Cross-adaptation between olfactory responses induced by two subgroups of odorant molecules

It has long been believed that vertebrate olfactory signal transduction is mediated by independent multiple pathways (using cAMP and InsP3 as second messengers). However, the dual presence of parallel pathways in the olfactory receptor cell is still controversial, mainly because of the lack of information regarding the single-cell response induced by odorants that have been shown to produce InsP3 exclusively (but not cAMP) in the olfactory cilia. In this study, we recorded activities of transduction channels of single olfactory receptor cells to InsP3-producing odorants. When the membrane potential was held at -54 mV, application of InsP3-producing odorants to the ciliary region caused an inward current. The reversal potential was 0 +/- 7 mV (mean +/- SD, n = 10). Actually, InsP3-producing odorants generated responses in a smaller fraction of cells (lilial, 3.4%; lyral, 1.7%) than the cAMP-producing odorant (cineole, 26%). But, fundamental properties of responses were surprisingly homologous; namely, spatial distribution of the sensitivity, waveforms, I-V relation, and reversal potential, dose dependence, time integration of stimulus period, adaptation, and recovery. By applying both types of odorants alternatively to the same cell, furthermore, we observed cells to exhibit symmetrical cross-adaptation. It seems likely that even with odorants with different modalities adaptation occurs completely depending on the amount of current flow. The data will also provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

Whole-cell recordings and photolysis of caged compounds in olfactory sensory neurons isolated from the mouse

Gene manipulation and molecular biological techniques for the study of olfaction are well developed in mice, while electrophysiological properties of mouse olfactory sensory neurons have been less extensively investigated. We used the whole-cell voltage-clamp technique in mouse isolated olfactory sensory neurons to investigate both voltage-gated and transduction currents. Voltage-gated currents were composed of transient inward currents followed by outward currents with transient and sustained components. Of the tested olfactory sensory neurons, 12% responded to the odorant cineole with an inward current. Caged compounds were introduced into the cytoplasm through the patch pipette and flash photolysis of caged cyclic nucleotides activated an inward current in 94% of the cells. When the flash was localized at the cilia, the response latency, rising time and duration were shorter than when the flash illuminated the soma. The amplitude of the photolysis response was dependent on light intensity and the relation was fitted by the Hill equation, with a Hill coefficient of 3.2. These results demonstrate that it is possible to obtain recordings in the whole-cell configuration from olfactory sensory neurons isolated from the mouse and that voltage-gated currents and transduction properties are largely similar to those of amphibians.     

Identification of second messenger mediating signal transduction in the olfactory receptor cell

One of the biggest controversial issues in the research of olfaction has been the mechanism underlying response generation to odorants that have been shown to fail to produce cAMP when tested by biochemical assays with olfactory ciliary preparations. Such observations are actually the original source proposing a possibility for the presence of multiple and parallel transduction pathways. In this study the activity of transduction channels in the olfactory cilia was recorded in cells that retained their abilities of responding to odorants that have been reported to produce InsP3 (instead of producing cAMP, and therefore tentatively termed "InsP3 odorants"). At the same time, the cytoplasmic cNMP concentration ([cNMP]i) was manipulated through the photolysis of caged compounds to examine their real-time interactions with odorant responses. Properties of responses induced by both InsP3 odorants and cytoplasmic cNMP resembled each other in their unique characteristics. Reversal potentials of currents were 2 mV for InsP3 odorant responses and 3 mV for responses induced by cNMP. Current and voltage (I-V) relations showed slight outward rectification. Both responses showed voltage-dependent adaptation when examined with double pulse protocols. When brief pulses of the InsP3 odorant and cytoplasmic cNMP were applied alternatively, responses expressed cross-adaptation with each other. Furthermore, both responses were additive in a manner as predicted quantitatively by the theory that signal transduction is mediated by the increase in cytoplasmic cAMP. With InsP3 odorants, actually, remarkable responses could be detected in a small fraction of cells ( approximately 2%), explaining the observation for a small production of cAMP in ciliary preparations obtained from the entire epithelium. The data will provide evidence showing that olfactory response generation and adaptation are regulated by a uniform mechanism for a wide variety of odorants.     

Metabotropic glutamate receptor expression in olfactory receptor neurons from the channel catfish,Ictalurus punctatus

Metabotropic glutamate receptors (mGluRs) were identified in olfactory receptor neurons of the channel catfish, Ictalurus punctatus, by polymerase chain reaction. DNA sequence analysis confirmed the presence of two subtypes, mGluR1 and mGluR3, that were coexpressed with each other and with the putative odorant receptors within single olfactory receptor neurons. Immunocytochemical data showed that both mGluR subtypes were expressed in the apical dendrites and some cilia of olfactory neurons. Pharmacological analysis showed that antagonists to each mGluR subtype significantly decreased the electrophysiological response to odorant amino acids. α-Methyl-L -CCG1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a known antagonist to mGluR3, and (S)-4-carboxyphenylglycine (S-4CPG), a specific antagonist to mGluR1, each significantly reduced olfactory receptor responses to L -glutamate. S-4CPG and MCCG reduced the glutamate response to 54% and 56% of control, respectively, which was significantly greater than their effect on a neutral amino acid odorant, methionine. These significant reductions of odorant response by the antagonists, taken with the expression of these receptors throughout the dendritic and ciliated portions of some olfactory receptor neurons, suggest that these mGluRs may be involved in olfactory reception and signal transduction. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 94–104, 1998     

Maturation-associated increase in IP3 receptor type 1: role in conferring increased IP3 sensitivity and Ca2+ oscillatory behavior in mouse eggs

Maturation of mouse oocytes is accompanied by an increase in sensitivity to inositol 1,4,5-trisphosphate (IP(3))-mediated release of intracellular calcium. To test the hypothesis that the maturation-associated 1.5- to 2.0-fold increase in the mass of the type 1 IP(3) receptor (IP(3)R-1) confers this increase in IP(3) sensitivity, we employed RNA interference to prevent this change in IP(3)R-1 protein level. Microinjection into germinal vesicle (GV)-intact oocytes of dsRNA corresponding to the IP(3)R-1 sequence resulted in a >90% reduction in the amount of maternal IP(3)R-1 mRNA and prevented the maturation-associated increase in the mass of the IP(3)R-1 protein. These injected oocytes matured to metaphase II, and there was no effect on the maturation-associated increases in p34(cdc2)/cyclin B kinase and MAP kinase activities or the global pattern of protein synthesis. IP(3)-induced cortical granule exocytosis was significantly decreased in these eggs when compared with controls previously injected with enhanced green fluorescent protein (EGFP) dsRNA. Following insemination, the IP(3)R-1 dsRNA-injected eggs displayed significantly fewer Ca(2+) transients than controls, and the duration of the first Ca(2+) transient was about half that of controls. These results support the hypothesis that the maturation-associated increase in the mass of IP(3)R-1 confers the increase in IP(3)-sensitivity that is observed following oocyte maturation and is necessary for the proper Ca(2+) oscillatory pattern following insemination.     

Protein kinase C and receptor kinase gene expression in olfactory receptor neurons

Recent biochemical evidence indicates that protein kinase C (PKC) and G-protein-coupled receptor kinases (GRKs) are involved in olfactory signal termination and desensitization. The polymerase chain reaction (PCR) was used to investigate the expression of PKC and GRK genes in olfactory tissue and in isolated olfactory receptor neurons from channel catfish (Ictalurus punctatus). Sequence analysis of cloned PKC PCR products showed that the α, β, δ, ϵ, and τ isotypes were expressed in olfactory tissue. Sequence analysis of PCR products obtained from isolated olfactory receptor neurons showed that PKCβ and PKCδ were expressed in the receptor cells. A 600-bp GRK PCR product was obtained from isolated olfactory neurons that shared 86% and 92% amino acid sequence identity to the mammalian β-adrenergic receptor kinase gene products βARK1 and βARK2, respectively. Go6976, a specific inhibitor of calcium-regulated PKC activity, completely inhibited odorant-stimulated PKC activity in isolated olfactory cilia. This result suggested that odorant-stimulated PKC activity is mediated by the calcium-sensitive PKCβ isotype. Taken together, these results are consistent with the conclusion that PKCβ and βARK mediate odorant receptor phosphorylation and olfactory signal termination. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 387–394, 1997     

IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) serve to discharge Ca²⁺ from ER stores in response to agonist stimulation. The present review summarizes the role of these receptors in models of Ca²⁺-dependent apoptosis. In particular we focus on the regulation of IP₃Rs by caspase-3 cleavage, cytochrome c, anti-apoptotic proteins and Akt kinase. We also address the evidence that some of the effects of IP₃Rs in apoptosis may be independent of their ion-channel function. The role of IP₃Rs in delivering Ca²⁺ to the mitochondria is discussed from the perspective of the factors determining inter-organellar dynamics and the spatial proximity of mitochondria and ER membranes.     

Stimulation of olfactory receptors alters regulation of [Cai] in olfactory neurons of the catfish (Ictalurus punctatus)

Summary Intracellular calcium was measured in single olfactory neurons from the channel catfish (Icatalurus punctatus) using the fluorescent Ca²⁺ indicator fura 2. In 5% of the cells, olfactory stimuli (amino acids) elicited an influx of calcium through the plasma membrane which led to a rapid transient increase in intracellular calcium concentration. Amino acids did not induce release of calcium from internal stores in these cells. Some cells responded specifically to one stimulus (l-alanine,l-arginine,l-norleucine andl-glutamate) while one cell responded to all stimuli. An increase in intracellular calcium could also be elicited in 50% of the cells by direct G-protein stimulation using aluminum fluoride. Because the fraction of cells which respond to direct G-protein stimulation is substantially larger than the fraction of cells responding to amino acids, we tested for possible damage of receptor proteins due to exposure of the olfactory neurons to papain during cell isolation. We find that pretreatment with papain does not alter specific binding ofl-alanine andl-arginine to olfactory receptor sites in isolated olfactory cilia. The results are discussed in terms of their relevance to olfactory transduction.     

Binding of IRBIT to the IP3 receptor: determinants and functional effects

IRBIT has previously been shown to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in an IP3-sensitive way. So far it remained to be elucidated whether this interaction was direct or indirect, and whether it was functionally relevant. We now show that IRBIT can directly interact with the IP3R, and that both the suppressor domain and the IP3-binding core of the IP3R are essential for a strong interaction. Moreover, we identified a PEST motif and a PDZ-ligand on IRBIT which were critical for the interaction with the IP3R. Furthermore, we identified Asp-73 as a critical residue for this interaction. Finally, we demonstrated that this interaction functionally affects the IP3R: IRBIT inhibits both IP3 binding and IP3-induced Ca2+ release.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert & Zhao, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca(2+)-activated Cl(-) channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body. In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors. The suction pipette technique was later adapted to also record from ORNs. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.     

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     

D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation

Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.     

ATP and adenosine trigger the interaction of plasma membrane IP3 receptors with protein kinase A in oviductal ciliated cells

We have demonstrated that adenosine did not produce any change of intracellular free Ca²⁺ concentration ([Ca²⁺]i) in oviductal ciliated cells; however, it increased the ATP-induced Ca²⁺ influx through the activation of protein kinase A (PKA). Uncaging of IP₃ and cAMP triggered a larger Ca²⁺ influx than did IP₃ alone. Furthermore, the IP₃ effect was abolished by Xestospongin C, an IP₃ receptor blocker. Whole-cell recordings demonstrated the presence of an ATP-induced Ca²⁺ current, and the addition of adenosine increased the peak of this current. This effect was not observed in the presence of H-89, a PKA inhibitor. Using excised macro-patches of plasma membrane, IP₃ generated a current, which was higher in the presence of the catalytic PKA subunit and this current was blocked by Xestospongin C. We show here that activation of plasma membrane IP₃ receptors directly triggers Ca²⁺ influx in response to ATP and that these receptors are modulated by adenosine-activated PKA.     

Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor

Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP(3)R have been reported; those of the IP(3)-binding core (IP(3)R(CORE)) with bound IP(3), and the suppressor domain. This study examines the structural effects of ligand binding on an IP(3)R construct, designated IP(3)R(N), that contains both the IP(3)-binding core and the suppressor domain. Our circular dichroism results reveal that the IP(3)-bound and IP(3)-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP(3) has a large effect on the stability of IP(3)R(CORE), it has little effect on IP(3)R(N), indicating that the suppressor domain is critical to the stability of IP(3)R(N). The NMR data for IP(3)R(N) provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP(3)-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP(3)R(N) undergoes a change in average conformation in response to IP(3) binding and the presence of Ca2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP(3) binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set.     

Differential development of odorant receptor expression patterns in the olfactory epithelium: a quantitative analysis in the mouse septal organ

The rodent olfactory epithelium expresses more than 1000 odorant receptors (ORs) with distinct patterns, yet it is unclear how such patterns are established during development. In the current study, we investigated development of the expression patterns of different ORs in the septal organ, a small patch of olfactory epithelium predominantly expressing nine identified ORs. The presumptive septal organ first appears at about embryonic day 16 (E16) and it completely separates from the main olfactory epithelium (MOE) at about postnatal day 7 (P7). Using in situ hybridization, we quantified the densities of the septal organ neurons labeled by specific RNA probes of the nine abundant OR genes from E16 to postnatal 3 months. The results indicate that olfactory sensory neurons (OSNs) expressing different ORs have asynchronous temporal onsets. For instance, MOR256-17 and MOR236-1 cells are present in the septal organ at E16; however, MOR0-2 cells do not appear until P0. In addition, OSNs expressing different ORs show distinct developmental courses and reach their maximum densities at different stages ranging from E16 (e.g. MOR256-17) to 1 month (e.g. MOR256-3 and MOR235-1). Furthermore, early onset does not correlate with high abundance in adult. This study reveals a dynamic composition of the OSNs expressing different ORs in the developing olfactory epithelium.     

Linking integrin to IP(3) signaling is important for ovulation in Caenorhabditis elegans

Signals from germ and myoepithelial sheath cells initiate ovulation in Caenorhabditis elegans. The coordinated dilation and contraction of spermatheca lead to subsequent fertilization of oocyte. Either the dominant negative mutant pat-3 beta integrin or disruption of talin expression block ovulation. Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans. Role of beta pat-3 integrins in development and function of Caenorhabditis elegans muscles and gonads, suggesting that the interaction between the cell and the extracellular matrix (ECM) is also important for ovulation. Here, we report that integrin plays an essential role in fertility via IP(3) signaling. Sterility caused by RNAi of pat-3 and ECM molecules was suppressed by increased IP(3) signaling. Our data suggest that the cell-ECM interaction controls ovulation via IP(3) signaling.