Specificity of odorant-evoked inhibition in lobster olfactory receptor neurons

Lobster olfactory receptor neurons, like those of many animals, use two modes of olfactory signaling, excitation and inhibition to code olfactory information. Inhibition appears to act through two distinct ionic mechanisms. Here we show that neither ionic mechanism is odor-specific, providing further support for the emerging understanding that there are no inhibitory odorants per se, but rather that the action of a particular odorant is inherent in the olfactory receptor cell on which an odorant acts.     

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.     

Elements of the olfactory signaling pathways in insect antennae

Owing to their enormous ability to recognize airborne molecules, insects have long been used as model systems for studying various aspects of olfaction. Modern biological techniques have opened new avenues for exploring the molecular mechanisms underlying the complex signaling processes in chemosensory neurons. Biochemical and molecular analyses have allowed the identification of molecular elements of the olfactory reaction pathways and have shed light on mechanisms that account for the sensitivity and specificity of the chemosensory system.     

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).     

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     

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.     

Olfactory marker protein (OMP) exhibits a beta-clam fold in solution: implications for target peptide interaction and olfactory signal transduction

Olfactory marker protein (OMP) is a ubiquitous, cytoplasmic protein found in mature olfactory receptor neurons of all vertebrates. Electrophysiological and behavioral studies demonstrate that it is a modulator of the olfactory signal transduction pathway. Here, we demonstrate that the solution structure of OMP, as determined by NMR studies, is a single globular domain protein comprised of eight beta-strands forming two beta-sheets oriented orthogonally to one another, thus exhibiting a "beta-clam" or "beta-sandwich" fold: beta-sheet 1 is comprised of beta3-beta8-beta1-beta2 and beta-sheet 2 contains beta6-beta5-beta4-beta7. Insertions include two, long alpha-helices located on opposite sides of the beta-clam and three flexible loops. The juxtaposition of beta-strands beta6-beta5-beta4-beta7-beta2-beta1-beta8-beta3 forms a continuously curved surface and encloses one side of the beta-clam. The "cleft" formed by the two beta-sheets is opposite to the closed end of the beta-clam. Using a peptide titration series, we have identified this cleft as the binding surface for a peptide derived from the Bex1 protein. The highly conserved Omega-loop structure adjacent to the Bex1 peptide-binding surface found in OMP may be the site of additional OMP-protein interactions related to its role in modulating olfactory signal transduction. Thus, the interaction between the OMP and Bex1 proteins could facilitate the interaction between OMP and other components of the olfactory signaling pathway.     

ROS perception in Arabidopsis thaliana: the ozone-induced calcium response

Ozone is responsible for more crop losses than any other air pollutant. The changes in gene expression, which occur in plants in response to ozone, have been well characterized, yet little is known about how ozone is perceived or the signal transduction steps that follow. The earliest characterized response to ozone is an elevation in cytosolic-free calcium, which takes place within seconds of exposure. In this study, the calcium response to ozone was investigated in Arabidopsis thaliana seedlings using a variety of fumigation protocols. Ozone elicited distinct calcium responses in the aerial tissue and roots of seedlings. The calcium response in the cotyledons and leaves was biphasic and sensitive to the rate at which the ozone concentration increased. The response in the root was monophasic and insensitive to the rate of increase in ozone concentration. Experiments utilizing inhibitors of antioxidant metabolism demonstrated that the magnitude of the first peak in calcium in the aerial tissues was dependent upon the redox status of the plant. Seedlings were shown to be able to distinguish between ozone and hydrogen peroxide, producing a calcium signal in response to one of these reactive oxygen species (ROS) when they had become refractory to the other. Pre-treatment with ozone altered the calcium response to hydrogen peroxide and vice versa, indicating that the calcium response to a given ROS may reflect the stress history of the plant. These data suggest ROS signalling is more sophisticated than previously realized and raise questions over current models of ozone perception.     

Flexible categorization in the mouse olfactory bulb

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Origin of basal activity in mammalian olfactory receptor neurons

Mammalian odorant receptors form a large, diverse group of G protein-coupled receptors that determine the sensitivity and response profile of olfactory receptor neurons. But little is known if odorant receptors control basal and also stimulus-induced cellular properties of olfactory receptor neurons other than ligand specificity. This study demonstrates that different odorant receptors have varying degrees of basal activity, which drives concomitant receptor current fluctuations and basal action potential firing. This basal activity can be suppressed by odorants functioning as inverse agonists. Furthermore, odorant-stimulated olfactory receptor neurons expressing different odorant receptors can have strikingly different response patterns in the later phases of prolonged stimulation. Thus, the influence of odorant receptor choice on response characteristics is much more complex than previously thought, which has important consequences on odor coding and odor information transfer to the brain.     

Spontaneous calcium transients regulate neuronal plasticity in developing neurons

Calcium ions play critical roles in neuronal differentiation. We have recorded transient, repeated elevations of calcium in embryonic Xenopus spinal neurons over periods of 1 h in vitro and in vivo, confocally imaging fluo 3-loaded cells at 5 s intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a double exponential decay, whereas waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura 2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease, whereas the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials and also utilize intracellular calcium stores. Waves are produced by a mechanism that does not involve classic voltage-dependent calcium channels. Spikes are required for expression of the transmitter GABA and for potassium channel modulation. Waves in growth cones are likely to regulate neurite extension. The results demonstrate the roles of a novel signaling system in regulating neuronal plasticity, that operates on a time scale 10⁴ times slower than that of action potentials. © 1995 John Wiley & Sons, Inc.     

Evidence for glutamate-mediated activation of hippocampal neurons by glial calcium waves

Communication from astrocytes to neurons has recently been reported by two laboratories, but different mechanisms were thought to underlie glial calcium wave activation of associated neurons. Neuronal calcium elevation by glia observed in the present report is similar to that reported previously, where an increase in neuronal calcium was demonstrated in response to glial stimulation. In the present study hippocampal neurons plated on a confluent glial monolayer displayed a transient increase in intracellular calcium following a short delay after the passage of a wave of increased calcium in underlying glia. Activated cells displayed action potentials in response to glial waves and showed antineurofilament immunoreactivity. Finally, the N-methyl-D -aspartate glutamate receptor antagonist DL -2-amino-5-phosphonovaleric acid and the non-NMDA glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione significantly reduced the responsiveness of neurons to glial calcium waves. Our results indicate that hippocampal neurons growing on hippocampal or cortical astrocytes respond to glial calcium waves with elevations in calcium and increased electrical activity. Furthermore, we show that in most cases this communication appears to be mediated by ionotropic glutamate receptor channels. © 1995 John Wiley & Sons, Inc.     

Ca2+ stabilizes the membrane potential of moth olfactory receptor neurons at rest and is essential for their fast repolarization

The role of Ca(2+) in insect olfactory transduction was studied in the moth Spodoptera littoralis. Single sensillum recordings were made to investigate in vivo the role of sensillar Ca(2+) on the electrophysiological properties of sex pheromone responsive olfactory receptor neurons (ORNs). Lowering the sensillar Ca(2+) concentration to 2 x 10(-8) M increased ORN spontaneous firing activity and induced long bursts of action potentials (APs) superimposed on spontaneous negative deflections of the transepithelial potential. We inferred that Ca(2+) stabilizes the membrane potential of ORNs, keeping the spontaneous firing activity at a low and regular level. Neither the amplitude and kinetics of the rising phase of sensillar potentials (SPs) recorded in response to pheromone stimuli nor the AP generation during stimulation depended on the extracellular Ca(2+) concentration. Thus, extracellular Ca(2+) is not absolutely necessary for ORN response. Partial inhibition of responses with a calmodulin antagonist, W-7, also indicates that intracellular Ca(2+) contributes to the ORN response and suggests that Ca(2+) release from internal stores is involved. In 2 x 10(-8) M Ca(2+), the repolarization of the SP was delayed when compared with higher Ca(2+) concentrations. Therefore, in contrast to depolarization, ORN repolarization depends on extracellular Ca(2+). Ca(2+)-gated K(+) channels identified from cultured ORNs with whole-cell recordings are good candidates to mediate ORN repolarization.     

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     

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.     

Intracellular calcium mobilization and neurite outgrowth in mammalian neurons

Cultured adult rat dorsal root ganglion (DRG) neurons were used to study depolarization-induced Ca²⁺ mobilization and the effects of intracellular Ca²⁺ depletion on neurite outgrowth. Cytoplasmic and nuclear Ca²⁺ signals were visualized in dissociated DRG neurons using confocal scanning laser microspcopy and the Ca²⁺ indicator dye fluo-3. The depolarization-induced Ca²⁺ signals were highest in neurons during the first few days in culture, prior to neurite extension; during this time nuclear signals exceeded those of the cytoplasm severalfold. After several days in culture, neurons began to arborize, depolarization-induced Ca²⁺ signals became attenuated, and nuclear signals no longer exceeded those of the cytoplasm. Elevated Ca²⁺ signals were dependent upon both Ca²⁺ influx and intact intracellular Ca²⁺ stores, indicating that the signals are generated by calcuim-induced calcium release (CICR). Thapsigargin, an endoplasmic reticulum Ca²⁺ ATPase inhibitor, depleted intracellular Ca²⁺ stores and blocked the induction of the large nuclear Ca²⁺ signals. Treating DRG neurons briefly with thapsigargin (200 nM for 20 min) shortly after plating reduced subsequent neuritogenesis, impyling that intact Ca²⁺ stores are necessery for initiating neurite outgrowth. Immunostaining of DRG neurons with antibodies to Ca²⁺ /calmodulin-dependent kinase II (CaM kinase II) demonstrated that this enzyme is present in the nucleus at early times in culture. These observations are consistent with the idea that CICR triggered by Ca²⁺ entry subsequent to depolarization may elicit neurite outgrowth by activating nuclear enzymes appropriate for such outgrowth. © 1994 John Wile & Sons, Inc.     

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.