Neuroscience Letters, 41 (1983)145-150 Elsevier Scientific Pubfishers Ireland Ltd.

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BRUCE R. JOHNSON and JELLE ATEMA

Boston University Marine Program, Marine Biological Laboratory, Woods Hole, MA 02543 (U.S.A.)

(Received July 5th, 1983; Revised version received and accepted July 29th, 1983)

Key words: Homarus americanus - Crustacea - chemoreception - olfaction - taste - narrow-spectrum chemoreceptors - amino acid receptors - hydroxy-L-proline receptors - taurine receptors

The present study shows that smell (antennular) receptor cells are as narrowly tuned to single com- pounds as taste (leg) receptor cells in the lobster. Antennular receptors responded best to hydroxy-L- proline (57070 of the 30 cells sampled) and taurine (240;'0). In the presence of 14 mher compounds in equimolar concentrations, the hydroxy-L-proline and taurine receptors showed a suppressed response to their best stimulus. Other cells had best responses to ammonium chloride, betaine, L-glutamate or L- proline. The results have implications for molecular receptor processes and for the neural basis of feeding behavior.

The lateral filament of the lobster's biramous antennules is a chemoreceptor oig.~.~-~'ought to function analogous to the vertebrate sense of smell [2]. In both hc 3~,~:id [6] and panulirid [13] lobsters, the lateral filament has been demonstrated to ~,gger search for and orientation to an odor source. Electrophysiological ex- periments have found chemoreceptors on the lateral filament which respond to low molecular weight compounds, particularly amino acids [/ , 9, 14], someofwhich are attractive as feeding stimuli in ttomarusamericanus [12]. Chemoreceptors specifical- ly sensitive to taurine and a fe'x o f its analogs have been found o n t h e antennules of the spiny lobster [7] but it is not known if other specialized chemoreceptor popvJations also occur. Despite the work on the behavorial function o f this chemosensory organ and the stimuli which excite it, we know little about the in- dividual chemoreceptor cells in the antennules. To understand the neural mechanisms of odor orientation in the lobster, the qualitative (spectral) and quan, titativeJdynamic) discrimination of chemical stimhii by the peripheral receptors must be clarified.

In the present study we survey the response spectra of the lateral filament chemoreceptors on the antennules of the American lobster, Homarus americanus, and compare their spectral properties with those of the walking leg chemoreceptors. Populations of chemoreceptors on the walking legs of Homarus have been described

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which have very restricted response spectra, each population responding maximally to either L-glutamate, L-glutamine, L-arginine, taurine, betaine or ammonium chloride [3]. Leg chemoreceptors are essential for the performance of normal feeding behavior in this species [5] and are analogous to the vertebrate sense of taste

[21. Details of the preparation chamber, dissection and extracellular recording pro-

cedure were ~ssentially those of Johnson and Ache [9] except for the use of a Homarus Ringer [8|. The temporal dilution profile of a 50 ~1 aliquot of chemical stimulus was inferred by measuring the change in conductance of flowing, deionized water (10 ml/min) after the injection of 50 td of a salt solution. The measured con- centration rapidly peaked within 1.5 sec to approximately 0.025 times the injected concentration and after 5 sec declined to approximately 50°7o of the peak concentration.

Single chemoreceptor cells were identified with a search stimulus (SS) composed of the following 15 compounds, prepared in artificial sea water (ASW), all at an in- jected concentration of 10 -a M: taurine, L-hydroxyproline, L-glutamate, L- glutamine, ammonium chloride, L-arginine, sucrose, ethanol, L-alanine, L-lysine, be- taine, L-aspartate, glycine, L-leucine and L-proline. At the measured concentration, the pH of all solutions was within 0.15 units of the ASW pH (7.9). The test stimuli were chosen as examples of compounds with high, medium and low stimulatory ability based on previous studies of Homarus chemoreceptors [3, 4, 141. All single SS components injected at 10 -4 M (applied peak at about 2.5 x 10 -6 M) and an ASW control were then tested individually to determine the response spectrum for a particular ceil. Stimuli were applied in a fixed order with every sixth stimulus the SS. Through individual test series it was noted that responses to subsequent SS injec- tions could increase by a mean of 60°7o (± 15.1 S.E.M., n=20) over previous responses to the SS. From this we assumed (for the time being without specific ex- perimental evidence) that normal responses to repeated stimuli could vary by as much as 60070. This allowed us to set our rejection criterion for preparation viability as the failure to obtain for every SS response at least 4007o of the initial SS response. The data reported here are only for single chemoreceptor cells successfully exposed to the entire series of 15 test compounds. Stimuli were introduced approximately every 1.5 min with vigorous flushing of the chamber between stimuli.

The total number of action potentials occurring in the first 5 sec following the onset of a response was used as a measure of stimulus effectiveness. Action poten- tials were either counted manually or with a window discriminator in conjunction with a rate-interval analyzer. Action potentials from individual chemoreceptors were discriminated on the basis of amplitude, wave form and relative latency after the test stimulus was introduced. We standardized for every cell its responses to single compounds against the average of the previous and subsequent SS response. Thus responses are reported relative to the SS response (10007e).

The complete series of compounds were successfully tested on 30 individual

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TEST STIMULI

Fig. 1. Response spectra of two chemoreceptor populations in the antennular filaments of H. americanus. One population responds best to hydroxy-L-proline (open bars, n= 17) and the other to taurine (hatched bars, n = 7). The bars represent the mean response + S.E.M. of all cells in each popula- tion to the test stimuli, standardized to the SS response (100%).

chemoreceptor cells in 10 lateral filaments from 10 lobsters. Almost all of the cells had very narrow response spectra (Fig. 1). Most cells (17) responded best to hydroxy-L-proline; 9 of these did not respond to the other test stimuli. Only two of the hydroxy-L-proline best cells responded appreciably to other compounds: one cell had a response of 151 °7o tO hydroxy-L-proline, 86°/o to taurine, 78°7o to L-aspartate and 62o7o to L-leucine; the other was almost equally stimulated by hydroxy-L-proline (64o70) and L-glutamate (54o]0). The second most frequently encountered cell type responded best to taurine (7 cells); their response spectra were as narrow as those of the previous type (Fig. 1).

Two other cells responded best to ammonium chloride (95°'/0) with only a weak response to other test stimuli (largest response 24o70, to L-alanine). One cell respond- ed only to L-glutamate (50o7o) and another only to betaine (78~o). One cell responded best to L-proline (92o70) but also gave a large response (83°/o) to L-aspartate and smaller responses to the other test compounds. Finally, one cell responded only to the SS mixture.

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The cells which responded best to hydroxy-L-proline and taurine dearly respond- ed better when their best compound was presented alone (2700'0 and 182% respec- tively) than within the SS mixture (100070 per definition) (Figs. I and 2). The dif- ference is significant in both cases (matched pair t-test, P<O.01). This suggests an inhibitory effect of the other compounds present despite the restricted response spectra. However, in other cells the stimulatory effectiveness of individual com- pounds was enhanced by other compounds: one of the hydroxy-L-proline best cells gave responses to L-glutamate and hydroxy-t.-proline which together almost equaled the SS response; the betaine and L-glutamate best cells were stimulated more by the SS; and one cell was stimulated only by the SS, i.e. the entire group of test stimuli.

These results demonstrate the frequent occurrence of primary chemoreceptor cells with narrow response spectra on the antennules of Homarus americanus: both smell and taste organs in this species comain receptor cell populations with best responses to L-glutamate, taurine, betaine and ammonium. In addition, the antennules contain a very prominent hydroxy-L-proline cell population, which was not encountered in the legs despite focused search [3]. The legs, however, contain L-glutamine and L- arginine cells not encountered in the antennules, but our small sample size does not exclude their presence. The receptor cell populations thus far described for legs and

SS I

A

O H - P r o 1

S S 2

T a u 2

Fig. 2. Extracellular records of two antennular chemoreceptors in H. americanus in response to the stan- dard stimulant (SS) and hydroxy-L-proline, cell 1, and the SS and taurine, cell 2. Time calibration, 500 msec. Stimulus on at arrow.

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antennules of H. americanus are among the most narrowly tuned chemoreceptor cells known, resembfing in their specificity pheromone receptors of saturniid moths [I0]. Such preparations make excellent tools for pharmacological studies of molecular receptor processes. The fact that simple amino acids are the key stimuli for these cells is a significant advantage for receptor binding studies.

We know that in this lobster-species the antennular and leg chemoreceptor organs perform a necessary function in food orientation [6], localization and manipulation [5]. To what advantage, then, does the lobster use narrowly tuned amino acid recep- tors in the search of food? Why do antennules but not legs show a prominent population of hydroxy-L-proline best cells? At present these questions cannot be answered. The thresholds and dynamic properties of the receptor cells need to be investigated along with studies on the behavorial function of narrowly tuned cells in orientation to and identification of food. Theoretically, narrowly tuned receptors should have better dynamic resolution in a noisy odor environment.

A most intriguing result of this study is that most, but not all of the narrowly tun- ed cells responded far more strongly to their best compound when presented alone than when presented in a mixture of 15 compounds in equimolar concentrations in- cluding the best compound. This contrasts sharply with behavorial results where mixtures are much more effective than single compounds in eliciting feeding behavior [I 1, 12]. This raises interesting questions about the central processing of chemosensory information. In addition, elucidating the mechanism of their response-suppressing effect may give significant information about primary chemoreceptor processes. Thus, the antennular chemosensory system of the lobster provides a model to examine not only molecular receptor processes, but also neural mechanisms of olfactory orientation and physiological processes underlying the behavioral differences in smell and taste of aquatic animals.

This work was supported l~y grants from the Whitehall Foundation and the Na- tional Science Foundation (BNS 8210434).

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