysis in slices of the olfactory epithelium to measure dose–response relations in the presence of various intracellular Ca2+ concentrations, ion selectivity, and blockage. We find that knockout of TMEM16A abolishes Ca2+-activated Cl− currents, demonstrating that TMEM16A is essential for these currents in supporting cells. Also, by using extracellular ATP as physiological stimuli, we found that the stimulation of purinergic receptors activates a large TMEM16A-dependent Cl− current, indicating a possible role of TMEM16A in ATP-mediated signaling. Altogether, our results establish that TMEM16A-mediated currents are functional in olfactory supporting cells and provide a foundation for future work investigating the precise physiological role of TMEM16A in the olfactory system.

TMEM16B is expressed in olfactory sensory neurons, but previous attempts to establish a physiological role in olfaction have been unsuccessful. Pietra et al. find that genetic ablation of TMEM16B results in defects in the olfactory behavior of mice and the cellular physiology of olfactory sensory neurons., The Ca2+-activated Cl− channel TMEM16B is highly expressed in the cilia of olfactory sensory neurons (OSNs). Although a large portion of the odor-evoked transduction current is carried by Ca2+-activated Cl− channels, their role in olfaction is still controversial. A previous report (Billig et al. 2011. Nat. Neurosci. http://dx.doi.org/10.1038/nn.2821) showed that disruption of the TMEM16b/Ano2 gene in mice abolished Ca2+-activated Cl− currents in OSNs but did not produce any major change in olfactory behavior. Here we readdress the role of TMEM16B in olfaction and show that TMEM16B knockout (KO) mice have behavioral deficits in odor-guided food-finding ability. Moreover, as the role of TMEM16B in action potential (AP) firing has not yet been studied, we use electrophysiological recording methods to measure the firing activity of OSNs. Suction electrode recordings from isolated olfactory neurons and on-cell loose-patch recordings from dendritic knobs of neurons in the olfactory epithelium show that randomly selected neurons from TMEM16B KO mice respond to stimulation with increased firing activity than those from wild-type (WT) mice. Because OSNs express different odorant receptors (ORs), we restrict variability by using a mouse line that expresses a GFP-tagged I7 OR, which is known to be activated by heptanal. In response to heptanal, we measure dramatic changes in the firing pattern of I7-expressing neurons from TMEM16B KO mice compared with WT: responses are prolonged and display a higher number of APs. Moreover, lack of TMEM16B causes a markedly reduced basal spiking activity in I7-expressing neurons, together with an alteration of axonal targeting to the olfactory bulb, leading to the appearance of supernumerary I7 glomeruli. Thus, TMEM16B controls AP firing and ensures correct glomerular targeting of OSNs expressing I7. Altogether, these results show that TMEM16B does have a relevant role in normal olfaction.

Calcium-activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium-activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction.

TMEM16A/ANO1 is a calcium-activated chloride channel expressed in several types of epithelia and involved in various physiological processes, including proliferation and development. During mouse embryonic development, the expression of TMEM16A in the olfactory epithelium is dynamic. TMEM16A is expressed at the apical surface of the entire olfactory epithelium at embryonic day E12.5 while from E16.5 its expression is restricted to a region near the transition zone with the respiratory epithelium. To investigate whether TMEM16A plays a role in the development of the mouse olfactory epithelium, we obtained the first immunohistochemistry study comparing the morphological properties of the olfactory epithelium and nasal glands in TMEM16A-/- and TMEM16A+/+ littermate mice. A comparison between the expression of the olfactory marker protein and adenylyl cyclase III shows that genetic ablation of TMEM16A did not seem to affect the maturation of olfactory sensory neurons and their ciliary layer. As TMEM16A is expressed at the apical part of supporting cells and in their microvilli, we used ezrin and cytokeratin 8 as markers of microvilli and cell body of supporting cells, respectively, and found that morphology and development of supporting cells were similar in TMEM16A-/- and TMEM16A+/+ littermate mice. The average number of supporting cells, olfactory sensory neurons, horizontal and globose basal cells were not significantly different in the two types of mice. Moreover, we also observed that the morphology of Bowman’s glands, nasal septal glands and lateral nasal glands did not change in the absence of TMEM16A. Our results indicate that the development of mouse olfactory epithelium and nasal glands does not seem to be affected by the genetic ablation of TMEM16A.

In vertebrate olfactory transduction, a Ca(2+)-dependent Cl(-) efflux greatly amplifies the odorant response. The binding of odorants to receptors in the cilia of olfactory sensory neurons activates a transduction cascade that involves the opening of cyclic nucleotide-gated channels and the entry of Ca(2+) into the cilia. The Ca(2+) activates a Cl(-) current that, in the presence of a maintained elevated intracellular Cl(-) concentration, produces an efflux of Cl(-) ions and amplifies the depolarization. In this review, we summarize evidence supporting the hypothesis that anoctamin 2/TMEM16B is the main, or perhaps the only, constituent of the Ca(2+)-activated Cl(-) channels involved in olfactory transduction. Indeed, studies from several laboratories have shown that anoctamin 2/TMEM16B is expressed in the ciliary layer of the olfactory epithelium, that there are remarkable functional similarities between currents in olfactory sensory neurons and in HEK 293 cells transfected with anoctamin 2/TMEM16B, and that knockout mice for anoctamin 2/TMEM16B did not show any detectable Ca(2+)-activated Cl(-) current. Finally, we discuss the involvement of Ca(2+)-activated Cl(-) channels in the transduction process of vomeronasal sensory neurons and the physiological role of these channels in olfaction.

A conformational conversion of the cellular prion protein (PrP(C)) is now recognized as the causal event of fatal neurodegenerative disorders, known as prion diseases. In spite of long-lasting efforts, however, the physiological role of PrP(C) remains unclear. It has been reported that PrP(C) is expressed in various areas of the olfactory system, including the olfactory epithelium, but its precise localization in olfactory sensory neurons (OSNs) is still debated. Here, using immunohistochemistry tools, we have reinvestigated the expression and localization of PrP(C) in the olfactory epithelium of adult congenic mice expressing different PrP(C) amounts, that is, wild-type, PrP-knockout, and transgenic PrP(C)-overexpressing animals. We found that PrP(C) was expressed in OSNs, in which, however, it was unevenly distributed, being detectable at low levels in cell bodies, dendrites and apical layer, and more abundantly in axons. We also studied the involvement of PrP(C) in the response of the olfactory epithelium to odorants, by comparing the electro-olfactograms of the 3 mouse lines subjected to different stimulation protocols. We found no significant difference between the 3 PrP genotypes, supporting previous reports that exclude a direct action of PrP(C) in the early signal transduction activity of the olfactory epithelium.

Ca2+-activated Cl− channels play relevant roles in several physiological processes, including olfactory transduction, but their molecular identity is still unclear. Recent evidence suggests that members of the transmembrane 16 (TMEM16, also named anoctamin) family form Ca2+-activated Cl− channels in several cell types. In vertebrate olfactory transduction, TMEM16b/anoctamin2 has been proposed as the major molecular component of Ca2+-activated Cl− channels. However, a comparison of the functional properties in the whole-cell configuration between the native and the candidate channel has not yet been performed. In this study, we have used the whole-cell voltage-clamp technique to measure functional properties of the native channel in mouse isolated olfactory sensory neurons and compare them with those of mouse TMEM16b/anoctamin2 expressed in HEK 293T cells. We directly activated channels by rapid and reproducible intracellular Ca2+ concentration jumps obtained from photorelease of caged Ca2+ and determined extracellular blocking properties and anion selectivity of the channels. We found that the Cl− channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and DIDS applied at the extracellular side of the membrane caused a similar inhibition of the two currents. Anion selectivity measured exchanging external ions and revealed that, in both types of currents, the reversal potential for some anions was time dependent. Furthermore, we confirmed by immunohistochemistry that TMEM16b/anoctamin2 largely co-localized with adenylyl cyclase III at the surface of the olfactory epithelium. Therefore, we conclude that the measured electrophysiological properties in the whole-cell configuration are largely similar, and further indicate that TMEM16b/anoctamin2 is likely to be a major subunit of the native olfactory Ca2+-activated Cl− current.

A Ca2+-activated Cl− current constitutes a large part of the transduction current in olfactory sensory neurons. The binding of odorants to olfactory receptors in the cilia produces an increase in cAMP concentration; Ca2+ enters into the cilia through CNG channels and activates a Cl− current. In intact mouse olfactory sensory neurons little is known about the kinetics of the Ca2+-activated Cl− current. Here, we directly activated CNG channels by flash photolysis of caged cAMP or 8-Br-cAMP and measured the current response with the whole cell voltage-clamp technique in mouse neurons. We measured multiphasic currents in the rising phase of the response at −50 mV. The current rising phase became monophasic in the absence of extracellular Ca2+, at +50 mV, or when most of the intracellular Cl− was replaced by gluconate to shift the equilibrium potential for Cl− to −50 mV. These results show that the second phase of the current in mouse intact neurons is attributed to a Cl− current activated by Ca2+, similarly to previous results on isolated frog cilia. The percentage of the total saturating current carried by Cl− was estimated in two ways: 1) by measuring the maximum secondary current and 2) by blocking the Cl− channel with niflumic acid. We estimated that in the presence of 1 mM extracellular Ca2+ and in symmetrical Cl− concentrations the Cl− component can constitute up to 90% of the total current response. These data show how to unravel the CNG and Ca2+-activated Cl− component of the current rising phase.

In the pore of homomeric cyclic nucleotide-gated (CNG) channels, Ca(2+) and Mg(2+) bind to a set of glutamate residues, which in the bovine olfactory CNG channel are located at position 340. However, native CNG channels from olfactory sensory neurons are composed by the assembly of three different types of subunits, each having a different residue -- glutamate, aspartate or glycine -- at the position corresponding to the binding site for external Ca(2+) and Mg(2+). We co-expressed the wild-type principal alpha subunit with its mutants E340G and E340D in different combinations in Xenopus laevis oocytes, and measured Ca(2+) and Mg(2+) blockage in excised outside-out membrane patches. The comparison between our results and data from native olfactory CNG channels indicates that the presence of all three residues -- glutamate, aspartate and glycine -- in the different subunits, is necessary to restore the sensitivity to external Ca(2+) and Mg(2+) measured in native channels.

We used immunodeficient mice, whose dorsomedial olfactory region was permanently damaged by dichlobenil inoculation, to test the neuroregenerative properties of transplanted human adipose tissue-derived stem cells after 30 and 60 days. Analysis of polymerase chain reaction bands revealed that stem cells preferentially engrafted in the lesioned olfactory epithelium compared with undamaged mucosa of untreated transplanted mice. Although basal cell proliferation in untransplanted lesioned mice did not give rise to neuronal cells in the olfactory mucosa, we observed clusters of differentiating olfactory cells in transplanted mice. After 30 days, and even more at 60 days, epithelial thickness was partially recovered to normal values, as also the immunohistochemical properties. Functional reactivity to odorant stimulation was also confirmed through electro-olfactogram recording in the dorsomedial epithelium. Furthermore, we demonstrated that engrafted stem cells fused with mouse cells in the olfactory organ, even if heterokaryons detected were too rare to hypothesize they directly repopulated the lesioned epithelium. The data reported prove that the migrating transplanted stem cells were able to induce a neuroregenerative process in a specific lesioned sensory area, enforcing the perspective that they could become an available tool for stem cell therapy.

Adaptation to odorants begins at the level of sensory receptor cells, presumably through modulation of their transduction machinery. The olfactory signal transduction involves the activation of the adenylyl cyclase/cyclic AMP second messenger system which leads to the sequential opening of cAMP-gated channels and Ca2+-activated chloride ion channels. Several reports of results obtained from in vitro preparations describe the possible molecular mechanisms involved in odorant adaptation; namely, ordorant receptor phosphorylation, activation of phosphodiesterase, and ion channel regulation. However, it is still unknown whether these putative mechanisms work in the intact olfactory receptor cell. Here we investigate the nature of the adaptational mechanism in intact olfactory cells by using a combination of odorant stimulation and caged cAMP photolysis which produces current responses that bypass the early stages of signal transduction (involving the receptor, G protein and adenylyl cyclase). Odorant- and cAMP-induced responses showed the same adaptation in a Ca2+-dependent manner, indicating that adaptation occurs entirely downstream of the cyclase. Moreover, we show that phosphodiesterase activity remains constant during adaptation and that an affinity change of the cAMP-gated channel for ligands accounts well for our results. We conclude that the principal mechanism underlying odorant adaptation is actually a modulation of the cAMP-gated channel by Ca2+ feedback.

The rodent vomeronasal organ plays an important role in many social behaviors. Using the calcium imaging technique with the dye fluo-4 we measured intracellular calcium concentration changes induced by the application of sulfated steroids to neurons isolated from the vomeronasal organ of female mice. We found that a mix of 10 sulfated steroids from the androgen, estrogen, pregnanolone, and glucocorticoid families induced a calcium response in 71% of neurons. Moreover, 31% of the neurons responded to a mix composed of 3 glucocorticoid-derived compounds, and 28% responded to a mix composed of 3 pregnanolone-derived compounds. Immunohistochemistry showed that neurons responding to sulfated steroids expressed phosphodiesterase 4A, a marker specific for apical neurons expressing V1R receptors. None of the neuron that responded to 1 mix responded also to the other, indicating a specificity of the responses. Some neurons responded to more than 1 individual component of the glucocorticoid-derived mix tested at high concentration, suggesting that these neurons are broadly tuned, although they still displayed strong specificity, remaining unresponsive to high concentrations of the ineffective compounds.

The olfactory system excels in both discrimination and detection of odorants. In mammals, it reliably discriminates more than 3000 structurally diverse odorant molecules and has an amazingly high sensitivity that allows the detection of very low amounts of specific odorant molecules. In addition, the olfactory system has the capability to adapt to ambient odorants, allowing the recognition of a broad range of stimuli. The discrimination among different odorants is achieved by using hundreds of receptors, activated with a combinatorial code. Olfactory transduction uses a canonical second messenger system providing two critical attributes: amplification and high signal-to-noise characteristics, giving the system its remarkable detector capabilities. In this review, we present an introduction to the basic molecular mechanisms of olfactory transduction in olfactory sensory neurons.

1. Olfactory receptor cells were isolated from the adult tiger salamander Ambystoma tigrinum and the current in response to odorant stimuli was measured with the whole-cell voltage-clamp technique while odorants at known concentrations were rapidly applied for controlled exposure times. 2. Three odorants, cineole, isoamyl acetate and acetophenone, were first applied at 5 x 10(-4) M. Out of forty-nine cells tested, 53% responded to one odorant only, 22% to two odorants and 25% to all three odorants. 3. The amplitude of the current in response to a given odorant concentration was found to be dependent on the duration of the odorant stimulus and reached a saturating peak value at 1.2 s of stimulus duration. 4. The current measured at the peak of the response for odorant steps of 1.2 s as a function of odorant concentration was well described by the Hill equation for the three odorants with Hill coefficients higher than 1 and K1/2 (odorant concentration needed to activate half the maximal current) ranging from 3 x 10(-6) to 9 x 10(-5) M. 5. It is concluded that olfactory receptor cells are broadly tuned and have a low apparent affinity for odorants, integrate stimulus information over time, and have a narrow dynamic range.

In recent years, considerable progress has been achieved in the comprehension of the profound effects of pheromones on reproductive physiology and behavior. Pheromones have been classified as molecules released by individuals and responsible for the elicitation of specific behavioral expressions in members of the same species. These signaling molecules, often chemically unrelated, are contained in body fluids like urine, sweat, specialized exocrine glands, and mucous secretions of genitals. The standard view of pheromone sensing was based on the assumption that most mammals have two separated olfactory systems with different functional roles: the main olfactory system for recognizing conventional odorant molecules and the vomeronasal system specifically dedicated to the detection of pheromones. However, recent studies have reexamined this traditional interpretation showing that both the main olfactory and the vomeronasal systems are actively involved in pheromonal communication. The current knowledge on the behavioral, physiological, and molecular aspects of pheromone detection in mammals is discussed in this review.

The herbicide dichlobenil selectively causes necrosis of the dorsomedial part of olfactory neuroepithelium (NE) with permanent damage to the underlying mucosa, whereas the lateral part of the olfactory region and the nasal respiratory mucosa remain undamaged. We investigated here whether human umbilical cord blood CD133+ stem cells (HSC) injected intravenously to nod-scid mice pretreated with dichlobenil may engraft the olfactory mucosa and contribute to the regeneration of the damaged NE. We tested HLA-DQα1 DNA and three human microsatellites (Combined DNA Index System) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the olfactory mucosa and bulb, at 7 and 31 days following HSC transplantation. Histology, immunohistochemistry, and lectin staining revealed the morphological recovery of the dorsomedial region of the NE in dichlobenil-treated mice that received HSC, contrasting with the lack of regeneration in similarly injured areas as these remained damaged in control nontransplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating olfactory area with chimeric cells. Electro-olfactograms in response to odorants, to test the functionality of the olfactory NE, confirmed the functional damage of the dorsomedial area in dichlobenil-treated mice and the functional recovery of the same area in transplanted mice. These findings support the concept that transplanted HSC migrating to the damaged olfactory area provide conditions facilitating the recovery from olfactory receptor cell loss. Disclosure of potential conflicts of interest is found at the end of this article.

Hyperpolarization-activated currents ( Ih) are present in several neurons of the central and peripheral nervous system. However, Ihin neurons of the vomeronasal organ (VNO) is not well characterized. We studied the properties of Ihin sensory neurons from acute slices of mouse VNO. In voltage-clamp studies, Ihwas identified by the characteristic kinetics of activation, voltage dependence, and blockage by Cs+or ZD-7288, two blockers of the Ih. Forskolin, an activator of adenylyl cyclase, shifted the activation curve for Ihto less negative potentials. A comparison of Ihproperties in VNO neurons with those of heterologously expressed hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, together with RT-PCR experiments in VNO, indicate that Ihis caused by HCN2 and/or HCN4 subunits. In current-clamp recordings, blocking Ihwith ZD-7288 induced a hyperpolarization of 5.1 mV, an increase in input resistance, a decrease in the sensitivity to elicit action potentials in response to small current injections, and did not modify the frequency of action potentials elicited by a large current injection. It has been shown that in VNO neurons some pheromones induce a decrease in cAMP concentration, but the physiological role of cAMP is unknown. After application of blockers of adenylyl cyclase, we measured a hyperpolarization of 5.1 mV in 11 of 14 neurons, suggesting that basal levels of cAMP could modulate the resting potential. In conclusion, these results show that mouse VNO neurons express HCN2 and/or HCN4 subunits and that Ihcontributes to setting the resting membrane potential and to increase excitability at stimulus threshold.

Organotypic cultures of the mouse olfactory epithelium connected to the olfactory bulb were obtained with the roller tube technique from postnatal mice aged between 13 and 66 days. To test the functionality of the cultures, we measured electroolfactograms (EOGs) at different days in vitro (DIV), up to 7 DIV, and we compared them with EOGs from identical acute preparations (0 DIV). Average amplitudes of EOG responses to 2 mixtures of various odorants at concentrations of 1 mM or 100 microM decreased in cultures between 2 and 5 DIV compared with 0 DIV. The percentage of responsive cultures was 57%. We also used the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to trigger the olfactory transduction cascade bypassing odorant receptor activation. Average amplitudes of EOG responses to 500 microM IBMX were not significantly different in cultures up to 6 DIV or 0 DIV, and the average percentage of responsive cultures between 2 and 5 DIV was 72%. The dose-response curve to IBMX measured in cultures up to 7 DIV was similar to that at 0 DIV. Moreover, the percentage of EOG response to IBMX blocked by niflumic acid, a blocker of Ca-activated Cl channels, was not significantly different in cultured or acute preparations.

Ca-activated Cl channels are an important component of olfactory transduction. Odor binding to olfactory receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca concentration by Ca entry through cyclic nucleotide-gated (CNG) channels. Ca activates a Cl channel that leads to an efflux of Cl from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl channel remains elusive. Recent evidence has indicated that bestrophins are able to form Ca-activated Cl channels in heterologous systems. Here we have analyzed the expression of bestrophins in the mouse olfactory epithelium and demonstrated that only mouse bestrophin-2 (mBest2) was expressed. Single-cell RT-PCR showed that mBest2 was expressed in OSNs but not in supporting cells. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and colocalized with the main CNGA2 channel subunit. Electrophysiological properties of Ca-activated Cl currents from native channels in dendritic knob/cilia of mouse OSNs were compared with those induced by the expression of mBest2 in HEK-293 cells. We found the same anion permeability sequence, small estimated single-channel conductances, a Ca sensitivity difference of one order of magnitude, and the same side-specific blockage of the two Cl channel blockers commonly used to inhibit the odorant-induced Ca-activated Cl current in OSNs, niflumic acid, and 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonate (SITS). Therefore, our data suggest that mBest2 is a good candidate for being a molecular component of the olfactory Ca-activated Cl channel.

The vomeronasal system is involved in the detection of pheromones in many mammals. Vomeronasal sensory neurons encode the behaviorally relevant information into action potentials that are directly transmitted to the accessory olfactory bulb. We developed a model of the electrical activity of mouse basal vomeronasal sensory neurons, which mimics both the voltage-gated current properties and the firing behavior of these neurons in their near-native state, using a minimal number of parameters. Data were obtained by recordings with the whole-cell voltage-clamp or current-clamp techniques from mouse basal vomeronasal sensory neurons in acute slice preparations. The resting potential ranged from � 50 to � 70 mV, and current injections of less than 2–10 pA induced tonic firing in most neurons. The experimentally determined firing frequency as a function of injected current was well described by a Michaelis–Menten equation and was exactly reproduced by the model, which could be used in combination with future models that will include details of the mouse vomeronasal transduction cascade.

In the recent article by Lin and collaborators (2008), they show that solitary chemosensory cells dispersed in the respiratory epithelium of the mouse nasal cavity could comprise a new transduction system for some noxious stimuli. Chemical sensitivity in the nasal cavity is not limited to smelling

Adrenaline modulates signaling by the olfactory epithelium. A new study suggests that this effect may result from channel phosphorylation in olfactory receptor neurons.

The detection of odorant molecules begins in the nasal epithelium where olfactory sensory neurons interact with odorant stimuli from the external world and produce electrical signals that are transmitted to the brain. Olfactory sensory neurons are small bipolar neurons with several fine cilia. The binding of odorant molecules to odorant receptor proteins in the cilia triggers an enzymatic cascade that leads to the onset of an inward ionic current, depolarization of the membrane and generation of action potentials that are conducted along the neuron’s axon to the olfactory bulb, where further processing of odorant information occurs (for reviews see Refs. [1,11,14,15]).

Signal transduction in vertebrate photoreceptors and olfactory receptor neurons (see for review Torre et al., 1995) involves the activation of non-selective cationic channels directly and cooperatively gated by cyclic nucleotides (CNG channels).

Many sensory systems have evolved signal detection capabilities that are limited only by the physical attributes of the stimulus. For example, 'hair' cells of the inner ear can detect displacements of atomic dimensions. Likewise, both in vertebrates and in invertebrates photoreceptors can detect a single photon. The olfactory stimulus also has a quantal unit, the single odorant molecule. Insects are reportedly able to detect a single pheromone molecule, whereas quantal responses in vertebrate olfactory receptor cells have not been reported yet. Psychophysical measurements indicate that a minimum of 50 odorant molecules are necessary for human olfactory detection, suggesting that an individual receptor may be activated by a single odorant molecule. We report here measurements of current fluctuations induced by odorants that suggest a quantal event of about 0.3-1 pA, presumably triggered by the binding of a single odorant molecule.

Sensory transduction in both the visual and the olfactory system involves ion channels that are opened directly by cyclic nucleotides. Both the absorption of light and the binding of many, but not all, odorants trigger a chain of enzymatic events that culminate in a change in the concentration of a cyclic nucleotide, a decrease in the concentration of cyclic GMP (cGMP) in the visual system, and an increase in the concentration of cyclic AMP (cAMP) in the olfactory system. Alterations in intracellular levels of cyclic nucleotides in these systems affect the activity of cyclic nucleotide-activated channels located in the plasma membrane. In the visual system closing of these channels leads to hyperpolarization of the cell, whereas in the olfactory system opening of cyclic nucleotide-activated channels results in depolarization. These cyclic nucleotide-induced alterations in the membrane potential represent the first step in neural excitation. Since cyclic nucleotide-activated channels mediate the final steps of the visual and olfactory transduction cascades and initiate the flow of ions that triggers excitation of the cell, they represent the molecular switches that control sensory perception. These observations and recent discoveries of additional members of the cyclic nucleotide-activated channel family in cardiac pacemaker cells1 and cells of the pineal gland2 have focused considerable attention on this expanding new family of ion channels that are directly activated by second messengers.

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