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

Brain Research 771 Ž1997. 292–304

Research report

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 Michele F. Burgess 1, Charles D. Derby

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Department of Biology and Center for Neural Communication and Computation, Georgia State UniÕersity, P.O. Box 4010, Atlanta, GA 30302-4010, USA Accepted 17 June 1997

Abstract 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 ŽP.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 Ž1992. 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 K d values Ž1 nM and 3 mM. 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. q 1997 Elsevier Science B.V. Keywords: Chemoreception; Mixture; Binding; Coding; Crustacea; Panulirus

1. Introduction The olfactory system of the Caribbean spiny lobster Panulirus argus is sensitive to a variety of behaviorally relevant odorant molecules, including amino acids, nucleotides, and quaternary ammonium ions, which are recognized by receptors located on dendrites of their olfactory receptor neurons w4,7,18,30x. Biochemical studies directly examining odorant binding properties of these receptors reveal separate binding sites for the odorants taurine, adenosine-5X-monophosphate, D-alanine, and L-alanine w41,46,47x. These odorant–receptor interactions are specific, saturable and reversible, and the specificity of bind) Corresponding author. Fax: q1 Ž404. 651-2509; E-mail: [email protected] 1 Present address: Integrated Laboratory Systems, Atlanta Federal Center, 61 Forsyth St., EPA Region 4, Waste Division OTS, Atlanta, GA 30303-3104, USA.

0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 1 6 - 0

ing largely reflects physiological specificity, suggesting that these sites are receptors involved in olfactory transduction. Another important odorant molecule for P. argus is L-glutamate, since it is present in their food w7–9x, is detected by at least two classes of receptor neurons in their olfactory organ w15,20,56x, and stimulates searching behavior w21x. We are interested in exploring the olfactory L-glutamate receptors of P. argus towards understanding their binding characteristics and how they contribute to the coding of glutamate and mixtures containing glutamate. The study of olfactory L-glutamate receptors of P. argus is aided by the fact that L-glutamate is an important excitatory neurotransmitter in the central and peripheral nervous systems of many organisms. Consequently, a great deal is known about the pharmacology, molecular biology, and physiology of a diverse set of L-glutamate receptors. For example, distinct types of L-glutamate receptors in the mammalian brain have been identified based on pharmaco-

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logical characterization using specific agonists and antagonists w64x. There are two general types of glutamate receptors in mammals: ionotropic receptors, which are ligandgated ion channels and include the NMDA receptorrchannel; and metabotropic receptors, which are coupled through G-proteins to second-messenger effector systems. Invertebrate nervous systems also use L-glutamate to mediate a variety of synaptic processes and thus have specific postsynaptic receptor proteins w13,31,51,52,58,63x. Examples include receptors in neuromuscular junctions of arthropods w58x, CNS of snails w36,43x, and optic ganglia of crustaceans w49x. However, invertebrate L-glutamate receptors appear to differ in structure and function from their mammalian counterparts based on the physiological effectiveness of specific mammalian L-glutamate agonists and antagonists. Further similarities and differences between mammalian and invertebrate L-glutamate receptors are becoming evident via molecular techniques w13,16,31x. L-Glutamate-sensitive receptors involved in detecting food-related chemicals also have been partially characterized in a number of species, including paramecia w50,61x, hydra w 2,25 x , crustaceans w 14,19,20,29 x , fish w6,23,35,48,57x, and mammals w12,22x. Hence, characterization of the L-glutamate olfactory receptors in P. argus is important not only in understanding the role of these receptors in the transduction of this important biological molecule, but also in exploring the comparative biology and evolutionary diversity among a functionally diverse set of peripheral and central L-glutamate receptors. In our study, we have used a radioligand-binding assay and a membrane-rich preparation from dendrites of olfactory receptor neurons of P. argus to identify two sites with different affinities for L-glutamate and with features of olfactory receptors. Binding of L-glutamate to both sites can be completely inhibited by high concentrations of NMDA and L-cysteine. Other L-glutamate analogs and odorants can only partially inhibit L-glutamate binding to these sites. Physiological studies of L-glutamate-best olfactory receptor neurons show that NMDA and L-cysteine both function as partial agonists and antagonists of these glutamate receptors.

2. Materials and methods 2.1. Animals For radioligand receptor-binding experiments, adult Caribbean spiny lobsters Ž Panulirus argus . with carapace length ) 80 mm were collected from commercial fish houses in the Florida Keys. Lateral filaments of the antennules Žolfactory organ. were removed from live animals and placed in tubes containing Tris buffer with the following composition Žin mM.: 50 KCl, 10 Tris base, 320 sucrose, 12.9 CaCl 2 , 23.1 MgCl 2 , and 25.6 MgSO4 , pH 7.8. The antennules were frozen at y808C. For electro-

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physiological experiments, females and males with 40–80 mm carapace length were collected in the Florida Keys and transported to our laboratory where they were held in aquaria containing Instant Ocean w and were fed shrimp and squid. 2.2. Chemicals Stimuli used in our study were the odorant compounds and glutamate analogs listed in Table 1. These odorant compounds were chosen because they are present in natural prey of P. argus w7,9x and they are physiologically and behaviorally active w18,20,21x. All odorant compounds were purchased from Sigma and were ) 99% pure. These glutamate analogs were selected because they have been used to characterize specific types of metabotropic and ionotropic glutamate receptors of vertebrates and invertebrates. The analogs were obtained from Research Biochemical Inc., with the exception of CCG-I which was purchased from Tocris. For radioligand receptor-binding assays, wL- 3 Hxglutamic acid Žspecific activity, 25.0 Cirmmol. ŽAmersham. was used. Stock solutions of odorants and analogs were made up in modified Panulirus argus saline Žs MOPS buffer. at 10 mM, pH 7.4, and frozen in aliquots at y808C Žexcept L-cysteine which was made on the day of the experiment.. Stimuli were serially diluted with MOPS buffer at the beginning of each experiment. MOPS buffer contained Žin mM.: 480 NaCl, 10 KCl, 17 MgCl 2 , 17 Table 1 Chemical stimuli Odorants X X Adenosine-5 -monophosphate Ž5 -AMP. Ammonium chloride ŽNH 4 . Betaine ŽBET. L-Cysteine ŽCYS. L-Glutamate ŽGLU. Glycine ŽGLY. L-Proline ŽPRO. D,L-Succinate ŽSUCC. Taurine ŽTAU. Glutamate analogues Metabotropic agonists trans- "-1-Amino-1,3-cyclopentanedicarboxylic acid ŽACPD. Ž".-Ibotenic acid ŽIBO. ŽŽ2 S,3S,4S .-a-Carboxycyclopropyl.-glycine ŽCCG-I. Non-NMDA ionotropic agonists Ž".-a-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid ŽAMPA. Kainic acid ŽKA. Žq.-Quisqualic acid ŽQA. NMDA ionotropic agonist N-methyl-D-aspartic acid ŽNMDA. Metabotropic antagonist 6-Cyano-7-nitroquinoxaline-2,3-dione ŽCNQX. NMDA ionotropic antagonist L-Žq.-2-Amino-5-phosphonopropionic acid ŽL-AP-5. Agonist at glycine site of NMDA receptor D-Serine ŽSER.

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CaCl 2 , 21 Na 2 SO4, 1.7 glucose, 3.5 MOPS, with pH adjusted to 7.4. The pH and ion concentration used in this buffer are equal to those found in the hemolymph of spiny lobsters w11,17x. MOPS buffer was used instead of other buffer solutions because results of preliminary experiments showed that the specific binding of wL- 3 Hxglutamate was moderately enhanced in the presence of both MOPS and sodium Ždata not shown.. For electrophysiological experiments, aliquots of stock odorants and NMDA were prepared at 10 mM in artificial sea water ŽASW. at pH 8.1 w11x and frozen at y808C Žexcept L-cysteine which was made on the day of the experiment.. For each experiment, stock solutions of stimuli were serially diluted with ASW, which was also used as the control stimulus. 2.3. Biochemical experiments Our techniques were similar to those used in previous studies of P. argus olfactory membrane w41,46,47x. 2.3.1. Membrane preparation The paired lateral filaments of the antennules contain aesthetasc sensilla w24,26x. Located within the lumen of each aesthetasc sensillum are the dendrites of the olfactory receptor neurons. The aesthetasc sensilla were manually removed from the antennule and placed in MOPS buffer at 48C. To extract the dendritic membrane, the sensilla were manually homogenized in a glass–glass homogenizer and then sonicated Ž5 times for 5 s each on setting 10 of a Fisher Sonic Dismembrator Model 50 with a titanium alloy microtip.. The sonication was followed by centrifugation at 6000 = g for 10 min. The resultant pellet ŽP1. contained cuticle and the supernatant ŽS1. contained dendritic membrane and other cellular components. The S1 was removed and set on ice. The P1 was resuspended in MOPS buffer and centrifuged again at 6000 = g for 10 min. The S1 was removed and combined with the previously collected S1 and centrifuged at 150 000 = g for 30 min. The subsequent supernatant ŽS2. was discarded and the pellet ŽP2. was resuspended in 1 ml MOPS buffer and centrifuged again at 150 000 = g for 30 min. The P2 was resuspended in MOPS buffer to form the P2 fraction, which contained dendritic membrane of olfactory receptor neurons. All procedures for the membrane preparation were carried out at 48C. 2.3.2. Binding assay To assess the binding activity of the olfactory tissue, aliquots of the P2 fraction were incubated with wL3 x H glutamate at 1 mM Žunless specified otherwise. in MOPS buffer at 48C. Membrane from about 3.5 antennules Žca. 15 mg of protein. was typically incubated for 60 min in wL- 3 Hxglutamate in a total volume of 50 ml. Bound radioligand was separated from free radioligand by rapid filtration under vacuum through 0.45 mm pore-size cellu-

lose acetate filters ŽType HAWP, Millipore.. Prior to use, the filters were presoaked in 0.3% polyethylimine diluted in MOPS buffer and washed three times with 5 ml of ice-cold buffer. Following filtration, the filters were removed and dissolved in 1.3 ml of ethylene glycol monomethylether for 30 min prior to addition of scintillation fluid. Radiolabel content was assayed in Ecolite Žq. ŽICN Biomedical, Inc.. using a Beckman LS6500 liquid scintillation counter with a counting efficiency of 66%. Total binding and non-specific binding were characterized by incubation of P2 and wL- 3 Hxglutamate in the absence Žfor total binding. or presence Žfor non-specific binding. of 1 mM unlabeled L-glutamate. Specific binding was the difference between total binding and non-specific binding. Protein concentrations were determined according to Bradford w5x using bovine serum albumin as the standard. Binding data were analyzed by non-linear regression using Inplot ŽGraphPad Inc.. and LigandrKinetic ŽBiosoftrElsevier.. 2.3.3. Association and dissociation experiments Association experiments were performed to determine the time of incubation necessary to achieve steady-state equilibrium binding. Aliquots of the P2 fraction were incubated in 1 mM wL- 3 Hxglutamate for 0, 0.5, 1, 2, 5, 10, 15, 20, 30, 45, 60, and 75 min, with Žfor non-specific binding. or without Žfor total binding. 1 mM unlabeled L-glutamate. The resulting data were fitted to a non-linear regression using a single-exponential term equation w44,65x. Dissociation experiments determined the reversibility of binding. Aliquots of P2 fractions were incubated with 1 mM wL- 3 Hxglutamate for 60 min, at which time 1 mM unlabeled L-glutamate was added. Incubation then continued for 0, 1, 2, 3, 4, 5, 10, 20, 30, and 60 min, followed by filtration. The resulting data were fitted to non-linear regressions. 2.3.4. Saturation experiments Saturation experiments were performed to determine if the association of L-glutamate to olfactory membrane represented binding to receptors or was due to internalization. wL- 3 HxGlutamate binding was determined using concentrations of 10y8 to 10y4 M. To determine the binding affinity Ž K d . and the binding capacity Ž Bmax . for wL3 x H glutamate at equilibrium, the specific binding data were analyzed by non-linear regression. The data were tested with one-site and two-site models w44,65x. 2.3.5. Inhibition experiments To measure the ability of odorants and glutamate analogs to displace wL- 3 Hxglutamate from specific binding sites, aliquots of the P2 fraction were incubated with 1 mM wL- 3 Hxglutamate plus increasing concentrations of potential inhibitors. Specific binding in the absence of unlabeled glutamate was defined as 100%, and binding in the presence of inhibitor was expressed as a percentage of this

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value. Non-specific binding was determined from aliquots of P2 incubated in the presence of 1 mM wL- 3 Hxglutamate plus 1 mM unlabeled L-glutamate. The binding data were fitted to one-site and two-site inhibition equations. An F-test was used to determine the best fit of the same data set to the two equations w44x. IC 50 values were calculated for each inhibitor, if possible. 2.4. Electrophysiological experiments Our aim in electrophysiological experiments was to determine the ability of NMDA and L-cysteine to function as agonists or antagonists at L-glutamate receptor sites on olfactory receptor neurons ŽORNs. that respond maximally Ži.e. ‘best’. to L-glutamate. Odorants ŽTable 1. were also tested to establish the response spectrum of L-glutamate ORNs, and thereby allowing us to infer the sensitivity of receptors that are functionally expressed on these neurons. 2.4.1. Single-unit extracellular recordings This preparation has been described in detail elsewhere w17x. The lateral filament of the antennule of the spiny lobster was excised and the distal end was placed in a Teflon w tube in which ASW was continuously flowing at 10 cmrs. The proximal end was inserted into a lucite recording chamber containing P. argus saline. The antennular nerve was exposed and the antennular artery was cannulated and perfused with oxygenated P. argus saline. Fine-tipped suction electrodes were used to record action potentials from single axons in the nerve bundles. A valve in the olfactometer injected a 6-s pulse of the stimulus into the ASW flow. Responses were recorded on tape and analyzed using Data-Pac II ŽRun Technologies Inc... Responses were measured as the number of spikes induced by a stimulus during a 500-ms interval following onset of the response, which occurred ca. 1 s after stimulus delivery. The 500-ms time bin was chosen because it is a typical time interval between successive flicks of the lateral antennules w24x. The delay between stimulus delivery and onset of the response was held constant for all stimulations of individual ORNs. For each ORN, the greatest response delay for the best stimulus was calculated and used as the time to begin counting for those stimuli that evoked little or no neural activity. 2.4.2. Experimental protocol L-Glutamate-sensitive ORNs were identified by an increase in spiking rate when presented with L-glutamate at 10y4 M. Then, each cell’s response spectrum was characterized by recording responses to the nine odorants ŽTable 1. at 10y4 M presented in random order. Only those cells for which L-glutamate was the most excitatory odorant Ži.e. glutamate-best cells. were used in this study. After testing the odorants at 10y4 M, the following stimuli were tested in random order: L-glutamate at concentrations of 10y4 , 10y5 , and 10y6 M, NMDA and L-cysteine at 10y4 and

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10y5 M, and binary mixtures of 10y6 M L-glutamate plus 10y4 or 10y5 M of NMDA or L-cysteine. The concentrations of L-glutamate, NMDA and L-cysteine were the same as those used in the inhibition binding assays. An interval of 2–2.5 min between stimulations was used to avoid adaptation. Each stimulus was tested at least twice on each cell. L-Glutamate and ASW were presented at every fifth stimulation to evaluate the responsiveness of the cell. If the response to any presentation of L-glutamate was 50% greater or less than the original response to glutamate, the data for that cell were discarded. 2.4.3. Data analysis Responses to single chemicals and mixtures were compared using ANOVA. Agonism was evaluated by comparing responses to single odorants and the ASW control. To determine if NMDA or L-cysteine antagonized the response to L-glutamate, the response to each binary mixture containing L-glutamate was compared to two values: Ž1. the response of the more effective component of the mixture; and Ž2. the response calculated using a non-competitive model – sum-of-responses model w1x using the equation R AB s R wAx q R wBx where R AB s the response to the blend of odorants A and B corrected for the response to the control stimulus ASW, and R wAx and R wBx s the ASW-corrected responses to odorants A and B, respectively, at their concentration in the blend. A response to the mixture that is less than the response to the more effective component clearly indicates antagonism. If the responses to both of the components are excitatory, a response to the mixture that is less than the response calculated by the sum-of-response model suggests that the two excitatory components do not have independent actions.

3. Results 3.1. Radioligand receptor-binding study 3.1.1. Kinetic experiments: olfactory L-glutamate binding sites haÕe characteristics of chemoreceptors The association and dissociation of 1 mM wL3 x H glutamate to the P2 fraction of olfactory tissue are shown in Fig. 1. The association curve ŽFig. 1A. showed that specific binding was rapid and saturable. Association data were fitted by a single-exponential equation with a half-association time Ž t 1r2 . of 11.6 min Ž r 2 s 0.992.. Binding reached equilibrium by 60 min. Dissociation data ŽFig. 1B. showed rapid dissociation of L-glutamate from olfactory tissue. The dissociation data were fitted by a single-exponential non-linear regression with a half dissociation time Ž t 1r2 . of 2.9 min Ž r 2 s 0.971.. Radioligand was largely dissociated by 10 min and completely dissociated by 40 min. This rapid, saturable, and reversible

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Fig. 3. Inhibition of 1 mM wL- 3 Hxglutamate binding to P2 fraction of olfactory membrane in the presence of varying concentrations of unlabeled L-glutamate. Each point is the mean"S.E.M. of six experiments, each performed in triplicate. Data were normalized to the maximum value for that experiment, represented by 100% specific binding.

association is consistent with the idea that wL- 3 Hxglutamate binds to sites on membrane of olfactory cells.

Fig. 1. Kinetic analysis of ŽA. association and ŽB. dissociation of wL- 3 Hxglutamate and the P2 fraction of olfactory membrane. Values are the means"S.E.M. from four association and two dissociation experiments each run in triplicate. Data were normalized to the maximum value for each experiment, represented by 100%. Data were fitted to pseudo first-order equations w65x with an association rate constant of 0.06 miny1 and a dissociation rate constant of 0.24 miny1 .

3.1.2. Saturation experiments: two sites with different affinities for L-glutamate In three experiments run in triplicate, specific binding of wL- 3 Hxglutamate was saturable and best fitted by a two-site model. Results from a representative experiment are shown in Fig. 2. For the three experiments, the mean " S.E.M. values of K d and Bmax for the high-affinity bind-

Fig. 2. A: non-linear saturation plot of specific binding of wL- 3 Hxglutamate to the P2 fraction of the olfactory membrane. Values are the means" S.E.M. from triplicate measurements from one experiment. Specific binding data were best fitted by a two-site model Ž r 2 s 0.989, F-test, F Ž6,8. s 24.2, P s 0.0009.. B: Rosenthal transformation of data in A. For the high-affinity site GLUA , K d s 3.72 " 1.02 nM and Bmax s 0.603 " 4.60 fmolrmg protein; for the low-affinity site GLUB , K d s 3.33 " 3.45 mM, Bmax s 12.75 " 54.85 fmolrmg protein.

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ing sites GLUA are: K d s 1.1 " 0.82 nM, Bmax s 0.64 " 0.58 fmolrmg protein, and for the low-affinity sites GLUB are K d s 3.3 " 4.4 mM, Bmax s 13.42 " 12.93 fmolrmg protein. 3.1.3. Self-inhibition experiments: two sites with different affinities for L-glutamate The P2 fraction of olfactory membrane was incubated in 1 mM wL- 3 Hxglutamate plus concentrations of unlabeled

Fig. 4. Inhibition of 1 mM wL- 3 Hxglutamate binding to P2 in the presence of varying concentrations of unlabeled ŽA. ammonium chloride Žv ., ŽB. X adenosine-5 -monophosphate ŽI., and ŽC. L-cysteine Ž^.. The solid curve represents the L-glutamate self-inhibition curve, from Fig. 3. Each point represents the mean"S.E.M. of triplicate determinations from four experiments in A, six experiments in B, and five experiments in C. The L-cysteine curve is not significantly different from L-glutamate curve Žtwo-way ANOVA, F Ž11,12. s1.84, stimulus effect, P s 0.176..

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ranging from 10y1 6 to 10y3 M. The resulting data were fitted better by a two-site competition model Ž r 2 s 0.808 . than a one-site competition model Ž F Ž142,151. s 139, P - 0.0001; Fig. 3.. The IC 50 values and percentage of total glutamate sites bound were 0.22 fM and 23.5% for the high-affinity site ŽGLUA . and 5.3 mM and 76.5% for the low-affinity site ŽGLUB .. L-glutamate

3.1.4. Inhibition by odorants: a diÕersity of effects Inhibition studies were performed to investigate the inhibition of L-glutamate binding by behaviorally relevant odorant molecules. As described below, odorants differed in their ability to inhibit L-glutamate binding: most odorants showed either one- or two-site partial inhibition, but L-cysteine caused two-site and complete inhibition. Inhibition data for NH 4 , L-proline, D,L-succinate, and taurine were best fitted by one-site, partial inhibition curves Ž r 2 ) 0.99.. The inhibition curve for NH 4 is shown as an example ŽFig. 4A.. The IC 50 values for NH 4 , L-proline, D,L-succinate, and taurine were ) 1 mM, with ca. 30% inhibition at the highest concentration tested Ž1 mM.. However, concentrations in the nanomolar range were effective as inhibitors. Inhibition data for 5X-AMP, betaine, and glycine were best fitted by two-site, partial inhibition curves Ž r 2 ) 0.99. and demonstrated 34–65% inhibition at 1 mM. The inhibition curve for 5X-AMP is shown as an example ŽFig. 4B.. The IC 50 values and the percentage of the total glutamate receptor sites bound by an inhibitor for the high- and low-affinity sites respectively are as follows: 5X-AMP, 12.7 pM Ž16.9%. and 6.5 mM Ž30.6%.; betaine, 1 fM Ž25.6%. and 18 mM Ž38.4%.; glycine, 56.7 fM Ž7.48%. and 66 mM Ž50%.. Inhibition data for L-cysteine also showed a two-site fit Ž r 2 s 0.995, F Ž11,13. s 12.0, P s 0.0059; Fig. 4C.. However, unlike the other odorants, 1 mM L-cysteine completely inhibited wL- 3 Hxglutamate binding. The calculated IC 50 values for L-cysteine were 0.193 pM Ž29% of the total binding sites. and 9.8 mM Ž71% of the total binding sites. for the high- and low-affinity sites, respectively. 3.1.5. Inhibition by L-glutamate analogs: a diÕersity of effects A variety of glutamate analogs ŽTable 1. were tested over a range of concentrations as inhibitors of 1 mM wL- 3 Hxglutamate binding to characterize the structure– function relationship for these olfactory L-glutamate receptors and to compare their specificity with that of glutamate receptors in other animals. Most showed one-site and partial inhibition, but one analog ŽNMDA. caused two-site and complete inhibition, as described below. The L-glutamate analogs AMPA and ACPD inhibited L-glutamate binding by a maximum of 35–45% and failed to fit any type of function, include one- or two-site curves

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0.956, F Ž12,14. s 16.4, P s 0.0004; Fig. 5C.. The IC 50 values and percentage of glutamate receptor sites inhibited were 2.6 fM Ž33%. for the high-affinity site and 20.7 mM Ž57%. for the low-affinity site. The IC 50 values for NMDA and L-glutamate differ by a log unit for the high-affinity sites and by a half-log unit for the low-affinity site, suggesting that NMDA may be a competitive ligand for both glutamate sites, but with a slightly lower affinity than L-glutamate. The selective vertebrate metabotropic antagonist CNQX and the selective ionotropic antagonist L-AP-5 failed to inhibit the specific binding of wL- 3 Hxglutamate by greater than 10%, even at 1 mM Ždata not shown.. D-Serine, an agonist at the glycine binding site of NMDA receptors, failed to enhance the binding of L-glutamate Ždata not shown.. 3.2. Inhibition by binary mixtures of L-glutamate analogs

Fig. 5. Inhibition of 1 mM wL- 3 Hxglutamate binding by unlabeled ŽA. kainate Že., ŽB. ibotenate Ž`., and ŽC. NMDA ŽB.. The solid curve represents the L-glutamate self-inhibition curve from Fig. 3. Each point represents the mean"S.E.M. of triplicate determinations from four experiments in A and B and of seven experiments in C.

or linear regressions. The L-glutamate analogs QA and KA inhibited glutamate by a maximum of 25–31% at millimolar concentrations, and the inhibition data were fitted by a one-site inhibition curve. Data for KA are shown as an example ŽFig. 5A.. The metabotropic L-glutamate agonists CCG-I and IBO were similar to QA and KA in that their inhibition data were best fitted by a one-site curve, but their maximum inhibition values were greater, approximately 37–47% at 1 mM. IBO is given as an example in Fig. 5B. NMDA, an agonist for the ionotropic glutamate receptor subtype, completely inhibited wL- 3 Hxglutamate binding and was best fitted by a two-site competition curve Ž r 2 s

3.2.1. Non-NMDA analogs bind to a similar subset of glutamate receptor sites The results in the previous section suggest that NMDA may bind to two subsets of L-glutamate receptors: one site that can also be occupied by any of several non-NMDA analogs, and a second site that is insensitive to non-NMDA analogs. We asked whether or not the former site was composed of more than one site by examining inhibition by mixtures of non-NMDA analogs. If non-NMDA analogs bind to the same sites, then we would expect that the analogs would act competitively, and therefore a mixture of the analogs would act as a higher concentration of either component. However, if the non-NMDA analogs bind to different receptor sites, then the inhibitory effects of the two should add when they are combined as a mixture. This experiment was performed using three mixtures whose components were at equimolar concentrations: ACPDq AMPA, ACPDq KA, and AMPAq KA. Between 10y7 and 10y3 M, the amount of inhibition by individual analogs or binary mixtures was relatively constant for each stimulus, as is shown for KA in Fig. 5A. The mean values for percentage of inhibition over this concentration range were the following: KA, 22%; AMPA, 31%; ACPD, 43%; ACPDq AMPA, 26.3%; AMPAq KA, 19%; ACPDq KA, 14%. Thus, since the inhibition values for the binary mixtures of non-NMDA analogs were not greater than the inhibition value for either component and were much less than the sum of the inhibition values for the components, we conclude that these glutamate analogs bind to the same subset of L-glutamate receptor sites rather than each binding to different sites. 3.2.2. NMDA and non-NMDA analogs compete for one site but NMDA is specific for a second Lglutamate site Binary mixtures were used to test the hypothesis that non-NMDA analogs bind to a subset of the NMDA-sensi-

L-glutamate

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tive L-glutamate receptors. We compared the ability of equimolar binary mixtures of NMDAq ACPD, NMDAq AMPA, and NMDAq KA, and of NMDA, ACPD, AMPA, and KA alone, to inhibit wL- 3 Hxglutamate binding to the P2 fraction. The inhibition data for these binary mixtures were best fitted by two-site curves, as was the case for NMDA alone. Between 10y8 and 10y6 M, the amount of inhibition by the binary mixtures Ž13–34% inhibition. was generally similar to the inhibition by either NMDA Ž22%. or the non-NMDA component of the mixture in this concentration range Ž12–43% inhibition.. Thus, the inhibitory effects of the components of the binary mixtures were not additive. Above 10y6 M, inhibition by the mixtures was dominated by NMDA, which was the more effective inhibitor in this concentration range. Results for the mixture of NMDAq KA are given in Fig. 6 as an example. These results suggest that NMDA and non-NMDA analogs compete for one set of L-glutamate sites, but NMDA solely inhibits a second L-glutamate site. 3.2.3. Inhibition by mixtures of L-glutamateq NMDA shows that NMDA is a competitiÕe inhibitor of L-glutamate binding The amount of inhibition of 1 mM wL- 3 Hxglutamate binding in varying concentrations of unlabeled L-glutamate plus 10y7 M NMDA was measured to determine whether or not NMDA and L-glutamate compete for the same receptor sites. This concentration of NMDA was selected because it completely inhibits the GLUA site without inhibiting the GLUB site ŽFig. 5C.. Thus, if NMDA and L-glutamate are competing for the same sites, we would expect that inhibition values for mixtures of L-glutamateq 10y7 M NMDA where the L-glutamate concentration is less than 10y7 M will be similar to the inhibition value for 10y7 M NMDA. On the other hand, inhibition values for mixtures of L-glutamateq 10y7 M NMDA where the L-

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Fig. 7. Inhibition of 1 mM wL- 3 Hxglutamate binding to P2 in the presence of varying concentrations of unlabeled L-glutamate plus 0.1 mM NMDA Ždotted line, I., varying concentrations of unlabeled L-glutamate alone Žsolid line, v ., and 0.1 mM NMDA Ž'.. Each point represents the mean"S.E.M. of triplicate determinations from seven experiments.

glutamate concentration is 10y6 M or greater will be similar to those for the L-glutamate inhibition curve over this same concentration range. The results ŽFig. 7. reveal that the inhibition data for the binary mixture of L-glutamate q 10y7 M NMDA were best fitted by a two-site competition curve Ž r 2 s 0.993. with K d values of 3.2 fM and 6.2 mM for the high- and low-affinity sites, respectively. Overall, the curves for L-glutamateq NMDA and for L-glutamate alone were statistically different Žtwo-way ANOVA: for both main effects, P F 0.00001.. The mixtures of L-glutamateq 10y7 M NMDA produced inhibition similar to that by the corresponding concentrations of L-glutamate alone for L-glutamate concentrations greater than 10y6 M, but more inhibition than L-glutamate at concentrations less than 10y6 M and more inhibition than 10y7 M NMDA alone. Together, these results suggest that NMDA competes with L-glutamate for the same binding sites, but, in addition, NMDA may be a partial, non-competitive inhibitor of L-glutamate binding. 3.3. Electrophysiological recordings show that NMDA and cysteine are mixed agonistsr antagonists

Fig. 6. Inhibition of 1 mM wL- 3 Hxglutamate binding to P2 in the presence of varying concentrations of unlabeled NMDA ŽB., kainate Ž^., and NMDA plus kainate Ž`.. Each point represents the mean"S.E.M. of triplicate determinations from three experiments for each binary mixture.

Responses were recorded from 11 glutamate-best ORNs. The response spectrum for each ORN is shown in Fig. 8. Comparison of the mean ASW-corrected responses to GLU, CYS, NMDA and binary mixtures ŽFig. 9. allows an evaluation of the agonistic and antagonistic properties of CYS and NMDA for the GLU responses. The responses to 100 mM CYS and NMDA were significantly greater than zero ŽFig. 9.. Furthermore, the responses to 100 mM CYS and NMDA were statistically similar to each other ŽANOVA, with Duncan’s test, P s 0.42. ŽFig. 9., but both were less than the response to 100 mM GLU ŽDuncan’s test, P - 0.001.. NMDA and CYS at

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10 and 100 mM were about half as excitatory as 1 mM GLU, and the responses to the two analogs were statistically similar to each other ŽDuncan’s test, P s 0.36. ŽFig. 9.. Thus, both CYS and NMDA are weak, but partial, agonists for these glutamate-best ORNs. The observed response to the binary mixture 1 mM GLU q 10 mM CYS was qualitatively less than the response to 1 mM GLU alone Ži.e. the more effective component, Fig. 9.. This is suggestive of some degree of antagonism, although these differences are not statistically different ŽDuncan’s test, P ) 0.05.. The responses to the binary mixtures containing NMDA Ž10 or 100 mM. q 1 mM GLU were qualitatively greater than the responses to GLU, but only the response to the binary mixture of GLU and 10 mM NMDA was significantly greater than that to GLU alone Ž P s 0.04.. The sum-of-responses model Žsee Section 2: Materials and methods. assumes that CYS, NMDA and GLU bind to different receptors and do not antagonize the responses of each other. Consequently, the modeled responses to these mixtures Žwhich are tested at concentrations below saturation levels. would be expected to be greater than the response to GLU alone Žsee Section 2.4.3.. Comparison of the response to each mixture with the response predicted from sum-of-responses models showed that the response to 10 mM CYS q 1 mM GLU was significantly less than the modeled response ŽDuncan’s test, P s 0.01., suggesting that CYS is not acting independently of GLU and may be an antagonist of GLU. On the other hand, the responses to

Fig. 9. Mean"S.E.M. responses of 11 glutamate-best ORNs to GLU Ž100, 10, and 1 mM., CYS Ž100 and 10 mM. and NMDA Ž100 and 10 mM. and the observed and predicted responses to mixtures of GLU Ž1 mM. and either CYS Ž100 and 10 mM. or NMDA Ž100 and 10 mM.. The values have been corrected for the response to ASW by subtraction. ) GLU response at concentration of 1 mM was significantly greater than both CYS and NMDA responses ŽANOVA, P F 0.05.. L-Cysteine Ž100 and 10 mM. and NMDA Ž100 and 10 mM. were not significantly different from each other. The closed circle Žv . above the mixture of 1 mM GLU plus 100 mM NMDA designates it as the only mixture whose observed response was significantly different from 1 mM GLU.. The open circle Ž`. indicates that the observed response to the mixture of 1 mM GLUq10 mM CYS was significantly less than its predicted response.

binary mixtures of 1 mM GLU and either 10 or 100 mM NMDA are only slightly below the sum of the responses to the components ŽDuncan’s test, P s 0.31.. This suggests that there is only a modest amount of antagonism between NMDA and GLU, certainly less than between CYS and GLU. Therefore, CYS appears to be the stronger antagonist of GLU.

4. Discussion 4.1. Olfactory glutamate receptor sites in the spiny lobsters

Fig. 8. Response spectra of 11 glutamate-best olfactory receptor neurons ŽORNs. from electrophysiological assays. Shown are the responses of the ORNs to single chemicals and mixtures. Cells are arranged by the best-compound classification. All chemicals were tested at 10y6 M unless otherwise indicated. G sGLU, C sCYS, Ns NMDA, NH s ammonium chloride; 4 s10y4 M, 5s10y5 M, 6 s10y6 M. Type and size of the dot represent responses as standardized to the best compound for each ORN. Closed dots represent excitatory responses; open dots represent inhibitory responses; no dot represents no response. Closed dots of 4 sizes are shown. From smallest to largest they represent responses 1–25%, 26–50%, 51–75%, and 76–100% of the maximum response of that cell. Open dots of one size are shown, representing inhibitory responses whose absolute values are 1–25% of the maximum response of that ORN.

Direct measurement of the association and dissociation of radiolabeled L-glutamate to dendritic membrane from olfactory receptor neurons in the antennules of the spiny lobster Panulirus argus revealed that the association is rapid, reversible, and saturable ŽFig. 1.. These features suggest that the association represents binding rather than internalization. Furthermore, saturation kinetic analysis revealed that the L-glutamate binding is of high affinity and that there are two sites with different affinities. Inhibition studies showed that the binding of L-glutamate to olfactory sites is fairly specific, but can be completely inhibited by L-cysteine and NMDA. Most odorants and L-glutamate analogs, especially the non-NMDA

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analogs, were only partial inhibitors of L-glutamate binding, even at millimolar concentrations ŽFigs. 4 and 5.. Only NMDA and L-cysteine inhibited L-glutamate binding by more than 65%. At 1 mM, both produced two-site inhibition curves and complete inhibition of 1 mM wL3 x H glutamate binding at 1 mM ŽFig. 4CFig. 5C, respectively., as did L-glutamate in self-inhibition studies ŽFig. 3.. These results suggest that both NMDA and L-cysteine are highly effective competitors at both affinity sites. Results of binding inhibition using mixtures of L-glutamate plus NMDA ŽFig. 7. suggest that NMDA may not only compete with L-glutamate for binding sites, but may also non-competitively antagonize L-glutamate binding. We examined electrophysiological responses of glutamate-excited cells to L-glutamate, L-cysteine, NMDA, and binary mixtures of L-glutamate plus either NMDA or L-cysteine to investigate the efficacy of NMDA and L-cysteine. The cells in this study were all glutamate-best cells and the concentrations of the stimulants were the same as those used in the radioligand-receptor binding study. The results suggested that both L-cysteine and NMDA are partial agonists and weak antagonists at the glutamate receptors, and that L-cysteine is more effective than NMDA as an antagonist ŽFig. 9.. The partial agonist and antagonist activities of cysteine and NMDA are consistent with the binding studies that show cysteine and NMDA inhibit glutamate binding. However, from the results of the binding studies, we expected cysteine and NMDA to be even more effective as either agonists or antagonists. This quantitative difference between the binding and physiological results may be due to several factors. First, by necessity, the glutamate receptors were maintained in different conditions in the biochemical and electrophysiological experiments. In electrophysiological experiments, the receptors were maintained in conditions as close as possible to in vivo conditions. In binding experiments, however, the receptors were present on membrane that was isolated from ORNs and tested in vitro as vesicles in a different buffer solution. These different conditions could affect their relative activities in the two assays. Second, in the binding study, we examined all glutamate-binding sites in this olfactory tissue. These sites included not only all glutamate olfactory receptor sites mediating excitation as in the electrophysiological study, but also glutamate receptors mediating inhibition, which are known in the olfactory system of P. argus w20x, and glutamate uptake sites involved in clearance of odorants from the sensillar lymph w59x. Finally, the binding sites in the biochemical study could have included other ‘spare’ receptors that are not coupled to transduction processes but that can still bind odorants w3,66x. On the other hand, in the physiology study, in which we examined one cell or cell type at a time, we focused on a smaller set of glutamate binding sites – only those sites coupled to transduction. In contrast to NMDA and L-cysteine, the odorants 5X-AMP, betaine, ammonium, succinate, taurine and the

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mammalian non-NMDA L-glutamate agonists AMPA, ACPD, kainate, CCG-I, ibotenate, and quisqualate were only partial inhibitors of L-glutamate binding and presumably bind only to a subset of the L-glutamate sites. A further indication of two subsets of glutamate binding sites, only one of which is inhibited by non-NMDA analogs, is the results of binding inhibition assays using binary mixtures of L-glutamate analogs ŽFig. 6.. In these experiments, mixtures of NMDAq non-NMDA L-glutamate analogs produced two-site inhibition curves. In contrast, mixtures of two non-NMDA L-glutamate analogs showed one-site inhibition curves, suggesting that they competed for the same subset of L-glutamate receptors. Similarities in the affinity and specificity of L-glutamate binding to olfactory tissue of P. argus with the efficacy of L-glutamate as defined in previous electrophysiological studies of ORNs of P. argus suggest that these binding sites may be receptors coupled to olfactory transduction. On average, L-glutamate-best ORNs have half-maximal responses at approximately 1 mM and response thresholds below 0.1 mM w20x. In addition, many L-glutamate-best olfactory receptor neurons show high specificity to Lglutamate w15,20x. The two L-glutamate binding sites with different affinities may be either two distinct types of molecules or two affinity states of the same molecule. Two affinity states occur in a number of receptor systems, including L-glutamate-mediated systems and sensory systems that are coupled to intracellular effector systems, such as protein kinases and G-proteins w27,33,34x. However, the difference in the affinities for the two L-glutamate sites – nearly six orders of magnitude – is much larger than that which usually occurs through allosteric interactions involving G-protein coupled receptors w10,40x. Thus, these results favor the presence of at least two different types of receptor sites for L-glutamate. These binding sites might represent a specific L-glutamate receptor, with a high affinity for glutamate, and a generalist receptor, with a relatively low affinity for glutamate. The binding results using odorants as inhibitors of glutamate binding ŽFig. 4. were consistent with this idea, since they showed only partial inhibition. The IC 50 value for L-glutamate self-inhibition ŽFig. 3. and the K d value from saturation binding experiments ŽFig. 2. correlated well for the low-affinity, but not the high-affinity L-glutamate binding site. We believe that, compared to the IC 50 value, the K d value is a better indicator of the affinity of L-glutamate for the high-affinity site. This is because in our inhibition studies, the unlabeled L-glutamate concentrations near the IC 50 value were so much lower than the concentration of radiolabeled Lglutamate Ž1 mM. that accurate measurements of inhibition were difficult. Our attempts to measure IC 50 values for the high-affinity site using wL- 3 Hxglutamate concentrations near the K d value were unsuccessful because the combination of the low concentration of this site in the olfactory tissue

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and the low radioligand concentration did not yield levels of binding above background using filtration techniques. Both L-glutamate sites could be functionally important to olfaction for spiny lobsters, given the concentrations of L-glutamate in their environment. The background concentration of L-glutamate in sea water is often in the nanomolar range w32,42x and in some water constitutes 7.7% of the total dissolved amino acid pool in sea water, whereas concentrations in tissue of potential prey are usually micromolar to millimolar w7–9x. Thus, biologically relevant concentrations of L-glutamate are normally between millimolar and nanomolar, and consequently the different glutamate receptors may be functionally important under diverse physiological conditions. Inhibition of glutamate binding by other odorants may have functional significance in determining the responsiveness of spiny lobster olfactory neurons to mixtures. This is supported by the finding that a model of olfactory neuron transduction that incorporates binding inhibition accurately predicted responses of binary mixtures, including those containing L-glutamate w15x. 4.2. Comparison of glutamate olfactory receptors of spiny lobster with L-glutamate receptors in other systems 4.2.1. NMDA receptors Olfactory glutamate binding sites of P. argus appear to be novel NMDA-sensitive glutamate receptors. Unlike the classic vertebrate NMDA receptors w37x, the L-glutamate olfactory receptors did not show enhanced binding of L-glutamate or NMDA when in the presence of glycine or D-serine and were not inhibited by L-AP-5. The fact that 3 L-cysteine and NMDA displaced wL- Hxglutamate at NMDA receptors in chick retina and rat CNS w45x and in the olfactory organ of P. argus suggests that these receptors may have some similarities in binding properties. The non-NMDA analogs, whether they are ionotropic or metabotropic, appear to share similar binding sites on the low-affinity receptor GLUB of P. argus. Therefore, GLUB may be a mixed L-glutamate receptor type that can bind many types of L-glutamate agonists. Evidence of mixed muscarinicrcholinergic in receptors of invertebrates has been reported w39x; however, there has been no previous evidence of a mixed ionotropicrmetabotropic L-glutamate receptor in invertebrates. 4.2.2. InÕertebrate L-glutamate receptors L-Glutamate receptors are common at neuromuscular junctions and CNS of invertebrates, but they differ in their pharmacology from each other and from the olfactory receptors of P. argus, suggesting a diversity of receptor types w13,28,51,52,60,63x. For example, glutamate receptors at crayfish neuromuscular junctions cause depolarization in response to quisqualate and to a lesser extent to

kainate and the metabotropic glutamate analogs ACPD and CCG-I w52–54x. Secondly, ibotenate activates glutamate receptors on the presynaptic terminal of neuromuscular junctions of crayfish and extrajunctional receptors on locust muscle w38,53x. Thirdly, ibotenate, quisqualate and the metabotropic glutamate analog, ACPD, also are active in molluscan preparations w36x. Finally, glutamate receptors at neuromuscular junctions of insects are activated by the L-cysteine derivative L-cysteate w62x. Such comparisons of the binding properties of the spiny lobster’s olfactory glutamate receptors with those of glutamate receptors of other invertebrates show that they are different. 4.2.3. Glutamate chemosensory receptors A variety of organisms, including unicellular organisms, cnidarians, aquatic crustaceans, fish, and mammals, have L-glutamate-sensitive chemoreceptors that typically mediate feeding or searching behavior Žsee Section 1: Introduction.. In crustaceans, L-glutamate elicits feeding responses and walking w7,8,21x. In addition, physiological recordings from chemoreceptive neurons in the walking legs w19x and third maxillipeds w14x of the American lobster Homarus americanus and walking legs of the crayfish Austropotamobius torrentium w29x reveal cells with fairly narrow tuning to L-glutamate. The glutamate chemoreceptors in these diverse invertebrate species have physiological efficacies in the same range as the olfactory L-glutamate receptors of P. argus, yet there appears to be a diversity of types of glutamate receptors across these species. The glutamate receptors of the Arctic char SalÕelinus alpinus w57x differ from those of crayfish and other fish w35,55x in that they are activated by L-cysteine. In this way, olfactory L-glutamate binding sites of P. argus are more similar to those of char than crayfish or catfish. Furthermore, Lglutamate receptors of P. argus are similar to glutamate receptors in many invertebrate and vertebrate species in that they do not exhibit high specificity of binding to one particular ligand type and they have broad physiological response profiles to mammalian glutamate analogs w2,22,29x. 4.3. Summary The data presented here suggest that there are at least two types of glutamatergic receptors in olfactory neurons of the antennules of spiny lobsters, both of which also bind NMDA and L-cysteine. These receptors have similarities and differences with glutamatergic receptors in internal tissues and chemosensory organs of other species. Our electrophysiological results on the efficacy of NMDA and L-cysteine suggest that these receptors function in olfactory transduction and that L-cysteine is a better antagonist at the L-glutamate receptors of L-glutamate-best olfactory neurons.

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Acknowledgements We thank Karl Wagner of Coral Keys Fisheries, Gary Nichols of Conch Key Fisheries, and the staff of the Florida Keys Marine Laboratory, Long Key, Florida Žespecially John Swanson, Bill Gibbs, Chris Humphrey, Anita Duckworth, and Trent Moore. for help with collecting antennules and live animals. We also thank W.H. Lynn and Kirby Olson for their assistance in the radioligand binding assays, and Timothy Bartness, Stuart Cromarty, Vitaly Vodyanoy, and William Walthall for critically reviewing previous versions of the manuscript. Supported by NIH DC000312 and NSF IBN-9109783.

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