Evidence for multiple calcium response mechanisms in mammalian olfactory receptor neurons

Olfactory receptor neurons employ a diversity of signaling mechanisms for transducing and encoding odorant information. The simultaneous activation of subsets of receptor neurons provides a complex pattern of activation in the olfactory bulb that allows for the rapid discrimination of odorant mixtures. While some transduction elements are conserved among many species, some species-specificity occurs in certain features that may relate to their particular physiology and ecological niche. However, studies of olfactory transduction have been limited to a relatively small number of vertebrate and invertebrate species. To better understand the diversity and evolution of olfactory transduction mechanisms, we studied stimulus-elicited calcium fluxes in olfactory neurons from a previously unstudied mammalian species, the domestic cat. Isolated cells from cat olfactory epithelium were stimulated with odorant mixtures and biochemical agents, and cell responses were measured with calcium imaging techniques. Odorants elicited either increases or decreases in intracellular calcium; odorant-induced calcium increases were mediated either by calcium fluxes through the cell membrane or by mobilization of intracellular stores. Individual cells could employ multiple signaling mechanisms to mediate responses to different odorants. The physiological features of these olfactory neurons suggest greater complexity than previously recognized in the role of peripheral neurons in encoding complex odor stimuli. The investigation of novel and unstudied species is important for understanding the mechanisms of odorant signaling that apply to the olfactory system in general and suggests both broadly conserved and species-specific evolutionary adaptations.     

Block by amiloride derivatives of odor-evoked discharge in lobster olfactory receptor neurons through action on a presumptive TRP channel

Amiloride and its derivatives inhibit a number of sensory transduction processes, including some types of chemosensory transduction. Here, we report that pyrazine derivatives of amiloride reversibly inhibit odorant-evoked activity in lobster olfactory receptor neurons. The potency sequence is as follows-(IC50, mM): 5-(N,N-hexamethylene)amiloride (0.015) approximately 5-(N-methyl-N-isobutyl)amiloride (0.02) approximately 5-(N-ethyl-N-isopropyl)amiloride (0.03) > 5-(N,N-dimethyl)amiloride (0.48); 3',4'-dichlorobenzamil (0.4), phenamil (0.5), and amiloride itself (2) are ineffective. The same derivatives with the similar potency sequence also block a presumptive transient receptor potential (TRP) channel that is the likely downstream target of phosphoinositide signaling in these cells. Our results suggest that pyrazine derivatives of amiloride are useful probes to study more detailed mechanisms of chemosensory transduction in this system and possibly in other chemosensory systems in which TRP channels are the known or suspected downstream effector.     

Odorant response of isolated olfactory receptor cells is blocked by amiloride

Summary Olfactory receptor cells were isolated from the nasal mucosa ofRana esculenta and patch clamped. Best results were obtained with free-floating cells showing ciliary movement. 1)On-cell mode: Current records were obtained for up to 50 min. Under control conditions they showed only occasional action potentials. The odorants cineole, amyl acetate and isobutyl methoxypyrazine were applied in saline by prolonged superfusion. At 500 nanomolar they elicited periodic bursts of current transients arising from cellular action potentials. The response was rapidly, fully and reversibly blocked by 50 m amiloride added to the odorant solution. With 10 m amiloride, the response to odorants was only partially abolished. 2)Whole-cell mode: Following breakage of the patch, the odorant response was lost within 5 to 15 min. Prior to this, odorants evoked a series of slow transient depolarizations (0.1/sec, 45 mV peak to peak) which reached threshold and thus elicited the periodic discharge of action potentials. These slow depolarizing waves were reversibly blocked by amiloride, which stabilized the membrane voltage between –80 and –90 mV. We conclude that amiloride inhibits chemosensory transduction of olfactory receptor cells, probably by blocking inward current pathways which open in response to odorants.     

Odorant-regulated Ca2+ gradients in rat olfactory neurons

Olfactory neurons respond to odors with a change in conductance that mediates an influx of cations including Ca2+. The concomitant increase in [Cai] has been postulated to play a role in the adaptation to maintained odorant stimulation (Kurahashi, T., and T. Shibuya. 1990. Brain Research. 515:261-268. Kramer, R. H., and S. A. Siegelbaum. 1992. Neuron. 9:897-906. Zufall, F., G. M. Shepherd, and S. Firestein. 1991. Proceedings of the Royal Society of London, B. 246:225-230.) We have imaged the distribution of [Cai] in rat olfactory neurons (RON) using the Ca2+ indicator fura-2. A large percentage of the RON (42%, n = 35) responded to odorants with an increase in [Cai]. About half of the responding neurons displayed an increase in [Cai] at the apical end of the cell, but not at the soma. Moreover, in those cells that responded to odors with a standing [Cai] gradient, the gradient could be maintained for long periods of time (minutes) provided that the cells were continuously stimulated. In contrast, K(+)-induced depolarization elicited a more homogeneous increase in [Cai]. The spatially inhomogeneous increase in [Cai] elicited by odorants in some cells has important implications for the role of Ca2+ in adaptation because channels and enzymes regulated by Ca2+ will be affected differently depending on their location.     

Inositol-trisphosphate-dependent calcium currents precede cation currents in insect olfactory receptor neurons in vitro

Specialized olfactory receptor neurons in insects respond to species-specific sex pheromones with transient rises in inositol trisphosphate and by opening pheromone-dependent cation channels. These channels resemble cation channels which are directly or indirectly Ca²⁺-dependent. But there appear to be no internal Ca²⁺ stores in the outer dendrite where the olfactory transduction cascade is thought to start. Hence, it remains to be determined whether an influx of external Ca²⁺ precedes pheromone-dependent cation currents. Patch clamp measurements in cultured olfactory receptor neurons from Manduca sexta reveal that a transient inward current precedes pheromone-dependent cation currents. A transient inositol trisphosphate-dependent Ca²⁺ current, also preceding cation currents with the characteristics of pheromone-dependent cation currents, shares properties with the transient pheromone-dependent current. These results match the biochemical measurements with the electrophysiological data obtained in insect olfactory receptor neurons.Abbreviations ORNs Olfactory receptor neurons - IP₃ Inositol-1,4,5-trisphosphate - I_t Transient pheromone-dependent current - I_ir Transient IP₃-dependent current     

Slowing of sodium current inactivation by ruthenium red in snail neurons

The effects of ruthenium red (RuR) were tested on the membrane currents of internally perfused, voltage-clamped nerve cell bodies from the snail Limnea stagnalis. Bath application of nanomolar concentrations of RuR produces a prolonged Na current that decays approximately 40 times slower than the normal Na current in these cells. The relationship between the reversal potential for the prolonged Na current and the intracellular concentration of Na+ agrees well with the constant-field equation, assuming a small permeability for Cs+. Because a strong correlation was found between the magnitude of the normal Na current and that of the prolonged Na current, it is concluded that the prolonged Na current flows through the normal Na channels. This conclusion is supported by the similar selectivities, voltage dependencies, and tetrodotoxin (TTX) sensitivities of these two currents. This action of RuR to slow the inactivation of the Na channel was not observed at concentrations below 1 nM, but was complete at 10 nM. When the concentration of RuR is increased to 0.1 mM, the Ca current in these cells is blocked; but at this high concentration RuR also reduces the outward voltage-dependent currents and resting membrane resistance. Therefore, RuR is not a good Ca blocker because of its lack of specificity. However, its action of slowing Na current inactivation is very specific and could prove to be useful in studying the inactivation of the Na channel.     

An enhanced olfactory marker protein immunoreactivity in individual olfactory receptor neurons following olfactory bulbectomy may be related to increased neurogenesis

Olfactory marker protein (OMP) is a 19-kD acidic protein found throughout the cytoplasm of mature olfactory receptor neurons (ORNs). Its function remains unknown. Following olfactory bulbectomy, the proportion of ORNs mature enough to express OMP declines greatly. However, in the few remaining mature ORNs, it has been observed that the intensity of OMP immunoreactivity (IR) appears to increase over that of ORNs on the unoperated side. We have now investigated this phenomenon quantitatively in rats subjected to unilateral olfactory bulbectomy. Results show that at all postbulbectomy survival periods examined quantitatively (3 days to 6 months), a significant decrease (19–37%) occurs in the transmission of incident light through OMP(+)-ORNs in bulbectomized versus unoperated olfactory epithelium (OE). Further, we also observed a consistent side-to-side difference in OMP IR in control unoperated animals. Possible explanations for these observations and their relation to the still unknown function of OMP are discussed. To test the possibility that OMP might serve a mitogenic role in the OE, recombinant OMP was added to organotypic explant cultures of fetal olfactory mucosa. Addition of OMP resulted in a dose-dependent increase in the density of bromodeoxyuridine-positive cells in the cultures, with a 50% increase occurring at the plateau OMP concentration of 25 nM. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 377–390, 1998     

Odour transduction in olfactory receptor neurons

The molecular mechanisms that control the binding of odorant to olfactory receptors and transduce this signal into membrane depolarization are reviewed. They are compared in vertebrates and insects for interspecific (allelochemicals) and intraspecific (pheromones) olfactory signals. Attempts to develop quantitative models of these multistage signalling networks are presented. Computational analysis of olfactory transduction is still in its infancy and appears as a promising area for future developments.     

Sodium/calcium exchanger in rat olfactory neurons

The chemo-electrical transduction process in olfactory neurons is accompanied by a rapid and transient increase in intracellular calcium concentrations. The notion that Na⁺/Ca²⁺ exchanger activities may play a major role in extruding calcium ions out of the cell and maintaining Ca²⁺ homeostasis in olfactory receptor cells was assessed by means of laser scanning confocal microscopy in combination with the fluorescent indicators Fluo-3 and Fura-Red. The data indicate that high exchanger acitivity, which was inhibited by amiloride derivatives, is located in the dendritic knob and probably in the olfactory cilia. This result was supported by experiments using specific antiserum raised against retinal Na⁺/Ca²⁺ exchanger protein which labelled an immunoreactive protein of 230 kDa in Western blots from olfactory tissue and strongly stained the ciliary layer of the olfactory epithelium.     

Calcium signalling in squid olfactory receptor neurons

Isolated squid olfactory receptor neurons respond to dopamine and betaine with hyperpolarizing conductances. We used Ca(2+) imaging techniques to determine if changes in intracellular Ca(2+) were involved in transducing the hyperpolarizing odor responses. We found that dopamine activated release of Ca(2+) from intracellular stores while betaine did not change internal Ca(2+) concentrations. Application of 10 mM caffeine also released Ca(2+) from intracellular stores, suggesting the presence of ryanodine-like receptors. Depletion of intracellular stores with 100 microM thapsigargin revealed the presence of a Ca(2+) store depletion-activated Ca(2+) influx. The influx of Ca(2+) through the store-operated channel was reversibly blocked by 10 mM Cd(2+). Taken together, these data suggest a novel odor transduction system in squid olfactory receptor neurons involving Ca(2+) release from intracellular stores. Copyright Copyright 1999 S. Karger AG, Basel     

Odor-evoked inhibition in primary olfactory receptor neurons

Odors can inhibit as well as excite lobster olfactory receptorcells. Inhibitory components of an odor mixture act within thenormal, first 500 ms odor sampling interval of the animal toreduce the peak magnitude and increase the latency of the netexcitatory receptor potential in a concentration-dependent manner.The intracellular effects are reflected in the propagated outputof the cell. The results argue that inhibitory odor input isfunctional in olfaction by potentially serving to increase thediversity of the neuronal patterns that are thought to be thebasis of odor discrimination.     

All or none cell responses of Ca2+-dependent K channels elicited by calcium or lead in human red cells can be explained by heterogeneity of agonist distribution

Summary We have studied the all or none cell response of Ca²⁺-dependent K⁺ channels to added Ca in human red cells depleted of ATP by incubation with iodoacetate and inosine. A procedure was used which allows separation and differential analysis of responding and nonresponding cells. Responding (H for heavy) cells incubated in medium containing 5mM K lose KCl and water and increase their density to the point of sinking on diethylphthalate (specific gravity=1.12) on centrifugation. Nonresponding (L for light) cells do not lose KCl at all. There is no intermediate behavior. Increasing the Ca concentration in the medium increases the fraction of cells which become H. No differences in the sensitivity to Ca²⁺ of the individual K⁺ channels were detected in inside-out vesicles prepared either from H or from L cells. The Ca content of H cells was higher than that of L cells. Cells depleted of ATP by incubation with iodoacetate and inosine sustain pump-leak Ca fluxes of about 15 mol/liter cells per hour. ATP seems to be resynthesized in these cells at the expense of cell 2,3-diphosphoglycerate stores at a rate of about 150 mol/liter cells per hour. Inhibition of 2,3-diphosphoglycerate phosphatase by tetrathionate increased 6–8 times the measured rate of uptake of external⁴⁵Ca. This was accompanied by an increase in the fraction of H cells. All or none cell responses of Ca²⁺-dependent K channels have also been evidenced in intact human red cells on addition of Pb. They have the same characteristics as those in responding and nonresponding cells. The detailed study of the kinetics of Pb-induced shrinkage of red cells suspended in medium containing 5mM K showed that changes of Pb concentration changed not only the fraction of H cells but also the rate of shrinkage of responding cells. H cells generated by Pb treatment contained significantly more lead than L cells. The above results suggest that the two all or none cell responses studied here can be explained by heterogeneity of agonist distribution among cells. Since pump-leak fluxes exist in both cases, differences of agonist distribution could be generated by heterogeneity of pumping among cells. This interpretation turns interest from K channels to Ca pumps to explain the heterogeneous behavior of red cells in response to a uniform stimulus.     

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.     

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P₂/IP₃ and PI(3,4,5)P₃, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.     

Inward rectification in rat olfactory receptor neurons.

Inwardly rectifying currents in enzymically dissociated olfactory receptor neurons of rat were studied by using patch-clamp techniques. Upon hyperpolarization to membrane potentials more negative than -100 mV, small inward-current relaxations were observed. Activation was described by a single exponential with a time constant that decreased e-fold for a 21 mV hyperpolarization. The current was not reduced by the external application of 5 mM Ba2+, but was abolished by the addition of 5 mM Cs+ to the bath solution. Increasing the external K+ concentration ([K+]o) to 25 mM dramatically enhanced the current without affecting the voltage range or the kinetics of activation. In 25 mM [K+]o, tail currents reversed at -26 mV, significantly more positive than the K+ equilibrium potential of -44 mV. These characteristics are consistent with those of a mixed Na+/K+ inward rectification that has been reported in several types of neuronal, cardiac and smooth muscle cells. The current may contribute to controlling cell excitability during the response to some odorants.     

Pathway for uncoupler-induced calcium efflux in rat liver mitochondria: inhibition by ruthenium red

The rate of uncoupler-induced Ca2+ efflux from rat liver mitochondria is increased by acetate and decreased by phosphate. This effect depends on a shift of the apparent Km, which is increased by phosphate and decreased by acetate, while the Vmax is not modified. The modification of the apparent Km by permeant anions presumably reflects changes in the concentration of matrix free Ca2+. A major part of uncoupler-induced Ca2+ efflux is sensitive to Ruthenium Red, the specific inhibitor of the Ca2+ uniporter , but an apparent insensitivity is observed when the H+ permeability is rate limiting in the process of Ca2+ efflux. The titer of uncoupler required for maximal stimulation of Ca2+ efflux increases with the Ca2+ load and may be 1-2 orders of magnitude higher than that required for maximal stimulation of respiration. On the other hand, when the uncoupler concentration is raised above 10(-6) M, the process of Ca2+ efflux becomes again Ruthenium Red insensitive. The Ruthenium Red inhibition of uncoupler-induced Ca2+ efflux is time dependent, in that the degree of inhibition exerted by low amounts of Ruthenium Red increases with the incubation time. Since the inhibition of the rate of Ca2+ influx undergoes a parallel relief, it is possible that Ruthenium Red moves from the cytosolic to the matrix side of the inner membrane. It is concluded that, in native mitochondria, uncoupler-induced Ca2+ efflux occurs via reversal of the uniport Ca2+ carrier, and not through activation of an independent pathway.     

Axonal targeting of olfactory receptor neurons in Drosophila is controlled by Dscam

Different classes of olfactory receptor neurons (ORNs) in Drosophila innervate distinct targets, or glomeruli, in the antennal lobe of the brain. Here we demonstrate that specific ORN classes require the cell surface protein Dscam (Down Syndrome Cell Adhesion Molecule) to synapse in the correct glomeruli. Dscam mutant ORNs frequently terminated in ectopic sites both within and outside the antennal lobe. The morphology of Dscam mutant axon terminals in either ectopic or cognate targets was abnormal. Target specificity for other ORNs was not altered in Dscam mutants, suggesting that different ORNs use different strategies to regulate wiring. Multiple forms of Dscam RNA were detected in the developing antenna, and Dscam protein was localized to developing ORN axons. We propose a role for Dscam protein diversity in regulating ORN target specificity.