Springer-Verlag 1997
J Comp Physiol A (1997) 180: 481–491
ORIGINAL PAPER
Stuart I. Cromarty · Charles D. Derby
Multiple excitatory receptor types on individual olfactory neurons: implications for coding of mixtures in the spiny lobster
Accepted: 20 December 1996
Abstract 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, b-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. Key words Chemoreception · Crustacea · Olfaction · Sensory coding · Mixture interactions Abbreviations AMP adenosine-5′-monophosphate ASW artificial sea water · Bet betaine · b-Ala b-alanine · Glu L -glutamate · Hyp hypotaurine · ICI
S.I. Cromarty (&) · C.D. Derby Department of Biology, Georgia State University, P.O. Box 4010, Atlanta, Georgia 30302, USA Tel.: +1- 404/651-1646, Fax: +1- 404/651-2509, e-mail:
[email protected]
independent component index · MDI mixture discrimination index · NH4 ammonium chloride · ORN olfactory receptor neuron · Tau taurine
Introduction The question of how the quality of mixtures is encoded by olfactory neurons is of central importance to understanding how animals perceive natural chemical signals. Individual olfactory cells can have multiple transduction pathways, including receptor sites, second messengers, and ion channels (Ache 1994; Ache and Zhainazarov 1995; Dionne and Dubin 1994). To understand how cells encode a mixture’s quality, it is necessary to know the mode of action of each component of that mixture – whether the components activate the same or different transduction pathways. For example, if all components of a mixture have their effects through identical receptors and transduction pathways, then the response of the cell to the mixture might be accurately modeled using the competitive interactions between the components. On the other hand, if the components of a mixture activate different receptors, then one would expect greater if not complete independence in the effects of the components. Thus, critical to understanding mixture coding is to know whether individual olfactory receptor neurons express one or several types of receptors. Many olfactory cells, particularly those responsive to non-pheromonal stimuli, are responsive to compounds belonging to more than one class (e.g., Getchell and Shepherd 1978; Holley 1991; Smith and Getz 1994). This breadth of tuning could result from each neuron having more than one class of receptors, such that differences in efficacies for the compounds could be attributed to different densities or affinities of the receptors. But broad tuning might also be a consequence of each cell expressing a single receptor type that has low binding specificity. Molecular approaches have led some to conclude that individual vertebrate olfactory neurons
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express one or a very limited number of types of candidate receptors (Lancet and Ben-Arie 1993; Ressler et al. 1993; Vassar et al. 1993; Ngai et al. 1993; Strotmann et al. 1995); unfortunately, the binding characteristics and functional expression of these candidate receptors are unknown. On the other hand, results from physiological and biochemical studies (Caprio and Byrd 1984; Kalinoski et al. 1989; Bruch and Rulli 1988; Ngai et al. 1993; Kang and Caprio 1995) have led Kang and Caprio (1997) to conclude that individual olfactory neurons of the catfish express more than one of the several known classes of receptors. Multiple receptor types are also likely in individual cells having two opposing (i.e. excitatory and inhibitory) transduction pathways, such as in lobsters (Hatt and Ache 1994; Boekhoff et al. 1994), squid (Lucero and Piper 1994), mudpuppy (Dionne and Dubin 1994) and toads (Morales et al. 1994). Additionally, single chemosensory neurons of the nematode Caenorhabditis elegans express many types of receptors (Troemel et al. 1995; Sengupta et al. 1996). Thus, the issue of how many different types of receptors are functionally expressed on individual ORNs, which is critical to our understanding of olfactory coding, is currently unresolved in most olfactory neurons and systems, including the spiny lobster. Much is known about coding of mixtures in the olfactory systems of the Caribbean spiny lobster Panulirus argus and the channel catfish Ictalurus punctatus, because they have been studied using a combination of electrophysiological, odorant-receptor binding, and behavioral approaches. For example, in both species, several types of olfactory receptors and transduction pathways have been identified, and individual cells can have more than one transduction cascade (spiny lobsters: Hatt and Ache 1994; Ache 1994; Ache and Zhainazarov 1995; Michel and Ache 1992, 1994; Daniel et al. 1994; Olson and Derby 1995; catfish: Miyamoto et al. 1992; Caprio and Byrd 1984; Kalinoski et al. 1989; Bruch and Rulli 1988; Ngai et al. 1993; Kang and Caprio 1995). These results have been helpful in selecting mathematical models of transduction to be used to compare measured neural responses to mixtures with values calculated using measured responses to the components. The mixture models are of two general types. In competitive models, it is assumed that the components bind to the same receptors and activate the same transduction cascades. These models include stimulus substitution (Hyman and Frank 1980; Derby et al. 1985) and mixture discrimination index, MDI (Hyman and Frank 1980; Caprio et al. 1989; Kang and Caprio 1991). In the second type of model, components are assumed to have independent effects and interact with different receptors and other transduction elements. Such models include response summation (Hyman and Frank 1980; Derby et al. 1985), polynomial models (Jakinovich 1982; Derby et al. 1991; Zimmer-Faust 1987) and independent component index, ICI (Hyman and Frank 1980; Caprio et al. 1989; Kang and Caprio 1991). More complex models
that include both types of events have also been developed (Ennis 1991; Malaka et al. 1995; Getz and Akers 1995; Daniel et al. 1996). Measured responses to mixtures that are less than expected from the models are often called ‘mixture suppression’, and responses greater than the modeled responses are called ‘mixture enhancement’. The identification of ‘suppression’ and ‘enhancement’ in any system, of course, depends on how they are empirically defined and the techniques used. In general, different conclusions about mixture coding have been reported for spiny lobsters and catfish. ‘Suppression’ is more often reported for spiny lobsters (Gleeson and Ache 1985; Derby et al. 1985, 1991; Ache et al. 1988; Daniel et al. 1996; Steullet and Derby 1997) and ‘enhancement’ more common in catfish (Caprio et al. 1989; Kang and Caprio 1991). However, there are several major differences in these studies on catfish (Caprio et al. 1989; Kang and Caprio 1991, 1997) and spiny lobsters (Derby et al. 1985, 1991; Daniel et al. 1996; Steullet and Derby 1997) that make it difficult to compare directly how they code mixtures. 1) Different recording techniques have been used for catfish and spiny lobsters. In all but one study of catfish (Kang and Caprio 1997), multi-unit recordings (i.e. electro-olfactograms and summed spiking responses) have been used; for the spiny lobster, single unit or cell recordings have been exclusively used. Single cell studies obviously allow greater resolution of cellular effects and mechanisms. 2) Experimental protocols and operational definitions of suppression and enhancement have been different. This can have several effects. The more obvious effect is on the predicted response values for the mixture and thus the final determination of mixture interactions. Another consequence is the types of cells that can be included in the analysis. For example, the MDI and ICI are powerful in allowing direct comparisons of responses to mixtures and their components (Kohbara and Caprio 1996). But they are limited in that they can be applied only to cases in which the components of a mixture can be matched to evoke excitatory neural responses of the same magnitude. Thus, in the catfish, for which MDI and ICI have been used in all but one study (Kang and Caprio 1997), we know much about coding of some cells and some types of mixtures but less about others. In the spiny lobster, cells for which the components of a mixtures are either excitatory, inhibitory, or neutral have been extensively studied. In fact, cells for which one component of a binary mixture is excitatory and the other is either inhibitory or neutral commonly show mixture suppression (Derby et al. 1988; Michel and Ache 1992, 1994; Daniel et al. 1996), as has been observed recently in a singleunit study on catfish olfactory receptor neurons (Kang and Caprio 1997). 3) In catfish, two classes of mixtures have been studied: those composed of compounds that compete for the same receptors/transduction pathways, and those that do not. Studies of spiny lobsters have been limited to mixtures of compounds that show largely non-competitive binding.
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Our study was designed to answer three questions. 1) Do single ORNs of the spiny lobster express more than one type of functionally-defined olfactory receptors? 2) What are the consequences of multiple receptors to coding of odorant mixtures? 3) Is coding of mixtures by the olfactory system of the spiny lobster and catfish similar when the same analytical techniques (MDI and ICI) and the same general class of cells (those excited equally by the components of the mixture) are used? We report that for individual ORNs that are excited by taurine, mixtures of response-matched components are more excitatory if the components are non-competitors rather than competitors. Our findings suggest that individual ORNs of spiny lobsters functionally express more than one type of receptor mediating excitation, responses to mixtures of competitive agonists are less than those to mixtures of non-competitive agonists, and coding of binary odorant mixtures is generally similar in spiny lobsters and catfish.
Materials and methods Animals Male and female spiny lobsters (Panulirus argus) were collected in the Florida Keys, shipped by air and held in the laboratory at 20–25 °C in 800-l tanks containing recirculating and aerated Instant Ocean. Animals were fed shrimp and squid.
Chemical stimulants The stimulants used in this study were adenosine-5′-monophosphate (AMP), L -glutamate (Glu), taurine (Tau), betaine (Bet), balanine (b-Ala), hypotaurine (Hyp), and ammonium chloride (NH4). All compounds were obtained from Sigma Chemical Co. The purity of the chemicals used was > 99%. Stock solutions of the single compounds were prepared in artificial sea water (ASW) (Cavanaugh 1964), adjusted to pH 8.1, and frozen in aliquots at 10)2 mol · l)1. Stimuli were serially diluted with ASW. ASW was used as the control stimulus. Appropriate dilutions of the single compounds were prepared each day from the stock solutions. These stimuli were selected because they are present in many natural prey items of spiny lobsters (Carr et al. 1996) and are physiologically or behaviorally active compounds for spiny lobsters (Derby et al. 1989) or are structural analogues of taurine. Based on their molecular structure and results of previous electrophysiological (Fuzessery et al. 1978; Derby et al. 1991) and binding experiments (Olson and Derby 1995; Gentilcore et al. 1996), these compounds were classified a priori into either of two classes. ‘Within-group’ compounds are structurally similar, compete for the same or highly cross-reactive receptor sites, and are approximately equally-effective agonists for the same olfactory receptor neurons. ‘Across-group’ compounds are structurally dissimilar, show little or no competition for the same receptor sites and do not excite the same olfactory neurons equally. Withingroup binary mixtures tested in this study consisted of Tau and either Hyp or b-Ala, whereas across-group mixtures included Glu, NH4, Bet, and AMP, each in a binary mixture with taurine.
The distal end of an excised lateral filament of an antennule was inserted into a Teflon tube attached to an olfactometer in which ASW was flowing at 9 cm · s)1. This rapid flow allowed for extensive access of the stimulus to the receptor sites on the chemosensory sensilla. The proximal end of the antennule was inserted into a separate recording chamber containing Panulirus saline. Several annuli were removed from the cut proximal end of the filament, thus exposing both the antennular nerve and artery. The artery was then cannulated, and the preparation was perfused with oxygenated Panulirus saline delivered directly into the artery through a glass cannula at a flow rate of 0.6–1.0 ml · min)1. Chemical stimuli were injected into the ASW flowing over the aesthetasc sensilla by activation of an electronic valve. The stimulus had a fast rise time and remained constant at a minimally diluted concentration over about 4 s. The electronic valve allowed for a determination of the beginning of the stimulation period and response latency, since a stimulation artifact was placed on each record. Consecutive stimulations were separated by approximately 2 min. Single-unit responses were recorded extracellularly from the exposed axons of ORNs in the saline dish by using fine-tipped glass suction electrodes (tip inner diameter ~ 2–5 lm), coupled to a differential AC amplifier. Activity was monitored on-line and recorded on videotape in digitized form. For analysis, records were down-loaded to a microcomputer, and spikes were sorted according to their waveforms using Data-Pac II (Run Technologies ). This allowed us to quantify the responses of single neurons to chemical stimuli since each extracellularly recorded neuron has a distinctive spike waveform. Typically, recordings contained spikes from one to three different neurons. For each stimulus and neuron, the numbers of spikes during 1000-, 2000- and 5000-ms periods following initiation of the response were counted as measures of the response intensity. However, only results for the 2000-ms period are reported, for two reasons. 1) Since the experimental protocol (see below) required testing stimuli at concentrations that evoked responses of equal magnitude and since many ORNs responded very little to all except one stimulus, the response magnitudes were often low and variable. This was particularly true for short sampling times. Consequently, summing spikes over 2000- and 5000 ms resulted in larger and less variable responses. 2) The time interval of 2000 ms is closer than 5000 ms to the odorant sampling frequency associated with antennular flicking behavior (Daniel and Derby 1991; Gleeson et al. 1993). Each ORN response was corrected for any spontaneous activity or response to the ASW control. Experimental protocol Only ORNs responsive to Tau at 10 lmol · l)1, which was the search compound, were used in this study. Once such neurons were identified, individual compounds were presented in random order at various concentrations (Table 1) to determine a concentrationresponse (C-R) curve for each ORN. For each cell, a target response magnitude (usually about one-half maximal response to the more effective compound, usually Tau) was selected. Then, based on the C-R curves, a concentration for each compound was selected that elicited this target response magnitude. We then measured responses to each compound at its equally effective concentration and at two times its equally effective concentration, and to binary mixtures of Tau plus one of the other stimuli at their respective equally effective concentrations. This protocol therefore represents a single ‘mixture protocol’ for Tau, another compound and a mixture of the two. During this protocol, ASW and Tau were presented regularly to insure continuous responsiveness of the cells. We performed as many mixture protocols as possible for each ORN, the number depending on how long we could record from the ORN and how many compounds activated that ORN.
Antennular preparation and electrophysiological recordings
Response indices for binary mixtures
Electrophysiological recording methods were similar to those published previously (Derby 1995) but are described briefly here.
For each ORN, two indices of response were calculated for each binary mixture. These were the MDI and the ICI (Hyman and
484 Frank 1980; Caprio et al. 1989). The MDI is calculated as the mean value of Rab =R2a and Rab =R2b , while the ICI is Rab =
Ra Rb . a and b represent the two chosen odors at the response-matched concentrations, while Ra and Rb represent the response magnitudes to a and b respectively. R2a and R2b represent the responses to the doubled concentrations of a and b respectively. Finally, Rab is the response of the binary mixture (formed by mixing equal aliquots of 2a and 2b), thus consisting of their response matched concentrations of a and b. Besides requiring that the components a and b of the mixture be tested at response-matched concentrations, the MDI and ICI also require that the components have parallel and nonsaturating concentration-response functions in the range that they are tested (a to 2a and b to 2b). This can be evaluated post-hoc from the measured response. If the two response-matched compounds a and b are pure competitive agonists (e.g., they do not allosterically antagonize or enhance the binding of each other to their respective receptors) and there is not inhibitory or amplifying cross-talk between the transduction pathways for the two, then one would expect a mixture of the two, ab, to act either at a concentration of 2a or 2b. In this case, the MDI value should be 1. Consequently an MDI value significantly < 1 or > 1, which has been called ‘mixture suppression’ and ‘mixture enhancement’ respectively (Caprio et al. 1989; Kang and Caprio 1991), indicates that one or more of the above assumptions are incorrect. However, since most assumptions can be controlled or tested, the possibilities can usually be limited to the components not acting as pure agonists. The ICI measures whether or not the components of a mixture activate a cell through independent pathways. If the two compounds are independent, then the response of the mixture would be equal to the sum of the response to the single components. Thus, ICI values less than or greater than 1 indicate a lack of independence between the two compounds of the mixture. This lack of independence could occur at any of the many steps in the transduction/ activation cascade for ORNs between binding and spiking. Data analysis The mean ± SEM values for MDI and ICI for each mixture were calculated. All MDI and ICI values for the different binary mixtures were combined into either the ‘within’ or ‘across-group’ categories (Fig. 3). A one-way of variance (ANOVA) with a post-hoc Tukey test was used to compare means. Values were considered significant at P < 0:05. The data were natural-log transformed prior to analysis because MDI and ICI values are ratio data. Since the analyses using untransformed and the natural-log transformed data yielded very similar MDI and ICI values, for simplicity all values reported in the Results were derived from the untransformed data.
Results A total of 50 ORNs was used in this study, each from a different antennule. All Tau-sensitive ORNs were responsive to Hyp and b-Ala when tested, but not all were responsive to Glu, NH4, AMP, and Bet when tested. An example of a recording is shown in Fig. 1. Binary mixtures comprising two different withingroup mixtures (Tau + Hyp, Tau + b-Ala) and four different across-group mixtures (Tau + Glu, Tau + NH4, Tau + AMP, Tau + Bet) were tested on taurine-sensitive ORNs (Table 1). Of the 50 ORNs, only three were tested with all six mixture protocols for either of two reasons: 1) equally effective concentrations could not be established for the components of some mixtures because one of the components was not sufficiently excitatory; or 2) the recording was lost or the cell’s
Fig. 1A,B Spike records from a representative olfactory receptor neuron in response to various single and binary odorants. Each record begins with the initiation of odor-evoked response and lasts for 2000 ms: A taurine and hypotaurine represent a within-group set of odorants. In this set, concentration a is 10)8 mol · l)1, 2a is 2 × 10)8 mol · l)1, b is 10)7 mol · l)1, and 2b is 2 × 10)7 mol · l)1; B taurine and glutamate represent an across-group set of odorants. In this set, concentration a is 10)8 mol · l)1, 2a is 2 × 10)8 mol · l)1, b is 10)4 mol · l)1, and 2b is 2 × 10)4 mol · l)1. Ra, Rb, and Rab represent the numbers of only large amplitude sorted spikes. The MDI and ICI values for the within-group were 0.98 and 0.60, respectively, and for the across-group the values were 1.44 and 0.94
responsiveness was significantly reduced before all mixture protocols could be tested. Consequently, 6–11 cells were tested for each mixture protocol. Calculation of MDI values requires that the two components of a mixture be tested at approximately equally-effective concentrations and their C-R functions be approximately parallel in the range of concentrations
485 Table 1 Represents concentrations of equally effective compounds (response matched) for individual taurine-sensitive ORNs. For example, t7h8 represents response matched concentrations of 10)7 mol · l)1 and 10)8 mol · l)1 for taurine and hypotaurine, respectively. n = number of ORNs tested with each odorant pair
Within-group compounds (n = 19) Tau/Hyp Tau/b-Ala Tau/Glu (n = 11) (n = 8) (n = 10) t7h8 t7h6 t7h6 t7h6 t7h6 t7h6 t7h6 t6h5 t5h6 t5h4 t8h7 Median t7h6
Tau/NH4 (n = 6)
t8b7 t6b5 t7b6 t7b6 t7b6 t8b6 t7b6 t7b6
t7g2 t8g4 t9g4 t8g4 t8g3 t8g4 t9g3 t9g5 t9g5 t9g3
t8a3 t6a3 t8a3 t6a3 t10a3 t8a3 t8a6 t9a5
t6b3 t7b5 t8b3 t7b4 t9b3 t6b5 t10b3
t8n3 t8n3 t7n6 t7n4 t10n3 t7n3
Median t7b6
Median t8g4
Median t8a3
Median t8b3
Median t8n3
tested. The first criterion was met because of our protocol. For each taurine-sensitive ORN, equally-effective concentrations for compounds were selected based on their measured C-R functions. These concentrations were usually within one order of magnitude for withingroup odorants but four to five orders of magnitude for across-group odorants (Table 1). This difference in sensitivity to within-group and across-group odorants is also represented by the average C-R functions for all compounds, at all concentrations and for all ORNs (Fig. 2). These results also show that averaged C-R functions have shallow slopes, as has been previously observed (Derby et al. 1985, 1991; Daniel et al. 1996; Steullet and Derby 1997). The second criterion – parallelism in C-R functions in the range where mixtures were tested – was typically also met. A doubling of the equally effective concentrations produced a similar increase for both members of an odorant pair as is shown by comparing the ratios R2a/ Ra and R2b/Rb for each odorant pair (Table 2). Ratio values equal to 1 indicate no increase in response with a doubling of the concentration, while values greater or less than 1 indicate an increase or decrease, respectively, with doubling. This analysis shows that the values of R2a/Raand R2b/Rb are very similar, and between 1.16 and 1.47. This means that a doubling of the concentration of most compounds produced a 16–47% increase in response. All ratios except for Glu and Bet were significantly greater then 0 (P < 0:05), and those for Glu and Bet showed a strong trend (0:05 < P < 0:10). No significant differences were found between the ratios for the odorant pairs (paired t-test, P > 0:20). Thus, the criterion of parallelism in C-R functions for the members of odorant pairs was met for most ORNs. These results also show that the C-R functions for individual cells are much steeper than for populations of cells (Fig. 2), thus demonstrating range fractionation. MDI values for the across-group binary mixtures were significantly greater than for the within-group mixtures (Figs. 1, 3A). Their mean ± SEM values are 1.49 ± 0.06 (n 31) and 1.07 ± 0.01 (n 19), respectively (ANOVA, F
1 48 9:14; P 0:004). There was ;
Across-group compounds (n = 31) Tau/Amp Tau/Bet (n = 8) (n = 7)
no overlap in the 95% confidence intervals for the averaged MDI values of the across-group (1.37–1.61) and the within-group (1.05–1.09) binary mixtures. MDI values for within-group mixtures were similar to each other and not significantly different from 1. MDI values for across-group mixtures were similar to each other and significantly greater than 1 (Fig. 3A). The averaged ICI value for the across-group mixtures (0.83 ± 0.03; n 31) was significantly higher than that for the within-group mixtures (0.64 ± 0.02; n 19) (ANOVA, F
1 48 8:80; P 0:005; Fig. 3B). There was no overlap in the 95% confidence intervals of the averaged ICI values for the across-group (0.77–0.89) and within-group (0.60–0.68) binary mixtures. Tukey posthoc tests revealed that the ICI values for all four acrossgroup mixtures were not different from each other but were significantly higher than the two within-group mixtures. The ICI values for all the mixtures, including the across-group, were significantly less than 1. ;
Discussion Multiple receptor types on individual olfactory neurons Our major conclusion is that individual ORNs of the spiny lobster can have at least two different types of receptor sites mediating excitation. This follows from our observation that the response of single ORNs to a mixture relative to those to its excitatory components is different for mixtures of within-group compounds, i.e., those that compete for the same receptors, as determined by binding assays (Olson and Derby 1995) than for mixtures of across-group compounds, i.e., those that do not compete for the same receptors (Figs. 1, 3). Furthermore, the response magnitude to mixtures is generally predictable from knowing whether or not those excitatory components compete for the same receptor sites. As expected, MDI values were ca. 1.0 for withingroup mixtures (taurine and either hypotaurine or balanine) and significantly greater than 1.0 for acrossgroup mixtures (Figs. 1, 3). An additional expected re-
486 Table 2 Comparison of values for R2a/Ra and R2b/Rb for different pair of odorants. These values represent a relative measure of the increase in response magnitude to a stimulus resulting from a doubling of its concentration. Values are mean ± 95% CL; n = # of
ORNs tested with each odorant pair. No significant differences between R2a/Ra and R2b/Rb for all odorant pairs were found. All values except those for Glu and Bet are significantly greater than 0
Within-group compounds Mixture (a + b)
n
R2a/Ra
R2b/Rb
Across-group compounds Mixture (a + b)
n
R2a/Ra
(Taurine + Hypotaurine) (Taurine + b-Alanine)
11 8
1.16 ± 0.14 1.19 ± 0.17
1.19 ± 0.17 1.24 ± 0.21
(Taurine (Taurine (Taurine (Taurine
10 8 7 6
1.47 1.28 1.20 1.29
sult was that ICI values were greater for across-group mixtures than for within-group mixtures. These results support the idea that individual ORNs in the spiny lobster’s olfactory system express more than one type of receptor, and the magnitude of the response to binary mixtures depends on whether the components bind to and activate the same or different receptor types. While the presence of more than one receptor type on single olfactory cells has been observed in a few other species (see Introduction), how common this expression pattern is and how patterns of receptor expression are controlled (Kudrycki et al. 1993) await further research. What is clear is that olfactory systems composed of cells expressing multiple receptor types and second messenger systems have the potential for encoding a greater diversity of stimuli, particularly complex stimuli such as mixtures, using fewer broadly tuned receptor types (Erickson 1967; Derby et al. 1989; Boekhoff et al. 1994). Fig. 2 Average concentrationresponse functions for individual odorants. Values in parentheses represent the number of ORNs used for each compound
+ + + +
Glutamate) AMP) Betaine) NH4)
± ± ± ±
R2b/Rb 0.26 0.24 0.19 0.25
1.21 1.24 1.16 1.23
± ± ± ±
0.24 0.22 0.18 0.21
Whether these results generalize to other types of ORNs of spiny lobsters in addition to taurine-sensitive ORNs, and whether ORNs excited by more than two compounds express more than two receptor types on it, remain to be experimentally determined; however, both are highly likely. The spiny lobster’s olfactory organ has numerous receptor types, as shown from binding and electrophysiological studies. These include receptors for each of the following: taurine, and structurally related analogues such as hypotaurine and b-alanine (Fuzessery et al. 1978; Olson and Derby 1995), adenosine-5′-monophosphate and structural analogues including 6′-chloropurine and xanthosine-5′-monophosphate (Derby et al. 1984; Olson and Derby 1995), glutamate, and structural analogue NMDA (Burgess and Derby 1995), L -arginine, D -arginine (Michel et al. 1993), ammonium, and cysteine (Derby et al. 1991). It is highly probable that an ORN excited by several molecules that belong to structurally
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distant compound groups has an equal number of different receptor types. In addition, since taurine-sensitive cells do not appear to be exceptional in most respects from other cells, we believe that they would be similar in the pattern of receptor expression as well. It is currently unresolved why two compounds that excite a single ORN through different receptors have highly different efficacies. The receptors for one compound may have a much higher affinity or density than receptors for the other compound, or that the coupling to second messengers is different for the different receptor types. We know that different affinity sites for taurine as well as for other compounds exist in the olfactory organ of spiny lobsters (Olson and Derby 1995), but the absolute and relative distributions of these sites on individual cells, including whether or not both highand low-affinity sites are expressed on single cells, are not known. Regarding second messengers, it is known that excitatory transduction can be mediated by odorant activation of receptors coupled to Gq-like G-proteins that in turn activate IP3 and IP4 gated channels (Fadool et al. 1995). In addition there are other channels that can effect excitation, and odorants can activate more than one type of channel in single cells (Hatt and Ache 1994; Zhainazarov and Ache 1995). So, it remains a possibility that odorants may have different efficacies on a cell due to different coupling to excitatory transduction cascades.
have ICI values much less than 1 and MDI values greater than 1. On the other hand, the major difference is that the ICI values for across-group mixtures are lower for spiny lobsters than those for catfish. The mean ICI value is 0.83 for spiny lobsters from single unit responses, and for catfish it is 0.94 from integrated spiking responses and 0.88 from electro-olfactogram responses (Table 3). The significance of these similarities and differences is discussed below. The finding that MDI values of within-group mixtures are ca. 1 for spiny lobsters and catfish indicates that the efficacy of excitatory components that interact with the same receptor sites can be explained by competitive agonism. There are no inhibitory mechanisms operating in these cells that significantly shape the magnitude of responses to these types of mixtures. The fact that some MDI values for within-group mixtures can be slightly greater than 1 (Table 3) in both species can be explained most parsimoniously by the two components interacting with a small set of different receptor sites in addition to their common receptor sites. An important observation is that for both spiny lobsters and catfish ICI values for across-group mixtures Table 3 Comparison of binary mixture index (MDI and ICI) values for the spiny lobster and the catfish olfactory system. Values represented are mean ± SEM Panulirus olfaction Binary mixtures
Ictalurus olfaction Binary mixtures
Interspecific similarities in mixture coding A major issue examined in our study is whether or not different species encode chemical mixtures using similar rules. Our study was designed to explore this issue, especially for the spiny lobster Panulirus argus and the channel catfish Ictalurus punctatus. Both of these two species have been used in a series of analyses of mixture coding, and one apparent difference in the conclusions from past studies is that mixture suppression is more common in spiny lobsters than catfish (see Introduction). Since different experimental and/or analytical designs were used, it has been unclear whether this represents a difference in interspecific coding schemes, or rather, results from technical and semantic differences. Thus, our study on spiny lobsters was designed to resolve this issue by replicating the design and analytical techniques used by Caprio et al. (1989) on catfish. It should be stressed that our results for spiny lobsters are based on responses of individual ORNs, whereas those for catfish are based on multi-unit responses, either integrated spiking responses or electro-olfactograms. Interesting similarities and differences in the results for spiny lobsters and catfish are apparent (Table 3). First, the similarities, which are most striking. For both species, mixtures of compounds that are thought to interact with the same receptors/transduction pathways have MDI values of about 1.0 and ICI values much less than 1.0, and mixtures of compounds that are thought to interact with different receptors/transduction pathways
MDI Within
Across
ICI Within
Across
a
1.07 ± 0.01 (19/6)
a f, g
1.49 ± 0.06 (31/6)
0.64 ± 0.02 (19/6) 0.83 ± 0.03 (31/6)
a
1.09 ± 0.05 (20/3) 1.05 ± 0.01 (152/10) 1.13)1.20 (9/2) 1.11)1.14 (29/2) 1.58 ± 0.06 (28/3) 1.43 ± 0.01 (238/12) 1.52)1.61 (17/4) 1.20)1.32 (27/4)
b
0.62 ± 0.04 (10/3) 0.62 ± 0.01 (67/10) 0.94 ± 0.04 (14/3) 0.88 ± 0.01 (112/12)
b
Cromarty and Derby (this study) – Single-unit recordings Caprio et al. (1989) – Integrated spike recordings Caprio et al. (1989) – EOG recordings d Kang and Caprio (1991) – Integrated spike recordings e Kang and Caprio (1991) – EOG recordings f Number of tests or cells g Number of different binary mixtures tested b c
c d e
c
488 Fig. 3A, B MDI (A) and ICI (B) values for across-group mixtures and within-group mixtures. Values are mean ± SEM. Numbers in parentheses represent number of ORNs used for each mixture. The mean MDI and ICI values (± SEM and sample size, n) for all across-group mixtures and within-group mixtures are shown on the right
are less than 1. This signifies that the components do not act completely independently of each other. This lack of independence between non-competing components of mixtures is one of the phenomena that has been called mixture suppression (Derby et al. 1985, 1991; Johnson et al. 1989; Getz and Akers 1995; Daniel et al. 1996; Steullet and Derby 1997). This lack of independence in effects of mixtures of across-group compounds has been seen in vertebrate taste systems (Hyman and Frank 1980; Vogt and Smith 1994; Kohbara and Caprio 1996). Possible reasons for this effect include the following. 1) The components of the mixture share elements of the excitatory transduction pathways at any step prior to spike initiation. This can include competition of different odorants for the same receptor sites, competition of different odorant-receptor complexes for the same G proteins, competition of activated G proteins for the same catalytic enzymes, and use of same second mes-
senger gated ionic channels by different odorants. 2) There exists antagonism between the components of the mixture, such as allosteric antagonism in the binding of the components to their respective receptors (Olson and Derby 1995). 3) There is inhibitory cross-talk between the excitatory transduction cascades activated by the odorants (Frings 1993). As noted above, the major difference in the results for spiny lobsters and catfish is that the ICI values for across-group compounds are lower for spiny lobsters than catfish. This may be a consequence of any of the mechanisms described in the preceding paragraph being more prevalent in the spiny lobster than catfish. However, some portion of it could be due to a difference in the recording methods used in the two studies – singleunit for the experiments on spiny lobsters versus multiunit for catfish. Multi-unit recording has a greater likelihood of detecting independence of effects and
489
consequently yielding higher ICI values, since multi-unit recordings can include responses which by definition are independent, i.e., from cells excited only by one compound or the other. The resolution to this issue awaits single-unit recordings of responses of catfish ORNs using the MDI/ICI protocol. Given these results for spiny lobsters and catfish, it is appropriate to ask whether or not it is possible for two excitatory compounds to ever have completely independent actions on olfactory receptor neurons. The answer to this appears to be affirmative. For some single neurons of spiny lobsters (Derby et al. 1985, 1991; Daniel et al. 1996) and catfish (Kang and Caprio 1997), the response to some binary mixtures is the sum of the responses to its components. In the lamprey, components of blends of steroid pheromones act independently as demonstrated by ICI values of 1.0. However, since the lamprey’s neural activity was measured by EOGs, it is possible, perhaps even likely, that this independence is a consequence of an olfactory system composed of narrowly tuned cells ORNs each of which has receptors for only one pheromone component (Li and Sorensen 1994). The experimental approach used in our study is only applicable where both of the mixture’s components are excitatory to a cell. It cannot be used for cells that are excited by one component and either not excited or inhibited by the other component. For such cells, other experimental approaches are necessary (Derby and Ache 1985; McBride 1989; Derby et al. 1991; Getz and Akers 1995; Malaka et al. 1995; Daniel et al. 1996; Steullet and Derby 1997). Since only compounds that elicit net excitation were used in the MDI/ICI studies with spiny lobsters and catfish, other types of olfactory neurons need to be considered when predicting mixture responses in lobsters. This is especially true since in both of these olfactory systems (Michel and Ache 1992, 1994; Ivanova and Caprio 1993; Derby et al. 1991; Ache 1995; Daniel et al. 1996; Kang and Caprio 1995, 1997), as in other olfactory systems (Dionne and Dubin 1994; Lucero and Piper 1994; Morales et al. 1994), individual neurons can be excited by some compounds and inhibited by others. Therefore, olfactory cells that are excited by some compounds, inhibited by others, and not affected by others need to be included in studies of coding of mixtures. In fact, in mixture studies with such cells in catfish (Kang and Caprio 1997) and spiny lobsters (Derby et al. 1985, 1991; Michel and Ache 1992; Boekhoff et al. 1994; Ache and Zhainazarov 1995; Daniel et al. 1996), components that by themselves are not stimulatory can suppress or mask the stimulatory activity of other compounds. In summary, ORNs of catfish and spiny lobster appear to use more or less similar rules when coding mixtures. Both species have multiple types of receptors on individual olfactory receptor neurons, although the density and/or affinity of the receptors may differ. A mixture of excitatory compounds that bind to the same receptor acts much like a concentration of either compound, and the components predictably do not have independent effects. For a mixture of excitatory com-
pounds that do not compete for the same receptors, the components tend to approach but not reach complete independence in their effects. This non-independence is slightly greater in spiny lobsters than catfish, which may be at least in part due to differences in recording techniques. Taken together with previous work on both spiny lobsters (Michel and Ache 1992; Boekhoff et al. 1994; Ache 1994; Derby et al. 1991; Daniel et al. 1996) and catfish (Kang and Caprio 1997) which shows that mixtures of excitatory and neutral or inhibitory compounds can lead to mixture suppression or masking, mixture coding in these two species is strikingly similar. This may be a consequence of similarities in the basic design features of invertebrate and vertebrate olfactory cells (Dionne and Dubin 1994; Ache 1994; Ache and Zhainazarov 1995). Acknowledgements We thank the Florida Keys Regional Marine Laboratory in Long Key, Florida, for supplying lobsters. This study was supported by a grant from NIH (R01DC00312). We thank Malia Schwartz and Drs. Marc Weissburg and Pascal Steullet for helpful comments on the manuscript.
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