A CUB-serine protease in the olfactory organ of the spiny lobster Panulirus argus.

csp, a gene encoding a protein with high sequence identity to trypsinlike serine protease and CUB domains, was identified from a cDNA library from the olfactory organ (antennular lateral flagellum) of the spiny lobster Panulirus argus. The full-length cDNA sequence of csp is 1801 bp, encoding a protein of 50.25 kD, with three domains: signal peptide, trypsinlike serine protease, and CUB (named for a class of compounds including Complement subcomponents Clr/Cls, Uegf, and Bone morphogenic protein-1). RT-PCR, Northern blots, and immunoblots showed that csp is predominantly expressed in the lateral flagellum and eyestalk. Immunocytochemistry showed that Csp is present in olfactory (aesthetasc) sensilla around auxiliary cells (glia that surround the inner dendrites of olfactory receptor neurons, ORNs) and ORN outer dendrites. We propose that Csp is expressed and secreted by auxiliary cells, associates with ORN cell membranes or extracellular matrix via the CUB domain, and has trypsinlike activity. In the eyestalk, Csp is associated with cells surrounding axons between neuropils of the eyestalk ganglia. Possible functions in the olfactory organ and eyestalk are discussed. To our knowledge, this is the first report from any olfactory system of a gene encoding a protein with serine protease and CUB domains.

Morphology and distribution of setae on the antennules of the Caribbean spiny lobster Panulirus argus reveal new types of bimodal chemo-mechanosensilla.

This study describes the morphology and distribution of setae on the lateral and medial flagella of the antennules of the spiny lobster Panulirus argus in an effort to identify antennular chemoreceptors in addition to the well-studied aesthetasc chemosensilla. Setae were examined using light and electron microscopy, and their distribution on flagellar annuli was analyzed. We identified ten setal types based on external morphology: hooded, plumose, short setuled, long simple, medium simple, short simple, aesthetasc, guard, companion, and asymmetric setae, with the last four types being unique to the "tuft" located on the distal half of the lateral flagellum. The three setal types whose ultrastructure was examined--hooded, long simple, and medium simple setae--had characteristics of bimodal (chemo-mechanoreceptive) sensilla. The antennules have four distinct annular types based on their setal complement, as shown by cluster analysis. This basic distribution of non-tuft setal types is similar for both lateral and medial flagella. Annuli in the tuft region have tuft setal types superimposed on a basic organization of non-tuft setal types. These results show that the antennules possess a diverse set of setae, that these setae have a highly ordered arrangement on the antennules, that at least four (and probably many more) of these setal types are chemosensilla, and suggest that most antennular chemosensilla are bimodally sensitive.

Postembryonic proliferation in the spiny lobster antennular epithelium: rate of genesis of olfactory receptor neurons is dependent on molt stage.

Olfactory systems undergo continuous growth and turnover in many animals. Many decapod crustaceans, such as lobsters and crayfish, have indeterminate growth, and in these animals, turnover of both peripheral and central components of the olfactory system occurs continuously throughout life. In this study, we examine the dynamics of olfactory receptor neuron (ORN) proliferation in the antennule of the Caribbean spiny lobster, Panulirus argus, using in vivo incorporation of the cell proliferation marker BrdU. We show that addition of ORNs occurs in a "proximal proliferation zone" (PPZ), which exists on the proximo-lateral margin of the existing ORN population. The PPZ is spatially and temporally dynamic in that it travels as a wave in the proximal and lateral directions in the antennule. This wave results in continuous addition of ORNs throughout the molt cycle. The rate of proliferation, as measured by the size and shape of the PPZ, changes depending on the animal's molt stage. The rate is highest during premolt and lowest during intermolt. ORNs are the most prominent cell-type produced in the PPZ, but other cell types, including glia, are also produced. Patches of proliferating epithelial cells occur immediately proximal to the PPZ, suggesting that neuronal and glial precursors reside in this region. Possible mechanisms for peripheral and central modulation of ORN development are discussed.

Selective ablation of antennular sensilla on the Caribbean spiny lobster Panulirus argus suggests that dual antennular chemosensory pathways mediate odorant activation of searching and localization of food.

In spiny lobsters and other decapod crustaceans, odorant-mediated searching behavior patterns are driven primarily by chemosensory neurons in the antennules. Two groups of antennular chemosensory neurons can be distinguished on the basis of the sensilla that they innervate and their central projections: those that innervate the aesthetasc sensilla on the lateral flagella and project into the glomerularly organized olfactory lobes, and those that innervate other (i.e. non-aesthetasc) sensilla on both lateral and medial flagella and project into the stratified and non-glomerularly organized lateral antennular neuropils. By ablating different groups of antennular sensory neurons or sensilla, we examined the role of aesthetasc and non-aesthetasc chemosensory neurons in regulating local searching behavior of Caribbean spiny lobsters, Panulirus argus, for food (squid) in a low-flow environment. The results show that odorant-mediated activation of searching and localization of food under these conditions requires only a subset of functional antennular chemosensory neurons, since neither aesthetasc chemosensory neurons nor non-aesthetasc chemosensory neurons are by themselves necessary for these types of behavior. However, ablation of aesthetasc chemosensory neurons together with subsets of non-aesthetasc chemosensory neurons from either the medial or lateral flagella impairs the ability of lobsters to locate the food. This reveals a large degree of functional redundancy but also some complementary functions between aesthetasc and non-aesthetasc chemosensory neurons, and hence between these dual antennular chemosensory pathways, in odorant-mediated searching behavior of lobsters under these conditions.

Learning from spiny lobsters about chemosensory coding of mixtures.

Studies of the peripheral olfactory system of the Caribbean spiny lobster Panulirus argus and related decapod crustaceans have helped us understand mechanisms of coding of mixtures, some of which are discussed in this review. Although the number of cells in the lobster's olfactory system is much lower than in vertebrate olfactory systems, it is a highly complex system. The receptor neurons (RNs) of this olfactory system are complex processors that cannot be categorized into discrete cell types, but rather have a diversity of response profiles. Each RN can have different types of receptor proteins, second messengers, and/or ion channels, which undoubtedly contributes to the functional diversity of these neurons and makes them complex peripheral integrators. The RNs probably encode information about the quality of mixtures as a distributed or population code, providing a basis for behavioral discrimination of natural food stimuli. Analysis of distributed codes for a series of blend ratios of binary mixtures reveals that the qualities of individual compounds are probably not lost when mixed. Such peripheral processing allows spiny lobsters to perceive complex odors as a set of elemental cues if the salience of the components is sufficiently high.

A spatiotemporal wave of turnover and functional maturation of olfactory receptor neurons in the spiny lobster Panulirus argus.

Olfactory receptor neurons (ORNs) of crustaceans are housed in aesthetasc sensilla that are located on the lateral flagellum of the antennule. We used young adult spiny lobsters to examine turnover of aesthetascs and functional maturation of their ORNs after molting. The proliferation zone for new aesthetascs is located in the proximal part of the aesthetasc-bearing region and progressively moves along a distoproximal axis. Older aesthetascs are lost in the distal part of the aesthetasc-bearing region. As a result, an aesthetasc may be shed three to six molts after it differentiates. Taurine-like immunoreactivity is elevated in ORNs of aesthetascs that have yet to emerge on the cuticular surface and thereafter decreases gradually and asynchronously. ORNs from the distalmost-developing aesthetascs lose taurine-like immunoreactivity immediately before sensillar emergence, whereas ORNs from the most proximal and lateral new aesthetascs retain taurine-like immunoreactivity throughout the intermolt stage after sensillar emergence. Furthermore, taurine-like immunoreactivity is inversely correlated with odor responsiveness. These results suggest that taurine-like immunoreactivity reveals immature ORNs and that their functional maturation is not synchronized with molting and may not be completed until many weeks after sensillar emergence. Our data suggest successive spatiotemporal waves of birth, differentiation and functional maturation, and death of ORNs.

Functional units of a compound nose: aesthetasc sensilla house similar populations of olfactory receptor neurons on the crustacean antennule.

The lateral flagellum of the antennule of the spiny lobster Panulirus argus houses more than 1,000 morphologically similar olfactory sensilla, called aesthetascs. By using a high-resolution activity labeling technique that depends on entry of agmatine into olfactory receptor neurons (ORNs) through cation channels during odor stimulation, we examined the distribution of different functional types of ORNs within and across mature aesthetascs. A significant number of ORNs in mature aesthetascs are labeled with agmatine during stimulation by single odorants, including adenosine-5'-monophosphate, ammonium chloride, cysteine, glycine, proline, and taurine. The percentage of ORNs per aesthetasc that was agmatine labeled during odor stimulation averaged 0.5-1.6% for single compounds and 4.6% for a 33-component mimic of oyster tissue. For most antennules and antennular regions studied, the percentage of agmatine-labeled ORNs by stimulation with single or complex odorants was statistically homogeneous across most or all aesthetascs. The extent of heterogeneity among mature aesthetascs was correlated with their age: extensive heterogeneity was observed only in the distal part of the flagellum containing the oldest aesthetascs and their ORNs. Thus, it appears that over most of the length of the aesthetasc-bearing region of the lateral flagellum, different and distinct functional types of aesthetascs do not exist. Rather, aesthetascs appear to be repetitive morphological and functional units in olfactory coding. However, because odor sensitivity of ORNs can change with the age of an aesthetasc, some development-related functional heterogeneity exists among aesthetascs.

High-resolution functional labeling of vertebrate and invertebrate olfactory receptor neurons using agmatine, a channel-permeant cation.

Methods are described for odor-stimulated labeling of olfactory receptor neurons (ORNs) of the freshwater zebrafish Danio rerio and the marine spiny lobster Panulirus argus. Permeation of a cationic molecule, 1-amino-4-guanidobutane ( = agmatine, AGB), through ion channels following odor stimulation, and its detection by an anti-AGB antibody, allow labeling of odor-stimulated ORNs. Parameters adjusted to optimize activity-dependent labeling included labeling medium ionic composition, stimulation times, and AGB concentration. For lobsters, 7% of ORNs were labeled by a complex odor, oyster mixture, under optimal conditions, which was stimulation for 5 s per min for 60 min with 20 mM AGB in artificial seawater with reduced sodium and calcium concentrations. AGB was a weak odorant for lobsters; it elicited only a small electrophysiological response from ORNs and labeled < 1% of the ORNs during stimulation with AGB in the absence of odors. For the zebrafish, stimulation for 10 s per min for 10 min with 5 mM AGB plus odorant (L-glutamine) in fish Ringer's solution was the optimal labeling condition, resulting in labeling of 17% of the olfactory epithelial area. Approximately 6% of the olfactory epithelium was labeled during stimulation with a control stimulus, AGB alone. This labeling by AGB alone suggests it is an olfactory stimulus for zebrafish; a conclusion supported by electrophysiological recordings. We used electrophysiological assays and channel blockers to examine, for each species, potential ion channels for entry of AGB into ORNs. These results show that AGB can be used as an activity-dependent label for chemoreceptor neurons of diverse phyla living in a range of environmental conditions.

Inhibitory receptor binding events among the components of complex mixtures contribute to mixture suppression in responses of olfactory receptor neurons of spiny lobsters.

Responses of olfactory receptor neurons of spiny lobsters Panulirus argus to two-component mixtures can be shaped by inhibitory events such as odor-activated hyperpolarizations and inhibition of odor-receptor binding (Daniel et al. 1996). In the current study, we extend this analysis to complex mixtures by examining responses of spiny lobster olfactory receptor neurons to mixtures containing up to seven odorants, consisting of adenosine-5'-monophosphate, ammonium, betaine, L-cysteine, L-glutamate, DL-succinate, and taurine. The response to a mixture was often less than the response to its most excitatory component. The effect of adding an excitatory odorant to a mixture depended on olfactory receptor neuron type, composition of the mixture, and which compound was added. In some cases the added excitatory compound had no effect or even decreased the mixture's response intensity, thus demonstrating nonlinear contributions of the components. Response intensities predicted by a noncompetitive model, which is most representative of these olfactory receptor neurons, were improved when the model included a term for empirical measurements of inhibitory binding interactions, suggesting that inhibitory binding interactions are one mechanism contributing to mixture suppression. This model's predictions were accurate for binary mixtures but not for larger mixtures, suggesting that additional inhibitory mechanisms are needed to account for mixture interactions in complex mixtures.

Complex binding interactions between multicomponent mixtures and odorant receptors in the olfactory organ of the Caribbean spiny lobster Panulirus argus.

Our study was designed to examine how components of complex mixtures can inhibit the binding of other components to receptor sites in the olfactory system of the spiny lobster Panulirus argus. Biochemical binding assays were used to study how two- to six-component mixtures inhibit binding of the radiolabeled odorants taurine, L-glutamate and adenosine-5'-monophosphate to a tissue fraction rich in dendritic membrane of olfactory receptor neurons. Our results indicate that binding inhibition by mixtures can be large and is dependent on the nature of the odorant ligand and on the concentration and composition of the mixture. The binding inhibition by mixtures of structurally related components was generally predicted using a competitive binding model and binding inhibition data for the individual components. This was not the case for binding inhibition by most mixtures of structurally unrelated odorants. The binding inhibition for these mixtures was generally smaller than that for one or more of their components, indicating that complex binding interactions between components can reduce their ability to inhibit binding. The magnitude of binding inhibition was influenced more by the mixture's precise composition than by the number of components in it, since mixtures with few components were sometimes more inhibitory than mixtures with more components. These findings raise the possibility that complex binding interactions between components of a mixture and their receptors may shape the output of olfactory receptor neurons to complex mixtures.

Two novel types of L-glutamate receptors with affinities for NMDA and L-cysteine in the olfactory organ of the Caribbean spiny lobster Panulirus argus.

A subset of olfactory receptor neurons of the Caribbean spiny lobster Panulirus argus possesses receptors for L-glutamate that can mediate both excitatory and inhibitory responses (P.C. Daniel, M.F. Burgess, C.D. Derby, Responses of olfactory receptor neurons in the spiny lobster to binary mixtures are predictable using a non-competitive model that incorporates excitatory and inhibitory transduction pathways, J. Comp. Physiol. A 178 (1992) 523-536). In this study, we have used biochemical and electrophysiological techniques to understand the role of these receptors in olfactory transduction, and to compare these olfactory glutamate receptors with peripheral and central L-glutamate receptors in other animals. Using a radioligand-binding assay with a membrane-rich preparation from the dendrites of olfactory receptor neurons, we have identified two types of binding sites for L-glutamate. Both sites showed rapid, reversible, and saturable association with radiolabeled L-glutamate, and their Kd values (1 nM and 3 microM) are effective in physiological studies of glutamate-sensitive olfactory neurons, suggesting these binding sites are receptors involved in olfactory transduction. Both sites were completely inhibited by high concentrations of NMDA and L-cysteine, and only partially inhibited by other L-glutamate analogs and odorants. Electrophysiological recordings from L-glutamate-best olfactory receptor neurons showed that NMDA and L-cysteine are both partial agonists and antagonists of glutamate receptors. Together, these results suggest the olfactory L-glutamate receptors of spiny lobsters are novel types of L-glutamate receptors that are functionally important in mediating olfactory responses.

Perireception in olfaction: molecular mass sieving by aesthetasc sensillar cuticle determines odorant access to receptor sites in the Caribbean spiny lobster Panulirus argus.

The responsiveness of chemoreceptor neurons depends on a combination of perireceptor and receptor events. Olfactory neurons of crustaceans are packaged into distinctive cuticular sensilla called aesthetascs. The cuticle of aesthetascs is thin and permeable, even though it does not contain any obvious surface pores or pore tubules. This suggests that this 'spongy' aesthetasc cuticle may act as a molecular sieve that restricts large odorant molecules from entering the sensilla and binding to the olfactory neurons. We examined whether this is so for the aesthetasc cuticle of the Caribbean spiny lobster Panulirus argus. We used a chromatographic column packed with aesthetasc cuticle and connected to a flow-through ultraviolet spectrophotometer to measure the elution times of ultraviolet-absorbent molecular mass markers between 165 and 2 x 10(6) Da. Molecules larger than approximately 8.5 kDa had similar elution times, indicating that they did not penetrate the cuticle. Molecules smaller than 8.5 kDa had longer elution times that were directly and inversely proportional to their molecular mass. These results suggest that aesthetasc cuticle excludes molecules larger than 8.5 kDa from having access to the olfactory receptor neurons. We conclude that the molecular sieving capacity of the aesthetasc cuticle of P. argus is a perireceptor mechanism that is a critical determinant of the types of molecules capable of stimulating its olfactory receptors.

Elemental and configural learning and the perception of odorant mixtures by the spiny lobster Panulirus argus.

The present study used a conditioning assay to investigate if the type of learning task that spiny lobsters (Panulirus argus) were required to perform influenced the way that they perceived odorant mixtures. Mixtures were composed of 2 food-related compounds (adenosine-5'-monophosphate, betaine, or L-glutamate) at concentrations that produced the same duration of searching behavior in unconditioned animals. Aversive conditioning of search behavior coupled with generalization testing was used to evaluate perceptual similarity between related mixtures. When animals were conditioned to stop searching to a binary mixture AX, they did not generalize significantly from this mixture to either of its components (A or X), or to a binary mixture containing one novel component (AY). However, when lobsters were conditioned to avoid AX but to continue responding to AY, they generalized between AX and X and between AY and Y. The results support the hypothesis that altering the salience of a mixture's components by giving them different reinforcement contingencies changed the way that the mixtures were perceived. As a result of such conditioning, animals perceived the mixture's components as separate elements, rather than as a configuration, and, as a consequence, animals generalized between binary mixtures and their most salient or predictive components.

Multiple excitatory receptor types on individual olfactory neurons: implications for coding of mixtures in the spiny lobster.

The aim of our paper was to investigate whether single olfactory receptor neurons (ORNs) of the spiny lobster Panulirus argus functionally express more than one type of receptor, examine the consequences of this on coding of mixtures, and compare principles of odorant mixture coding by spiny lobsters with that by the channel catfish, which has been studied extensively using the same experimental and analytical procedures (Caprio et al. 1989; Kang and Caprio 1991). We examined responses of individual taurine-sensitive ORNs to binary mixtures of excitatory compounds, either competitive agonists (taurine, beta-alanine, hypotaurine) or non-competitive agonists (taurine, L-glutamate, ammonium chloride, adenosine-5'-monophosphate). Responses to mixtures were compared to two indices: mixture discrimination index (MDI) and independent component index (ICI). Binary mixtures of competitive agonists had MDI values close to 1.0, as expected for competitors. Mixtures of non-competitive agonists had ICI values averaging 0.83, indicating the effects of the components are not independent. We conclude that individual olfactory cells of spiny lobsters can express more than one type of receptor mediating excitation, one of which typically has a much higher density or affinity, and that spiny lobster and catfish olfactory cells encode mixtures of two excitatory agonists using similar rules.

Coding of blend ratios of binary mixtures by olfactory neurons in the Florida spiny lobster, Panulirus argus.

The aim of this study was to investigate quality coding of blend ratios of binary mixtures by olfactory receptor cells in the spiny lobster. Three odorants (adenosine-5'-monophosphate, L-glutamate, and taurine) at 0.1-100 mumol.1(-1) and seven blend ratios of each of their binary mixtures at a total concentration of 100 mumol.1(-1) were used. The olfactory cells recorded (n = 48) evoked across-neuron patterns for single odorants that were well separated from each other. Across-neuron patterns varied with stimulus concentration but less than with stimulus type. Blend ratios of the three mixtures evoked across-neuron patterns that were orderly placed within a continuum between those elicited by the components. Mixture interactions, defined as a lack of independent effects by a mixture's components, occurred in 25, 24 and 37% of responses to blend ratios of glutamate/taurine, adenosine-5'-monophosphate/taurine, and glutamate/adenosine-5'-monophosphate, respectively. These mixture interactions did not have a large enough effect on the across-neuron patterns for the mixtures such they would be novel relative to those of the single components. These results suggest that despite mixture interactions the quality of individual compounds is not lost when mixed. This corroborates behavioral studies showing that spiny lobsters have the ability to elementally process odor mixtures.

Identification and partial characterization of putative taurine receptor proteins from the olfactory organ of the spiny lobster.

To explore the initial stages of olfactory transduction, we have used biochemical techniques to characterize proteins associated with the dendritic plasma membrane from the olfactory receptor neurons of the spiny lobster Panulirus argus. In particular, we have studied proteins that interact with taurine, an amino acid that is an important odorant for this species. The cross-linker bis(sulfosuccinimidyl)suberate (BS3) was used to covalently link [3H]-taurine to cell surface proteins on membrane from the aesthetasc (olfactory) sensilla of the lateral filament of the antennule. A radioligand-receptor binding assay was used to show that this cross-linkage was highly specific for taurine at 0.2 mM BS3. In inhibition studies, of all the unlabeled odorants tested at excess concentrations (taurine, L-glutamate, adenosine-5'-monophosphate), only taurine significantly inhibited the cross-linkage of [3H]-taurine to the membrane. Membranes containing cross-linked proteins were solubilized, and proteins were separated on SDS-PAGE and examined with autoradiography. Bands with molecular weights of 100, 82, 62, 51, and 34kD were evident on the gels. However, only the 100 and 62 kD bands were consistently labeled with [3H]-taurine, and this labeling was completely inhibited in the presence of excess unlabeled taurine but not adenosine-5'monophosphate. The taurine-evoked behavioral search response of spiny lobsters was significantly reduced following treatment of their antennules with BS3 + taurine as compared with animals treated with BS3 alone, suggesting that the taurine-labeled binding proteins include taurine receptor proteins involved in the first stage of olfactory transduction.

Generalization among related complex odorant mixtures and their components: analysis of olfactory perception in the spiny lobster.

We investigated the processing of odorant mixtures containing two to seven components by the spiny lobster Panulirus argus. The chemicals tested were food-related compounds that are attractive to spiny lobsters, and include adenosine-5'-monophosphate, betaine, L-cysteine, L-glutamate, DL-succinate, taurine, and ammonium. Components were tested at concentrations that produced search behavioral responses of equal magnitude in unconditioned animals. Responses of unconditioned animals to mixtures and their components reveal hypoadditivity, in which the response to a mixture is less than the sum of the responses to that mixture's components. Aversive conditioning coupled with generalization testing was used to evaluate generalization and hence perceptual similarity between related mixtures. Animals were conditioned to either an individual odorant, a four-compound mixture, or a seven-compound mixture, followed by generalization testing with submixtures or larger mixtures containing the conditioned stimulus. Animals tended not to generalize, but significant generalization between a more simple conditioned stimulus and more complex mixtures containing that conditioned stimulus occurred in 2 of 11 cases, and significant generalization between a conditioned mixture and its submixtures was observed in 4 of 9 cases. Both the number and chemical identity of components of mixtures may contribute to the degree of generalization between mixtures. Overshadowing, in which the ability to learn about a chemical is affected by simultaneous presentation of other chemicals, occurred in two of three cases. We discuss implications of these findings with respect to elemental and configural processing of odorant mixtures in the spiny lobster, possible neural mechanisms responsible for these results, and the potential utility of generalization and overshadowing to the spiny lobster's natural behavior.

Responses of olfactory receptor neurons in the spiny lobster to binary mixtures are predictable using a noncompetitive model that incorporates excitatory and inhibitory transduction pathways.

Coding of binary mixtures by a population of olfactory receptor neurons in the spiny lobster (Panulirus argus) was examined. Extracellular single-unit responses of 50 neurons to seven compounds and their binary mixtures were recorded. The ability of a noncompetitive model with correction for binding inhibition to predict responses to mixtures based on responses to their components was compared with the predictive abilities of other models. This model assumes that different compounds activate different transduction processes in the same neuron leading to excitation or inhibition, and it includes a term quantifying the degree to which binding of an odorant to its receptor sites is inhibited by other compounds. The model accurately predicted the absolute response magnitude of the population of neurons for 13 of 15 mixtures assessed, which is superior to the predictive power of any of the other models. The model also accurately predicted the across neuron patterns generated by the binary mixtures, as evaluated by multidimensional scaling analysis. The results suggest that there is no emergence of unique qualities for binary mixtures relative to components of these mixtures.

Mixture suppression without inhibition for binary mixtures from whole cell patch clamp studies of in situ olfactory receptor neurons of the spiny lobster.

Whole cell patch clamping was used to investigate mechanisms of mixture suppression for in situ olfactory receptor neurons (ORNs) of the spiny lobster Panulirus argus. We used a set of single compounds and binary mixtures that have been used in previous biochemical studies of receptor-odorant binding, electrophysiological studies of spiking output from ORNs, and behavioral studies. These odorants were adenosine 5'-monophosphate (AMP), betaine (Bet), L-cysteine (Cys), L-glutamate (Glu), taurine (Tau), ammonium chloride, D,L-succinate, binary mixtures of these compounds, as well as a 33-component artificial oyster mixture (AOM). For the 40 ORNs studied, these stimuli more frequently elicited inward than outward currents. AMP, Glu, Tau and Bet evoked the largest and most numerous inward currents; Cys most commonly evoked outward currents. Na+ was an important charge-carrying ion for the Glu-evoked response in one ORN and the Bet-evoked response in another ORN. Mixture suppression, defined conservatively in this study as cases where the response to a binary mixture was less than the response to the more excitatory component of that mixture, was observed in 6 ORNs. In all 6 cases, neither component of the mixture evoked an outward conductance (i.e. neither was inhibitory). Five of these cases of mixture suppression involved a mixture containing two excitatory compounds (i.e. producing inward conductances): four ORNs were excited by both Glu and AMP, and one ORN was excited by both Tau and Glu. One case of mixture suppression occurred for a compound (Tau) tha did not produce a current when presented alone but which when added to Bet suppressed the inward current generated by Bet. Mechanisms for these suppressions are discussed, including inhibition of receptor binding by the components of a binary mixture and effects on second messengers or ion channels.

Inhibition of taurine and 5'AMP olfactory receptor sites of the spiny lobster Panulirus argus by odorant compounds and mixtures.

1. The effects of the odorant compounds adenosine-5'-monophosphate (5'AMP), ammonium, betaine, L-cysteine, L-glutamate, DL-succinate, and taurine and of mixtures of these compounds on binding of taurine and 5'AMP to dendritic membrane from the olfactory organ of spiny lobsters (Panulirus argus) were quantified to evaluate the contribution of inhibition of odorant-receptor binding to the generation of physiological responses to mixtures. 2. Taurine binding sites belong to two affinity classes, while 5'AMP binding sites belong to a single affinity class. Binding of either taurine or 5'AMP was partially inhibited in an apparently noncompetitive, concentration dependent fashion by most odorant compounds, with 25-40% inhibition by 1 mM of odorant. Mixtures of two or more odorant compounds also inhibited binding of taurine or 5'AMP to its sites. However, the inhibition by mixtures was often significantly less than expected from the inhibition produced by a mixture's components assuming either a noncompetitive or competitive mechanism. 3. By including this binding inhibition between compounds into models for predicting physiological responses to mixtures from the responses to the components, the predictive power of the models is significantly improved. This result strongly suggests that binding inhibition can influence the physiological responsiveness of chemoreceptor cells to mixtures.