Guanosine 3',5'-cyclic monophosphate reduces the response of the Moth's olfactory receptor neuron to pheromone

The effects of the membrane-permeable dibutyryl guanosine 3', 5'-cyclic monophosphate (db-cGMP) on the bombykol-elicited receptor current and nerve impulse activity were studied using the open sensillum recording technique. db-cGMP was applied to the outer dendritic membrane of the olfactory receptor neuron of the moth Bombyx mori. db-cGMP reduced the amplitude of the overall receptor current activated by a pulse of strong pheromone stimuli as well as diminished the nerve impulse frequency elicited by continuously applied weak pheromone stimuli. The observed inhibition of the response to pheromone was due to size reduction of an elementary receptor current that elicits the nerve impulses and underlies the overall receptor current. It is suggested that cGMP is a factor which may adjust cell sensitivity to odour and play a role in olfactory adaptation.     

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.     

Background odour induces adaptation and sensitization of olfactory receptors in the antennae of houseflies

The presence of background odour was found to have a small but significant effect on the sensitivity of the antennal olfactory system of houseflies, Musca domestica Linnaeus (Diptera: Muscidae), to new pulses of odour. We show that cross-adaptation and cross-sensitization between a background odour of (+/-)-1-octen-3-ol and pulses of (+/-)-1-octen-3-ol, 2-pentanone and R-(+)-limonene can occur, confirming that olfactory receptor cells are sensitive to different odours. Background odour can increase the responses to low concentration odour pulses and decrease the responses to higher concentration odour pulses. It is suggested that background odour has a larger effect on olfactory receptor cells that respond with a tonic increase of spike frequency, giving information about the level of odour concentration, i.e. the 'static' environment. Cells that respond in a phasic way only provide information on the dynamics of the olfactory environment.     

Olfactory receptor cell activity under electrical polarization of the nasal mucosa in the frog. II. Responses to odour stimulation

The interactions between electrical polarizations of the olfactory epithelium and odour stimulations were investigated at the level of the extracellular spike activity of the receptor cells in the frog. 1. In most cases, surface positive polarizations enhanced the excitatory olfactory responses, negative polarizations suppressed these responses; both interactive effects were graded. 2. The response of receptor cells to electrical polarization was markedly reduced or suppressed for several seconds following olfactory stimulation. This effect and the time course of the recovery period depended on the nature and the concentration of the olfactory stimulus. 3. The decrease in electrical excitability seemed to be independent of whether the recorded neuron had responded or not to the prior olfactory stimulation. 4. It is suggested that the olfactory stimulation caused the total constant current to change its distribution in the different cell pathways. Changes in conductance induced by olfactory stimuli could implicate the supporting cells. 5. The experimental findings are discussed with reference to a model of receptor cell function that assumes a deep, axo-somatic localization of the action potential trigger-zone.     

Ca2+-activated K+ currents regulate odor adaptation by modulating spike encoding of olfactory receptor cells

The olfactory system is thought to accomplish odor adaptation through the ciliary transduction machinery in olfactory receptor cells (ORCs). However, ORCs that have lost their cilia can exhibit spike frequency accommodation in which the action potential frequency decreases with time despite a steady depolarizing stimulus. This raises the possibility that somatic ionic channels in ORCs might serve for odor adaptation at the level of spike encoding, because spiking responses in ORCs encode the odor information. Here I investigate the adaptational mechanism at the somatic membrane using conventional and dynamic patch-clamp recording techniques, which enable the ciliary mechanism to be bypassed. A conditioning stimulus with an odorant-induced current markedly shifted the response range of action potentials induced by the same test stimulus to higher concentrations of the odorant, indicating odor adaptation. This effect was inhibited by charybdotoxin and iberiotoxin, Ca2+-activated K+ channel blockers, suggesting that somatic Ca2+-activated K+ currents regulate odor adaptation by modulating spike encoding. I conclude that not only the ciliary machinery but also the somatic membrane currents are crucial to odor adaptation.     

Range and Absolute Odour Intensity Affect Olfactory Generalisation in the Honeybee

The human olfactory psychophysical literature is rich with anecdotal reports of variation in the perceived quality between weak and strong concentrations of the same odour (Wilson and Stevenson 2006). Psychophysical experiments using animals have also found similar effects of concentration on odour quality. The proboscis extension reflex (PER) is an appetitive Pavlovian conditioning assay that has been used to investigate olfactory stimuli in the honeybee. In this series of experiments I aim to identify the sensitivity of honeybees to changes in odorant concentration across the range of a honeybee’s sensorium (0.01–100 Pa). I compared generalisation to test odours that differed in molecular identity, odour concentration or both and found that large changes in odorant concentration (1,000 fold change) can produce greater shifts in perceptual similarity than a change in the odorant’s molecular structure. Our findings suggest a failure in concentration invariance when identical odours differ greatly in concentration. I also found poorer olfactory sensitivity (between identical odours of differing concentration) and acuity (between novel odours of identical concentration) at low odour concentrations (0.01 Pa).     

Odour discrimination by frog olfactory receptors: a second study

Single unit activity of olfactory neuroreceptors was recordedin frogs. Stimulations with 20 pure chemicals delivered at knownconcentrations elicited excitatory and/or inhibitory responsesin 60 of the 76 recorded units. The responses exhibited varioustime patterns, partly depending on stimulus intensity. Longlasting after-effects were observed. Out of a total of 1520odour trials, 317 excited and 33 inhibited the cells, leadingto a receptor overall responsiveness of 23%. Various degreesof individual selectivity were encountered in the receptors;the greatest number responded to seven of the 20 odorants. Amarked tendency to stimulate the same receptors was observedfor several odorants. Three groups could be evidenced: benzene,anisole, dichlorobenzene and bromobenzene; camphor and cineole;tert-butyl alcohol, cyclohexanone and cyclohexanol. Fatty acidstended to be grouped. Sulphurous compounds elicited few responses,except tiophenol. Most of the neuroreceptors responded to odorantsbelonging to more than one odour group.     

Odorant-induced hyperpolarization and suppression of cAMP-activated current in newt olfactory receptor neurons

Although many studies have reported that odorants can elicit inhibitory responses as well as excitatory responses in vertebrate olfactory receptor neurons, the cellular mechanisms that underlie this inhibition are unclear. Here we examine the inhibitory effect of odorants on newt olfactory receptor neurons using whole cell patch clamp recording. At high concentrations, odorant stimulation decreased the membrane conductance and inhibited depolarization. Various odorants (anisole, isoamyl acetate, cineole, limonene and isovaleric acid) suppressed the depolarizing current in a dose-dependent manner. Furthermore, one odorant could suppress the depolarization caused by another odorant. The depolarization caused by isoamyl acetate was inhibited by anisole in cells that were excited by isoamyl acetate but not by anisole. Odorants were able to hyperpolarize cells that were depolarized by cAMP-induced conductance. Given that this inhibitory effect of odorants can affect excitation caused by other odorants, we suggest that it might play a role in coding odorants in olfactory receptor neurons.     

Biogenic amines modulate olfactory receptor neurons firing activity in Mamestra brassicae

The modulatory effects of the biogenic amines octopamine and serotonin on pheromonal receptor neurons of Mamestra brassicae were investigated. The responses to sex pheromone components of two cells types (A and B) in single male long sensilla trichodea were monitored. Cell types A and B do not respond to the same compound. The response of type A to a pulse of the major sex pheromone component increased 5 min after octopamine injection. Responses of type B to other odorants increased after 30 min. In the absence of any pheromone stimulation the background firing activity of type A increased following octopamine injection. This background activity was used to evaluate the kinetics of octopamine and other biogenic amine effects on olfactory receptor neurons. Octopamine increased this background activity in a concentration- and time-dependent manner. Clonidine, an octopamine agonist, was shown to be more powerful in increasing the background activity of olfactory receptor neurons. The effects of octopamine and clonidine were hypothesized to arise from specific receptor activation as chlorpromazine (an octopamine antagonist) was shown to block the effect of octopamine. Serotonin, a known neuromodulator in most animal species, induced a reversible inhibition of spike firing. Altogether, these results indicate that biogenic amines can modulate the sensitivity of olfactory receptor neurons of moths either directly or by an action on adaptation.     

Electro-olfactogram and multiunit olfactory receptor responses to binary and trinary mixtures of amino acids in the channel catfish, Ictalurus punctatus

In vivo electrophysiological recordings from populations of olfactory receptor neurons in the channel catfish, Ictalurus punctatus, clearly showed that responses to binary and trinary mixtures of amino acids were predictable with knowledge obtained from previous cross-adaptation studies of the relative independence of the respective binding sites of the component stimuli. All component stimuli, from which equal aliquots were drawn to form the mixtures, were adjusted in concentration to provide for approximately equal response magnitudes. The magnitude of the response to a mixture whose component amino acids showed significant cross-reactivity was equivalent to the response to any single component used to form that mixture. A mixture whose component amino acids showed minimal cross-adaptation produced a significantly larger relative response than a mixture whose components exhibited considerable cross-reactivity. This larger response approached the sum of the responses to the individual component amino acids tested at the resulting concentrations in the mixture, even though olfactory receptor dose-response functions for amino acids in this species are characterized by extreme sensory compression (i.e., successive concentration increments produce progressively smaller physiological responses). Thus, the present study indicates that the response to sensory stimulation of olfactory receptor sites is more enhanced by the activation of different receptor site types than by stimulus interaction at a single site type.     

The effect of concanavalin A on the rat electro-olfactogram at various odorant concentrations.

We have studied the effect of concanavalin A (Con A) on the rat electro-olfactogram response to several odorants. Each odorant was applied over a range of concentrations. For hydrophobic odorants whose response was affected by Con A, the diminution in response was maximal at odorant concentrations of about 1 microM in the olfactory mucus. The (odour) concentration-dependence of the change is compatible with the idea that Con A inactivates one or more types of olfactory receptor that normally bind odorants with dissociation constants of the order of 100 nM. With hydrophilic odorants we had to apply concentrations very much higher than this to elicit any response from the system. At these high concentrations we could observe Con A-induced diminutions in response.     

Calcium,the two-faced messenger of olfactory transduction and adaptation

Exposure of olfactory receptor cells to odour stimulates the influx of Ca(2+) through cyclic nucleotide-gated channels into the small volume within the cilia, the site of olfactory transduction. The consequent rise in intraciliary Ca(2+) concentration has two opposing effects: activation of an unusual excitatory Cl(-) conductance, and negative feedback actions on various stages of the odour transduction mechanism. Recent studies are beginning to unravel how Ca(2+) performs this dual function, and how the spatial and temporal dynamics of Ca(2+) modulate the odour response. The feedback actions of Ca(2+) on different elements of the transduction cascade seem to occur on different timescales, and are therefore responsible for shaping different parts of the receptor current response to odour stimulation.     

Olfactory CNG channel desensitization by Ca2+/CaM via the B1b subunit affects response termination but not sensitivity to recurring stimulation

Ca2+/calmodulin-mediated negative feedback is a prototypical regulatory mechanism for Ca2+-permeable ion channels. In olfactory sensory neurons (OSNs), such regulation on the cyclic nucleotide-gated (CNG) channel is considered a major mechanism of OSN adaptation. To determine the role of Ca2+/calmodulin desensitization of the olfactory CNG channel, we introduced a mutation in the channel subunit CNGB1b in mice that rendered the channel resistant to fast desensitization by Ca2+/calmodulin. Contrary to expectations, mutant OSNs showed normal receptor current adaptation to repeated stimulation. Rather, they displayed slower response termination and, consequently, reduced ability to transmit olfactory information to the olfactory bulb. They also displayed reduced response decline during sustained odorant exposure. These results suggest that Ca2+/calmodulin-mediated CNG channel fast desensitization is less important in regulating the sensitivity to recurring stimulation than previously thought and instead functions primarily to terminate OSN responses.     

Electrophysiological responses of olfactory bulb neurons to odour stimuli in the frog. A comparison with receptor cells

Extracellular recordings were performed from olfactory bulbneurons in the frog. The odour stimuli were the same as thosepreviously used for studying the receptor cells in the sameanimal species and were delivered at similar concentrations(Revial et al., 1982). The general properties of the neuronresponses are presented and discussed with reference to homologousproperties of olfactory receptor cells. The response rates elicitedby different stimuli from the bulbar neurons were found to behighly correlated with those elicited from receptor cells. Theindividual cell selectivity was better in the bulb than in theolfactory epithelium. The olfactory bulb neurons seemed to improvethe discrimination between stimuli (enantiomers) poorly distinguishedby the receptor cells. Reducing odor concentration caused therate of suppressive response to decrease faster than that ofexcitatory ones, suggesting that the manifestations of inhibitoryprocesses in some neurons requires a high level of excitationin others.     

Perception of plant volatile blends by herbivorous insects--finding the right mix

Volatile plant secondary metabolites are detected by the highly sensitive olfactory system employed by insects to locate suitable plants as hosts and to avoid unsuitable hosts. Perception of these compounds depends on olfactory receptor neurones (ORNs) in sensillae, mostly on the insect antennae, which can recognise individual molecular structures. Perception of blends of plant volatiles plays a pivotal role in host recognition, non-host avoidance and ensuing behavioural responses as different responses can occur to a whole blend compared to individual components. There are emergent properties of blend perception because components of the host blend may not be recognised as host when perceived outside the context of that blend. Often there is redundancy in the composition of blends recognised as host because certain compounds can be substituted by others. Fine spatio-temporal resolution of the synchronous firing of ORNs tuned to specific compounds enables insects to pick out relevant host odour cues against high background noise and with ephemeral exposure to the volatiles at varying concentrations. This task is challenging as they usually rely on ubiquitous plant volatiles and not those taxonomically characteristic of host plants. However, such an odour coding system has the advantage of providing flexibility; it allows for adaptation to changing environments by alterations in signal processing while maintaining the same peripheral olfactory receptors.     

Differential dephosphorylation of the insulin receptor and its 160-kDa substrate (pp160) in rat adipocytes.

A permeabilized rat adipocyte model was developed which permitted an examination of: 1) insulin receptor autophosphorylation, 2) phosphorylation of a putative insulin receptor substrate of 160 kDa, pp160, and 3) the dephosphorylation reactions associated with each of these phosphoproteins. Rat adipocytes, preincubated with [32P]orthophosphate for 2 h, were exposed to insulin (10(-7) M) at the time of digitonin permeabilization. Phosphorylation of pp160 and autophosphorylation of the insulin receptor increased as a function of Mn2+ concentration in the media with near maximum responses at 10 mM. Maximum response was at least as large as the intact cell response to 10(-7) M insulin. In contrast, magnesium did not increase phosphorylation of pp160 although an increase in receptor autophosphorylation was observed. Autophosphorylation was preserved at digitonin concentrations of 20-100 micrograms/ml, but pp160 phosphorylation was negligible beyond 40 micrograms/ml. Our previous work demonstrated that the insulin receptor was associated with a phosphotyrosine phosphatase activity in permeabilized adipocytes (Mooney, R., and Anderson, D. (1989) J. Biol. Chem. 264, 6850-6857). The current permeabilized adipocyte model made possible an examination of the effects of phosphotyrosine phosphatase inhibitors, including several divalent metal cations (Zn2+, Co2+, and Ni2+), vanadate, and molybdate on both net phosphorylation of pp160 and autophosphorylation of the insulin receptor. Zn2+ at 100 microM, Ni2+ at 1 mM, and Co2+ at 1 or 5 mM increased insulin-dependent phosphorylation of pp160 at least 5-fold and autophosphorylation 2-fold. At higher concentrations of Zn2+ (1 mM) and Ni2+ (5 mM), however, no increase in phosphorylation of pp160 was observed and autophosphorylation was inhibited. Vanadate (1 mM) and molybdate (100 microM) increased insulin-dependent phosphorylation of pp160 by 3-fold when tested separately and 7-fold in combination. Insulin receptor autophosphorylation was increased 50% by each and 3-fold when the agents were combined. Dephosphorylation of pp160 and the insulin receptor was analyzed directly by permeabilizing prelabeled insulin-treated adipocytes in the presence of EDTA (10 mM). Dephosphorylation of pp160 was especially rapid with a t1/2 of approximately 10 s. The t1/2 for the insulin receptor was 37 s. Zn2+ at 1 mM (a concentration that inhibited the insulin receptor kinase) was a strong inhibitor of dephosphorylation, prolonging the rate of pp160 dephosphorylation more than 12-fold and insulin receptor dephosphorylation 3-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

The electrochemical basis of odor transduction in vertebrate olfactory cilia

Most vertebrate olfactory receptor neurons share a common G-protein-coupled pathway for transducing the binding of odorant into depolarization. The depolarization involves 2 currents: an influx of cations (including Ca2+) through cyclic nucleotide-gated channels and a secondary efflux of Cl- through Ca2+-gated Cl- channels. The relation between stimulus strength and receptor current shows positive cooperativity that is attributed to the channel properties. This cooperativity amplifies the responses to sufficiently strong stimuli but reduces sensitivity and dynamic range. The odor response is transient, and prolonged or repeated stimulation causes adaptation and desensitization. At least 10 mechanisms may contribute to termination of the response; several of these result from an increase in intraciliary Ca2+. It is not known to what extent regulation of ionic concentrations in the cilium depends on the dendrite and soma. Although many of the major mechanisms have been identified, odor transduction is not well understood at a quantitative level.     

Sequence matters – selective adaptation in electroantennographic response to binary odour mixtures by the Colorado potato beetle

Chemically mediated behaviour of insects is often strongly affected by mixtures of odour stimuli and their temporal characteristics. Both sensory transduction and central processing of odour mixtures can give rise to several different kinds of interaction, which can influence how individual components are perceived and processed. In particular, odour mixtures have been examined in model experiments as premixed binary mixtures in comparison with pure odour stimuli. Only in few experiments, the influence of the temporal structure of odour mixtures on odour perception has been taken into account. Natural odour stimuli often have a pulsed structure and may in general be superimposed on a background of irrelevant or interfering compounds, which can fluctuate with different frequencies, depending on their source. To achieve a better representation of these natural conditions, our odour mixing experiments apply a new kind of stimulation protocol: odours were not premixed but superimposed with a specific time pattern; one odour stimulus was presented as a longer persisting background and the second stimulus was a superimposed short test signal. To gain an overview of odour interaction patterns in the Colorado potato beetle by causing adaptation of one receptor population at naturally occurring levels of concentration and time intervals, electroantennographic recordings were made on excised antennae. A matrix of 12 stimulus compounds led to 132 pairs of compounds tested, each in the role of background and test stimulus. In 64 cases, the interaction was significantly different, when the role of background and stimulus was exchanged. Interaction patterns ranging from no interference (independence) to suppression were found and assigned to four clearly distinguishable types. We discuss that the observed effects of the presentation sequence in odour mixtures may contribute to the mechanisms of olfactory pattern recognition and olfactory contrast perception by insects.     

Influence of stimulus intensity on odour discrimination by olfactory bulb neurons as compared with receptor cells

Previously reported electrophysiological responses recordedfrom individual neurons in the olfactory bulb of frogs stimulatedwith odorous compounds were further analyzed using statisticalmethods. Five of the odorants were delivered at two concentrations.The pattern of discrimination among these odorants was investigatedwith the aid of the Pearson's correlation test and Benzecri's‘analyse des correspondances’. Special attentionwas paid to the incidence of odour concentration on this discriminationpattern. The results were compared with those of a similar studyperformed on receptor cells in the same experimental conditions.The comparison indicated that the information processing inthe olfactory bulb seems to improve discrimination between chemicallydifferent stimuli, especially those poorly discriminated byreceptor cell responses, whereas it protects this discriminationagainst a massive influence of the intensity of the stimuli.     

Neurophysiological responses of pheromone-sensitive receptor neurons on the antenna of Trichoplusia ni (Hubner) to pulsed and continuous stimulation regimens

Both the frequency and the temporal pattern of action potentialproduction in an insect olfactory receptor neuron are stronglyaffected by odorant composition and the time course over whichstimulus concentration varies. To investigate the temporal characteristicsof the neurophysiological responses of these neurons, we deviseda stimulus delivery system that allows us to repeatedly presentwell-mixed, constant concentration odor pulses with relativelysharp onsets and offsets. Here we compare neurophysiologicalresponses to several different stimulation regimens, includingpulses of different durations and repetition rates. During stimulationwith high concentrations of pheromone, the temporal patternof neural activity from olfactory receptor neurons on the antennaof Trichoplusia ni (Hübner) is characterized by an initialphasic period (100–200 ms), followed by a tonic periodwhich is typically maintained for the remaining duration ofthe stimulus. Different olfactory receptor neurons appear tovary among themselves in the relative distribution between thephasic and tonic portions of the overall discharge. During stimulationregimens involving rapid repeated pulses of odorants, a portionof the phasic response levels is preserved during each pulse.Consequently, T. ni males probably detect much of the fluctuationin concentration of pheromone that may normally occur downwindfrom the site of pheromone release.