Learned olfactory discrimination versus innate taste responses to amino acids in channel catfish (Ictalurus punctatus)

Physiology& Behavior,Vol. 55, No. 5, pp. 865-873, 1994 Copyright © 1994ElsevierScienceLtd Printedin the USA.All rights reserved 0031-9384/94$6.00 + .oo

Pergamon 0031-9384(93)E0028.O

Learned Olfactory Discrimination Versus Innate Taste Responses to Amino Acids in Channel Catfish (Ictalurus punctatus) TINE VALENTIN~I~, 1 SANDRA WEGERT 2 AND JOHN CAPRIO 3

Department o f Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803 R e c e i v e d 15 J u n e 1993 VALENTIN(L'I(~,T., S. WEGERT AND J. CAPRIO. Learned olfactory discrimination Versus innate taste responses to amino acids in channel catfish Octalums punctatus). PHYSIOL BEHAV 55(5) 865-873, 1994.--Intact channel catfish conditioned to the L-amino adds, proline, arginine, alanine, and lysine, discriminated these stimuli from all other amino acids tested. Behavioral structure-activity tests indicated that L-pipecolate was the only effective agonist of the L-proline conditioned response. For channel catfish in which one of the paired olfactory organs was surgically removed, the number of turns to the conditioned stimulus was 40% fewer than those of intact catfish; however, these semiosmic channel catfish discriminated the conditioned from nonconditioned stimuli, as evidenced by their responding to the conditioned amino acid, with a two- to threefold greater number of turns than to the nonconditioned amino acids. Irrespective of the number of conditioning trials attempted, catfish with both olfactory organs removed were unable to discriminate the conditioned from the nonconditioned stimuli. Chemical senses

Olfaction

Behavior

Catfish

Conditioning

A basic question in the study of the chemical senses is why there exists two major chemical senses in vertebrates? Of the five major senses, there are single auditory, visual, and somatosensory systems complementing the two chemical senses, taste and smell. That olfaction evolved to detect volatile compounds and taste to detect water soluble compounds is not a viable hypothesis because both systems are highly developed in fishes. The selective pressures that led to the development of the two systems were present in an aqueous habitat and were not influenced by much later events involving a transition of organisms from an aqueous to a terrestrial environment. Evidence that the two chemosensory systems are not redundant come primarily from anatomical and electrophysiological studies. Anatomically, olfactory receptors are bipolar neurons that connect directly to forebrain (i.e., olfactory bulb), whereas the epithelial taste receptor cells synapse onto nerve fibers that connect initially to hindbrain nuclei. Physiologically, taste and smell receptors of tetrapods detect quite different stimuli because air, the breathing medium for terrestrial organisms, selects for different physicochemical properties of chemical stimuli than does water in the case of aquatic vertebrates. Electrophysiological data indicate that fish are highly sensitive to amino acid stimuli, but the specificity of the two chemosensory systems can be rather different (7,8). Although anatomical and physiological differences between olfaction and taste in

Amino acids

Discrimination

fishes are well documented, functional differences as they pertain to the behavior of the organisms are scant. Historically, several different functions were assigned to the olfactory system of fishes. Olfaction was reported to be involved in: 1. the localization of food (18,28), 2. alarm behavior (12), 3. sex pheromone detection (13,31,33,34) including the control of male sexual behavior (36), 4. nonspecific arousal and specific recognition of chemical stimuli (14), 5. the formation of a chemical search image of prey (2) and 6. recognition between conspecifics (38). The concept that olfaction was required for formed associations between chemical stimuli and the release of specific behaviors could be concluded from functions 4 - 6 listed above. Additionai experiments confirmed such arelationship between the olfactory sense and chemosensory learning phenomena. Bullhead catfish that were previously trained to recognize glutamine and phenethyl alcohol lost this ability to distinguish these sameo compounds after their nares were plugged (1). In an aversive conditioning paradigm using electric shocks as a physiological stimulus to induce bradycardia in channel catfish in response to specific single amino acids, the conditioned bradycardia was less expressed in nose plugged than in intact catfish, indicating the

1 Present address: Department of Biology, University of Ljubljana, Ve~na Pot III, 61000 Ljubljana, Sloveuia. 2 Present address: Department of Pharmacology, Louisiana State University Medical Center, New Orleans, LA 70119. 3 To whom requests for reprints should be addressed. 865

866

VALENTIN(~I(~, WEGERT AND CAPRIO

role of olfaction in learning the conditioned chemical stimulus for the cardiac response (20). Also, heart rate in young Atlantic salmon was conditioned to L-cysteine through the olfactory system (22). Changes in brain nuclear RNA were observed following stimulation of olfactory receptors by catfish with morpholine and natural food odors (29). The increased levels of the brain nuclear RNA, the changed base ratios during exposure to odorants, and a reversibility of these changes to the pretest values when olfactory organs were no longer stimulated was a possible indication of a growth of synapses associated with a learning phenomenon. The goldfish conditioned to discriminate different amino acids lost their discriminatory ability for amino acids after a bilateral exclusion of olfactory pathways (44). According to these data, the long-term memory to nonfamiliar odors lasted more than 3 months. The present report, using intact, semiosmic and anosmic channel catfish in a food-reward conditioning paradigrn clearly documents that the learned discrimination of individual amino acids is dependent upon a functioning olfactory sense. METHOD

gion of the aquarium surface exhibiting high turbulence created by the aquarium's aeration system. A microelectrochemical probe that measured dopamine concentrations (21) was used in a subset of experiments to calibrate stimulus profiles and dilutions within the aquaria. Results of these tests indicated that the initial stimulus cloud was diluted by >500 times within 3 s of stimulus injection and by 1000-10,000 times by the time the stimulus eddies reached the fish resting on the bottom of the aquarium. In addition, the rising concentration gradient of dopamine towards the center of the stimulus eddy passed the redox electrode in less than 2 s and the total duration of the passage by the electrode of a dopamine containing stimulus eddy was < 4 s. Thus, receptor adaptation was not expected to be a limiting factor in these experiments. During the first minute of the behavioral response to the injected stimulus, the catfish was repeatedly stimulated by alternating on and off gradients of the stimulatory amino acid within the stimulus eddies. The distribution of the added chemicals became approximately homogeneous within 1 2 min subsequent to stimulus injection into the aquarium.

Conditioning Procedures

Animals and Surgical Procedures Channel catfish (12-20 cm) maintained in a local university pond, were transferred to individual 80 liter aquaria with black gravel substrate. Each aquarium was extensively aerated and filtered via a vertical biological gravitational filter containing black gravel and a 5 cm layer of activated carbon. To prevent the development of the catfish pathogen, Edwardsiella ictaluri, the fish were maintained at a temperature >30°C, which is above the tolerance limits of this bacterium. The catfish were regularly fed with cod muscle for a period of approximately 2 months prior to the start of the experiments. Animals were conditioned to one amino acid and then crosstested with several different amino acids to evaluate the specificity of the conditioned response. To provide semiosmic (single olfactory organ removed) and anosmic (both olfactory organs removed) animals, surgeries were performed under MS-222 (3-aminobenzoic acid ethyl ester; 1:8000 concentration) anesthesia. The duration of anesthesia prior to surgery was approximately 1 h. Channel catfish completely recovered from the MS-222 anesthesia in less than 10 min after transfer to anesthetic free water. Most of the catfish started to feed within a few hours after the surgery. Postsurgical treatment of the fish included careful inspections of the skin and, if indicated, an immediate application of the disinfectant, malachite green (180/zg/l) supplemented with potassium iodide (10 #g/l) to the aquarium water. The infected skin lesions, which occurred primarily due to the damage to the external body mucus during the surgery, were in most cases successfully treated by a single application of malachite green. All channel catfish that were by visual inspection apparently free of the disease caused by EdwardsieUa ictaluri survived the surgery and were in excellent health throughout the experimental testing. The chemosensory experiments were resumed on the operated animals within 1 - 2 months of the surgery.

Stimulus Delivery The finest products of Sigma, Aldrich, ICN, and Fluka were employed in this study, and products from different sources were crosstested for comparisons. Amino acid test solutions were prepared with charcoal-filtered, artesian Baton Rouge tap water and were tested within 40 rain of their formation. One milliliter of a 1 0 -2 M stimulus solution was delivered from a Pasteur pipette that was suspended above each aquarium and directed to the re-

Within the first day of transfer to the test aquaria, the channel catfish were fed cod or flounder muscle. Initially, the food was added to the aquaria and the room lights were turned off immediately thereafter in order to turn off the excitatory escape state and to provide for the maximal initial food intake. Within 1 week, however, all catfish fed uninhibitedly within a lighted room. An additional 3 weeks were necessary for all the test catfish to start responding regularly with food-searching behavior to single dissolved amino acids. During this time, the fish accepted the experimenter's presence so that no further escape responses were released by normal experimental manipulations in front of the aquaria. Presentations of large objects, including the experimenter's hands, from above the aquaria were avoided so as not to release the escape behavior (40). Three to five conditioning trials were run daily. A channel catfish was rewarded with a piece of fish muscle 90 s after the conditioning amino acid stimulus was injected into the aquarium. The stimulus eddies reached the catfish on the bottom of the aquaria within 6 - 3 0 s. In most cases, >45 conditioning trials were necessary for the conditioned swimming response to stabilize. The search swim was quantified by counting from super-VHS video recordings of the experiments the number of >90 ° turns made by the catfish within 90 s of stimulus application in a 80 1 aquarium. In later trials, the conditioning amino acid was alternatively presented with at least one other amino acid until the turning rate to the conditioned amino acid was twice that to the nonconditionedstimulus. To determine the specificity of the conditioned response, several other amino acids and their analogs were alternatively presented (crosstested) with the conditioned amino acid. The ability of semiosmic and anosmic channel catfish to discriminate different chemical stimuli was, thus, tested. The testing of anosmic and semiosmic channel catfish began 1 - 2 months after the surgery. Initially, operated catfish were exposed to the same conditioning paradigm using the same amino acid as before the surgery. After the conditioning trials were conducted that were comparable in number to those performed with intact animals, crosstests with a second amino acid were initiated. The initial crosstests were repeated 6 - 1 9 times as part of the discrimination training. To show discrimination following training, the differential response to the conditioned amino acid was required to be significantly greater than the response to the nonconditioned amino acids.

OLFACTORY DISCRIMINATION IN CATFISH

867

RESULTS

Conditioning After the conditioningprocedures (three to five sessions daily) began, channel catfish increased the length of the search swim as evidenced by an increase in the number of turns >90 ° within 90 s of stimulus delivery to the aquarium. In response to the conditioned stimulus, L-lysine hydrochloride, a rapid increase in the search swim response occurred during the first 20 trials and then stabilized after approximately 45 trials. The median number of turns during conditioning increased from less than 10 to more than 60 turns in 90 s (Fig. 1). The time course of the increase in the swimming response to the conditioned stimulus, L-alanine, was similar to that for other conditioned stimuli (Fig. 1, intact animals). During crosstests, the turning rate after stimulation with the conditioned stimulus further increased and became less variable between tests. Only during the first crosstest in intact channel catfish were the responses to the conditioned and nonconditioned amino acids not significantly different. The difference between the number of >90 ° turns in response to conditioned and nonconditioned amino acids increased during the eight subsequent discriminationtraining crosstests (Fig. 2A). The overall increase in the conditioned response to L-alanine was unexpectedly small in semiosmic animals (Fig. 1C). The number of turns increased only during the first 15-20 trials and no further increase in number of >90 ° turns was observed after additional conditioning sessions. The total turning rate in intact conditioned channel catfish was, thus, 30-50% greater than in semiosmic conditioned catfish (Fig. 1). Quite unexpectedly, the discrimi-

L-LYSINE INTACT 11.-,

natory ability of the conditioned, semiosmic channel catfish increased rapidly during eight L-alanine/L-argininediscrimination training trials (Fig. 2]3). Only the first crosstest in these catfish did not provide a significant difference between the conditioned and the nonconditioned behavioral responses. After the first novel stimulus, L-arginine, was not rewarded in the initial test, the response of semiosmic channel catfish to this compound rapidly diminished. Anosmic channel catfish, which had been conditioned to Larginine, lost the ability of conditioned discrimination following removal of the olfactory organs and failed to regain it during 40 repeated L-arginine conditioning trials (Fig. 2C); however, these catfish with subsequent conditioning attempts did gradually increase their behavioral response to nearly all amino acids tested. These fish responded to L-arginine with a median turning rate of 28 (interquartile range of 18-44) at the start of the crosstests. The first response to L-alanine, a novel stimulus in this experiment, was significantly greater than the response to L-arginine. With the exception of this initial test during the discrimination training, no significant differences occurred in the subsequent 17 trials in the responses to L-arginine and L-alanine.

Olfactory Discrimination of the Conditioned From the Nonconditioned Amino Acids Subsequent to the presentations (40-50 times each) of the conditioning stimuli, L-proline, L-arginine, L-lysine (Fig. 1), and L-alanine (Fig. 1), and a short discrimination training consisting of at least five crosstests (Fig. 2A,B), the conditioned stimuli

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MEDIAN AND INTERQUARTILERANGE OF TURNS FIG. 1. Conditioning of intact channel catfish to L-lysine (A) and L-alanine (B) revealed an increase in swimming responses which stabilized at a relatively high responsiveness levels after approximately 45 conditioningtrials. The swimming response increase measured in number of turns above 90° in semiosmic animals (nose unilaterally ablated) (C) was considerably smaller than in intact animals. Bars and lines indicate medians and interquartile ranges of turns above 90°, respectively,which occurred within 90 s of stimulus presentation (n = 11).

868

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MEDIAN AND INTERQUARTILE RANGE OF TURNS FIG. 2. Discriminationconditioning in intact channel catfish (A), in semiosmic channel catfish (B) and in anosmic channel catfish (C). Bars and lines are as in Fig. 1. Dots indicate statistical differences establishedby the Wilcoxon sum of ranks test between the conditioned and the nonconditionedstimuli. Numbers in parentheses adjacent to each stimulus indicate the number of trials a particular amino acid was tested. always stimulated fast swimming responses in intact channel catfish that lasted longer than the responses to the nonconditioned amino acids. The median number of turns to the conditioned stimuli, L-proline and L-arginine, was more than twice that to the nonconditioned stimuli (Fig. 4A,B). For the L-lysine conditioned catfish, all three series of erosstests yielded significant discrimination between the conditioned stimulus, L-lysine, and the other amino acids (Fig. 3). The turning rates in response to the different amino acids were reasonably consistent across the three repeated crosstest series. During the first crosstest, only a few compounds, such as o-lysine (on two occasions), L-citrulline and L-alanine, did not yield significantly smaller turning rates than the conditioned stimulus, L-lysine; however, all differences between the responses to the L-lysine conditioned stimulus and those to the nonconditioned amino acids were significantly different during the third crosstest series. The median responsiveness to the conditioned stimulus, L-lysine, was approximately twice that to the nonconditioned stimuli, results similar to those found for the other conditioned stimuli (L-proline, L-arginine, L-alanine). The responsiveness of the intact, L-alanine-conditionedchannel catfish to the nonconditionedamino acids was lower than that to L-alanine. Initially, the responses to D-alanine and glycine were nearly as large as those to L-alanine, but these differences became significantly different in the second crosstest series (Fig. 5). The ability to discriminate L-alanine from glycine was also tested in semiosmic catfish. After only three repeated crosstests, semiosmic channel catfish also showed significantly greater responses to L-alanine than to glycine (Fig. 6). To determine structure-activity relations, intact channel catfish conditioned to swim

approximately twice as much to the imino acid, L-proline, than to the neutral amino acids were crosstested with several L-proline analogs (Fig. 7). The only compound that provoked turning rates that were not significantly less than to L-proline was L-pipecolic acid. Betaine, L-azetidine-2-carboxylic acid and trans-hydroxyL-proline, which released greater numbers of turns than other compounds, were significantly less effective than L-proline in both the first and second crosstest series. Crosstests indicated that the responses to the L-arginine analogs tested were at most 50% of the L-arginine response, which was in all cases significantly different from the L-arginine response (Fig. 8). DISCUSSION

Conditioning in Intact and in Anosmic Channel Catfish The complex chemical search image formed in nature was in the laboratory substituted by a simple search image for a single substance. After the formation of a search image for a single substance in bullhead catfish (lctalurus nebulosus), the food search to chemically similar substances was equally intense as the behavioral response to the conditioned stimulus (42,43). Any amino acid that releases turning rates that are statistically not different than the turning rates released by the conditioned stimulus is, thus, considered a possible agonist of the amino acid to which the fish was conditioned. Bullhead catfish hiding in shelters did not respond with swimming behavior to nonconditioned amino acid stimuli if these stimuli were not agonists of the conditioned stimulus. In intact channel catfish, the swimming response to the conditioned amino acid was twice that to the other

OLFACTORY DISCRIMINATION IN CATFISH

869

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MEDIAN AND INTERQUARTILE RANGE OF TURNS FIG. 3. Repeated presentations of the same amino acids in L-lysine-conditioned channel catfish revealed reasonable repeatability of the behavioral results with the same group of 11 animals over a period of 3 months. Statistical tests are the same as indicated in Fig. 2. amino acids tested, whereas anosmic catfish failed to discriminate amino acids even after they were repeatedly exposed to the same standard conditioning paradigms as were intact fish. Semiosmic (one olfactory organ removed) catfish, however, were also capable of discriminating the conditioned amino acid from all other amino acids. Thus, at least one olfactory organ was necessary for the conditioned discrimination of chemical stimuli (Fig. 2). Although an increase in swimming activity did occur in anosmic L-PROLINE CONDITIONED L-ARGININE COONDITIONED L-PRO

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catfish in response to amino acids, the increased swimming activity was not conditioned amino acid specific. These results are similar to those found by Holland and Teeter (15) using a feeding bioassay where no change in response frequency was observed between intact and anosmic channel catfish. The results of both behavioral studies (the present and Holland and Teeter (15)) are consistent with the early findings of Bardach et al. (3) that ictalurid catfish can orient to an amino acid source by taste alone; however, both the present report using a food-search paradigm and that by Little (20) using a heart-rate conditioning paradigm clearly indicate that learned discrimination between amino acids cannot be acquired through the taste system alone. The report by Holland and Teeter (15), which used a cardiac conditioning paradigm, did not test for amino acid discrimination. These findings involving food search and escape behaviors (e.g., conditioned heart rate) do not exclude that in other behaviors [e.g., taste aversion (19)] discrimination by taste alone may occur. The physiological basis of how channel catfish and possibly other telcosts are capable of the olfactory discrimination of different amino acids is presently unknown. Although there is evidence for multiple receptor sites for amino acids in the channel catfish (4,9,10,17) and other fishes (30,32,37), how these receptor sites are distributed across the olfactory receptor neurons (ORNs) is also unknown. Presently, there is not a single quantitative study of the specificity of individual olfactory receptor neurons of any teleost to chemical stimuli. A recent preliminary study of the specificity of isolated ORNs of channel catfish to amino acids (16) indicated that single ORNs responded to different amino acids that were suggested from electrophysiological crossadaptation (9), receptor binding (4), and mixture (10,17) experiments to bind to independent receptor sites. How this translates into the ability of channel catfish to discriminate amino acids by olfaction is a topic for future experiments. The present study indicates that in olfaction the compound specific behavioral response is not innately related to a particular

870

VALENTIN(~IC, WEGERT AND CAPRI•

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stimulus. The perception of a particular smell and its influence on behavior is associated by learning to some behavioral experience leaving the use of the olfactory system in catfish in relation to amino acids as an open system. Thus, the particular function of the olfactory system in feeding behavior of channel catfish is learned. There is apparently no need in channel catfish for information lines (e.g., salty, sweet, L-alanine, L-arginine), typical for gustation, to be assigned by evolution to particular purposes for olfaction. For social functions, olfactory flexibility is extremely important because it gives no bias to scents of particular individuals until this bias is learned in encounters with these same individuals (27). Such an open, flexible system is also extremely

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FIG. 6. With the single exception of the first crosstest with glycine, unilaterally nose-ablated channel catfish discriminated glycine and Lproline from the conditionedstimulus, L-alanine.Dots indicate a significant difference to the initial glycine response, and squares indicate a dgnificant difference to the conditioned L-alanineresponse in crosstests ~n = 11). Numbers in parentheses adjacent to each stimulus indicate the aumber of trials a particular amino acid was tested.

suited for imprinting home stream odors in migratory salmons (35), which most probably return to their home stream based on learned olfactory information and not on predetermined and inherited properties, as is the case for feeding behavior patterns which are in the channel catfish directly released through the taste system (11,39,41). Because the swimming activity that occurred in response to olfactory detection of the conditioned amino acid was greater than the swimming activity in response to nonconditioned amino acids, the specificity of the olfactory organ was determined using these conditioning techniques. For more than 70 pairs of compounds crosstested two to three times each with different groups of 11 catfish, the response to the conditioned compound was, but with one exception, significantly greater than the response to the nonconditioned amino acid. The sole exception was the response to L-pipecolic acid in L-proline-conditioned channel catfish, which suggests that these two compounds may bind to the same receptor site(s); however, electrophysiological crossadaptation studies of olfactory receptor responses to these compounds are necessary for confirmation. It is interesting, however, that these same two compounds were recently indicated from electrophysiological crossadaptation experiments to be possible agonists of the proline taste receptor sites in the channel catfish. Correspondingly, a differential behavioral response between two compounds is possible only if at least two different receptors are involved, one being more specific to one of the pair than to the other compound. A differential response to each of the amino acids and to their analogs that were crosstested in the present report indicates a high diversity of receptor mechanisms for amino acids in catfish olfaction. This finding is consistent with the probable existence of a diversity of olfactory receptor proteins (5,24-26,30,37). A previous study in the channel catfish suggested the existence of at least four different olfactory receptor sites for amino acids, one each for amino acids that are acidic, basic, neutral with small side chains, and neutral with long side chains (9). This division of receptor sites for amino acids was confirmed in receptor binding studies (4) where only neutral amino acids with short side chains competed for the L-alanine binding site. An

OLFACTORY DISCRIMINATION IN CATFISH

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FIG. 7. Comparisonof swimming responses after stimulation with different analogs of L-prolinein Lproline-conditionedchannel catfish. Statistical tests are the same as indicated in Fig. 2 (n = 11). electrophysiological study of olfactory receptor responses to binary mixtures of amino acids also provided support for the independence of receptor sites for acidic, basic, and neutral amino acids in the channel catfish (10). Subsequent studies testing more complex mixtures of amino acids (i.e., mixtures consisting of up to 10 component amino acids) not only confirmed the independence of the acidic, basic, and neutral olfactory receptor sites for amino acids, but provided the additional evidence for the existence of multiple receptor sites for neutral amino acids (17). The results of the present behavioral experiments are consistent with the concept of multiple receptor sites, even for amino acids

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FIG. 8. Comparison of swimming responses after stimulation with analogs of L-arginine in L-arginine-conditionedchannel catfish. Statistical tests are the same as indicated in Fig. 2 (n = 11).

within the same category. The most recent discovery of a family of genes encoding odorant receptors in the channel catfish (24) further supports this view.

Taste-Mediated Response to a Novel Stimulus and OlfactionMediated Search Image for Single Amino Acids After conditioning procedures to single amino acids were completed in intact channel catfish, the response to the first novel amino acid did not differ significantly from that to the conditioned amino acid. In all later presentations, however, the response to the conditioned stimulus was significantly greater than the response to the nonconditioned stimulus. Also, anosmic catfish responded significantly more to the first presentation of a novel amino acid than to a previously frequently employed stimulns. In all later alternative presentations of both stimuli, however, the responses of anosmic catfish to the repeatedly reinforced amino acid and to the nonreinforccd amino acids were not significantly different. These results were the first indications that the response to a novel amino acid stimulus was most probably taste mediated and that the conditioned response to amino acids was olfactory mediated. The intriguing finding that the first novel chemical stimulus presented to the intact and anosmic catfish released unexpectedly large swimming responses indicates that arousal for searching activity exists also in anosmic catfish and that this arousal mechanism can be influenced by taste. The possibility that the response to chemical novelty in catfish is mediated exclusively by taste and that the conditioned searching activity is controlled by olfactory system requires further experiments. The swimming response to taste stimuli, such as L-alanine and L-arglnine, was relatively high in anosmic animals. This elevated response was possibly due to a decreased inhibition of feeding behavior derived from the following multiple procedures:

872

VALENTIN~I(~, W E G E R T A N D CAPRIO

1. several hundred stimulations with feeding stimuli during regular daily feeding sessions, 2. the conditioning of intact animals, 3. the discrimination training of intact animals, 4. the crosstesting of intact animals, 5. the feeding and later repeated presentations of an amino acid and food to the same anosmic animals after the surgery, and 6. the discrimination training of the ablated animals. Such a high number of food stimulus presentations and feeding bouts without the presence of conflicting key stimuli for escape behavior reduced the inhibition from the escape behavior and yielded a high swimming rate of anosmic catfish to presentations of amino acids and food extracts.

Klinokinesis and Olfaction The arousal function of olfaction, which is in part possibly derived from the long-term tonic activity of the olfactory system to a continuous presentation of a stimulus (6), serves to maintain catfish active for long periods of time. Increased swimming activity stimulated by olfaction increases the chances for fish to encounter food in nature. The swimming stimulated and maintained by olfaction generally lasts longer than the swimming activity stimulated by taste alone. One of the functions of olfaction,

therefore, appears to be to extend the duration of klinokinetic movements (39). For channel catfish conditioned to a single amino acid, the specific result of learning was the increase in the intensity and duration of the animal's klinokinetic movements which increased its chances of encountering food. This role of rhynencephalic learning for feeding purposes presumably yielded an advantage to fish, which were able to remember not only the visual and vibrational picture of their most frequently encountered prey, but were also able to learn and effectively use the chemical search image of the prey. The role of olfaction in the feeding behavior of channel catfish is to gain recognition of a food by experience which guides the fish to the food item, and not to release single patterns of feeding behavior. A general feeding arousal is largely influenced by the olfactory input. The arousal function for a particular behavior and the maintenance of excitation for this behavior greatly increases the chances for a suitable behavioral outcome in simple behavioral responses and in social interactions of the catfish. ACKNOWLEDGEMENTS This research was supported by NSF (BNS-8819772; IBN-9221891) and ONR (N00014-90-J-1583) grants to J.C. and a grant (P1-0114-487) from the Slovenian MZT to T.V. We also thank Dr. P. Moore for his assistance with the spatio-temporal analysis of the stimulus wave fronts.

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