Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
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ABSTRACT |
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Ogawa, K. and J. Caprio. Facial Taste Responses of the Channel Catfish to Binary Mixtures of Amino Acids. J. Neurophysiol. 82: 564-569, 1999. We investigated the neural processing of binary gustatory mixtures of amino acids by the facial taste system of the channel catfish, Ictalurus punctatus. In vivo electrophysiological recordings indicated that the magnitude of both integrated and single-unit facial taste responses to binary mixtures of amino acids was greatest if the components bound to independent receptor sites. Facial taste responses were obtained from 32 multiunit and 55 single taste fiber preparations to binary mixtures of amino acids whose components bind to independent taste receptor sites (group I) or to the same or highly cross-reactive taste receptor sites (group II). All component stimuli were adjusted in concentration to provide approximately equal response magnitude as determined by either the height of the integrated multiunit taste response or by the number of action potentials generated/3 s of response time/single taste fiber. The mixture discrimination index (MDI), defined as the response to the mixture divided by the average of the responses to the component stimuli, was calculated for each test of a binary mixture. MDIs of group I binary mixtures for both the integrated multiunit and single fiber data were significantly greater than those for either the control or group II binary mixtures. In a subset of multiunit recordings, the MDIs of a group I binary mixture across three log units of stimulus concentration were similar and significantly greater than those of a group II binary mixture. Analysis of the single fiber data also indicated that the MDIs of group I binary mixtures were significantly larger than those of group II binary mixtures for both alanine-best and arginine-best taste fibers; however, the MDIs of group I binary mixtures calculated from recordings from arginine-best taste fibers were significantly greater than those recorded from alanine-best taste fibers.
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INTRODUCTION |
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The natural stimuli of olfactory and gustatory
organs of animals are mixtures of chemicals. Perhaps the best-known
examples of the potency of blends of specific chemicals are insect
pheromones (Kaissling 1996; Tumlinson et al.
1989
; Vickers et al. 1991
). Numerous other
examples come from behavioral studies of aquatic animals, where
stimulus mixtures, rather than individual compounds, were documented to
account for a significant percentage of the potency of a natural
extract or complex synthetic mixture (Adron and Mackie
1978
; Carr 1976
; Carr and Chaney
1976
; Carr et al. 1977
, 1984
;
Elliott 1986
; Harada and Ikeda 1984
;
Harada and Matsuda 1984
; Hashimoto et al.
1968
; Zimmer-Faust et al. 1984
).
The few previous reports on olfactory receptor responses of the
freshwater channel catfish to binary and more complex amino acid
odorant mixtures indicated that the magnitude of the
electrophysiological (EOG and integrated neural) was predictable based
on knowledge of the relative independence of the receptor sites for the
component stimuli (Caprio et al. 1989; Kang and
Caprio 1991
). The relative independence of the receptor sites
for the component stimuli was based on the results of
electrophysiological cross-adaptation (Caprio and Byrd
1984
) and biochemical competitive binding (Bruch and
Rulli 1988
) experiments. Mixtures whose components showed little cross-adaptation or competition resulted in enhanced
electrophysiological responses compared with those mixtures whose
components cross-adapted or competed for the same receptor sites. An
enhanced taste response is defined here as a response to the binary
mixture that is significantly greater than the response to a 50:50
mixture of the equipotent components. These results clearly
demonstrated that one mechanism for response enhancement evoked by a
specific stimulus mixture was simply the simultaneous activation of
relatively independent receptor sites by the respective components in
the mixture. This basic principle derived from olfactory receptor
recordings to odorant mixtures in the channel catfish was also recently
shown to be directly applicable to predicting the magnitude of single olfactory receptor neurons that are excited by binary odorant mixtures
and their components in the spiny lobster (Cromarty and Derby
1997
) and to integrated taste recordings in the sea catfish Arius felis (Kohbara and Caprio 1996
). These
results suggest common principles of the chemosensory processing of
neural information for both crustaceans and vertebrates. The present
report on the channel catfish examines whether these same principles
can be used to predict the magnitude of the responses of single facial taste fibers to binary mixtures of amino acids and whether integrated multiunit data can predict the behavior of single taste fibers.
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METHODS |
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Experimental animals
A total of 74 channel catfish (Ictalurus punctatus; 10-130 g) tested in the present experiments were obtained from a local hatchery, kept in floating cages in a nearby university pond, and fed regularly with commercial catfish chow. The catfish, transported as needed to the Animal Care Facility in the Life Sciences Building, were maintained in a 250-l fiberglass aquarium filled with aerated, charcoal-filtered tap water at 25°C, and were used within 2 wk of transfer.
Experimental procedures
The catfish were immobilized with an intramuscular injection of
Flaxedil (gallamine triethiodide, 0.1 mg/100 g body wt; Davis and Geck
Dept., American Cyanamid, Pearl River, NY), wrapped in a wet tissue
paper, and secured to a wax block, which was placed in a Plexiglas
container. The gills were irrigated throughout the experiment with
aerated, charcoal-filtered tap water containing the anesthetic 0.05%
MS-222 (ethyl-m-aminobenzoate methane sulfonic acid). During the course
of an experiment, additional doses of Flaxedil and MS-222 were provided
as needed. Procedures for the surgical exposure of the
facial/trigeminal nerve complex to the maxillary barbel, stimulus
delivery, surgical isolation, and electrophysiological recordings of
both integrated (Wegert and Caprio 1991) and single fiber (Kohbara et al. 1992
; Michel and Caprio
1991
) taste activity were previously described. Responses of
single facial taste fibers during the initial 3 s of response were analyzed.
Stimuli
Tested were amino acids indicated to bind to independent taste
receptor sites (group I) and those indicated to bind to the same or
highly cross-reactive taste receptor sites (group II) (Wegert
and Caprio 1991). The experimental paradigm tested the same
single group I [L-alanine (Ala) + L-arginine
(Arg)] and group II [Ala + L-methionine (Met)] binary
mixtures and the control [a "pseudo-binary mixture" composed of
only Ala (i.e., Ala + Ala prepared with the identical protocol as the
"true" binary mixtures tested)] on both multiunit and single fiber
preparations. Additional group I [Arg + L-glutamic acid
(Glu)] and group II (Ala + Glu) binary mixtures were tested in the
multiunit experiments, and two other group I [Arg + L-proline (Pro); Ala + Pro] and group II [Ala + glycine
(Gly); Arg + L-
-amino-
-guanidino propionic acid
(AGPA)] mixtures were tested in the single-unit experiments. All amino
acids were Sigma grade (Sigma Chemical, St. Louis, MO). Fresh stock
solutions of individual amino acids, prepared at least once a week at
10 mM with charcoal-filtered tap water, were stored in glass bottles at
4°C and were diluted to the desired experimental concentration just
before testing. For the majority of the test solutions, the pH remained
between 8.0 and 8.5; for Glu, the pH was adjusted to 8.4 with NaOH. A
binary mixture of Ala + Arg, each at a final concentration of 5 × 10
5 M, was used to search for a responsive
nerve twig or fiber. Photodensitometry studies indicated that the peak
stimulus concentration delivered to the maxillary barbel was ~75% of
the concentration injected into the stimulus delivery system
(Kohbara et al. 1992
). The reported stimulus
concentrations in the text were not corrected for dilution.
Experimental protocol and the MDI
SERIES A. The two component amino acids for each of the tested binary mixtures were adjusted in concentration to provide approximately equal response magnitude [i.e., the whole log concentration of each component that resulted in the more similar (mostly <10% difference) response magnitude] based on either 1) the height of the stimulus-induced phasic displacement of integrated multiunit activity or 2) the number of action potentials produced by a single taste fiber within the first 3 s of response time (Table 1). After duplicate recordings of the responses to each of the two components, duplicate responses were obtained to the binary mixture comprising the components at half their original concentration because the binary mixture was obtained by mixing equal volumes of equipotent components. Duplicate responses to each of the components of the binary mixture were again recorded to confirm response reproducibility. Responses to this latter test of components were required to be within 20% of the magnitude of the first series or these results were discarded. Subsequently, the mixture discrimination index (MDI), defined as the averaged response to the mixture divided by the average of the responses to the component stimuli, was calculated for each tested binary mixture.
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SERIES B.
Whole log steps (106 M to
10
3 M) of Ala and Arg alone and as binary
mixtures with 1 mM Met were tested in five fish. The response to the
mixture divided by the response to Ala and Arg, respectively, was
calculated at each tested concentration.
Data analysis
All MDI values for the different binary mixtures were analyzed
by one-way ANOVA ( = 0.05). Because the MDI is ratio data, the
respective values were also natural log-transformed. Because no
significant differences between the analyses for the untransformed and
natural log-transformed data for the tests were indicated, all values
reported were derived from the untransformed data. Post hoc comparisons
were performed by the Duncan's Multiple Range test.
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RESULTS |
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Integrated facial taste activity
SERIES A. MULTIUNIT RECORDINGS: TASTE RESPONSES TO BINARY MIXTURES OF EQUIPOTENT COMPONENTS. A total of 85 tests of binary mixtures and 32 control tests of the "pseudo-mixture" (i.e., Ala + Ala) obtained from 21 catfish were studied. Forty-four tests of 2 group I mixtures (Ala + Arg; Glu + Arg; n = 17 nerve twigs), 41 tests of 2 group II mixtures (Ala + Met; Ala + Glu; n = 15 nerve twigs), and 32 control tests were performed (Fig. 1). MDIs for group I binary mixtures were significantly greater compared with those for group II binary mixtures (ANOVA, P < 0.05; F = 4.1121, df = 84). There was also no overlap in the 95% confidence intervals for the MDIs for group I (1.20-1.12; n = 44) and group II (1.11-1.01; n = 41) binary mixtures. However, MDI values for group I were not significantly different from each other (Duncan, P > 0.05); likewise, group II mixtures were not significantly different from each other (Duncan, P > 0.05; Fig. 2A).
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SERIES B. MULTIUNIT RECORDINGS: DOSE-RESPONSE EFFECTS.
MDI values to group I binary mixtures of 1 mM Met + Arg
(106 to 10
3 M) were
significantly larger than those to group II binary mixtures of 1 mM Met + Ala (10
6 to 10
3 M;
ANOVA, P < 0.05; F = 21.7455, df = 7; Fig. 3). There was also no overlap
in the 95% confidence intervals for the MDIs for the 1 mM Met + Arg
(1.26-1.12) and 1 mM Met + Ala (1.11-0.93) across the four log steps
of concentration. The responses to the binary mixture of Met + Arg
averaged 18.8 ± 3.7% (mean ± SD) greater than those to Arg
alone at each of the four log steps of concentration (10
6 to 10
3 M). The
responses to Met + 10
6 to
10
3 M Ala were not significantly different from
the responses to Ala alone (ANOVA, P > 0.05;
F = 0.0003, df = 7). There were also no
significant differences between the MDIs for group I binary mixtures
between series A (above) and B (ANOVA, P >0.05;
F = 0.2079, df = 47) tests, or for group II binary
mixtures between series A (above) and B (ANOVA, P > 0.05; F = 0.2142, df = 44) tests.
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Single facial taste fiber activity
RESPONSES TO BINARY MIXTURES COMPOSED OF EQUIPOTENT COMPONENTS. A total of 149 tests of binary mixtures were performed on 61 single facial taste fibers from 47 catfish (Figs. 2B and 4). One hundred sixteen tests of 3 group I mixtures [Ala + Arg (n = 48 fibers tested); Ala + Pro (n = 36); Arg + Pro (n = 32)], 33 tests of 3 group II mixtures [Ala + Met (n = 10); Ala + Gly (n = 12); Arg + AGPA (n = 11)] and 9 control tests [i.e., Ala + Ala (n = 9)] were performed. As found previously for the multiunit recordings, MDIs for group I binary mixtures were significantly greater than those for group II binary mixtures and for controls (ANOVA, P < 0.05; F = 2.8845, df = 148), and there was also no overlap in the MDI 95% confidence intervals for group I (1.21-1.14; n = 116) and group II (1.08-1.01; n = 33) binary mixtures. MDI values for group I binary mixtures were not significantly different from each other (Duncan, P > 0.05); similarly, MDI values for group II binary mixtures were not significantly different from each other (Duncan, P > 0.05). There were also no significant differences between the MDIs for the control and group II mixtures (Duncan, P > 0.05). In a comparison of the present single fiber and the previous integrated data, no significant differences occurred in MDIs 1) between group I single fiber and group I series A integrated data (ANOVA, P > 0.05; F = 0.1532, df = 159), or 2) between group II single fiber and group II series A integrated data (ANOVA, P > 0.05; F = 0.7157, df = 73).
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RESPONSES BY TASTE FIBER TYPE TO BINARY MIXTURES COMPOSED OF
EQUIPOTENT COMPONENTS.
An analysis of the MDI values based on responses by fiber type
[i.e., fibers most responsive to L-Ala (i.e., Ala-best)
vs. those most responsive to L-Arg (i.e., Arg-best)
(Kohbara et al. 1992)] was also performed. MDI values
for group I mixtures were significantly larger than those for group II
mixtures for both alanine (ANOVA, P < 0.05;
F = 4.2533, df = 90) and arginine (ANOVA, P < 0.05; F = 5.5911, df = 57)
fiber types (Table 2). In addition, there
was no overlap in the MDI 95% confidence intervals for group I and II
binary mixtures for the Arg-best (group I: 1.28-1.16; n = 52;group II: 1.14-0.87; n = 6)
taste fibers; however, there was a slight overlap in the MDI 95%
confidence intervals for group I (1.18-1.09; n = 64)
and group II (1.10-1.01; n = 27) binary mixtures for
the Ala-best fiber type. There were no significant differences in the
MDIs for group II mixtures between the Ala-best and Arg-best taste
fibers (ANOVA, P > 0.05; F = 1.0587, df = 32). However, MDIs for group I mixtures of the Arg-best
fibers were significantly greater than those for the Ala-best fibers
(ANOVA, P < 0.05; F = 5.1644; df = 115); there was a slight overlap in the 95% confidence intervals for
group I binary mixtures for Arg-best fibers (1.28-1.16;
n = 52) and Ala-best fibers (1.18-1.09;
n = 64).
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DISCUSSION |
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The present report of multiunit and single fiber taste responses
of the channel catfish, Ictalurus punctatus, to binary
mixtures is consistent with recent reports of multiunit facial taste
activity in the sea catfish, Arius felis (Kohbara and
Caprio 1996) and hamster (Hyman and Frank 1980
),
and multiunit and electroolfactogram (EOG) responses of olfactory
receptor neurons in the channel catfish (Caprio et al.
1989
; Kang and Caprio 1991
). These reports
collectively indicate that the magnitude of multiunit responses to
binary and more complex olfactory and gustatory stimulus mixtures is
greater if the component stimuli bind simultaneously to relatively
independent receptor sites than to the same or highly cross-reactive sites.
Although an investigation of integrated multiunit taste responses to
binary mixtures of amino acids was previously performed in the sea
catfish, Arius felis, it was unknown as to how single taste
fibers respond to group I and group II binary mixtures. The enhanced
integrated multiunit taste activity recorded from the sea catfish to
group I mixtures (Kohbara and Caprio 1996) could have
resulted from the simultaneous activation of different populations of
taste fibers. Thus a major objective of the present experiments was to
determine whether single taste fibers could evoke the magnitude of
enhanced taste activity observed in the integrated multiunit
recordings. Also, because the amino acid-sensitive facial taste system
of the channel catfish is composed of two major types of fibers (i.e.,
alanine-best and arginine-best) (Davenport and Caprio
1982
; Kohbara et al. 1992
) based on their
different amino acid specificities, another major objective was to
determine whether there were response differences to binary mixtures
dependent on the fiber type sampled.
The present study clearly shows that MDIs of group I mixtures were
significantly greater than those of group II mixtures, confirming that
one mechanism for gustatory enhancement is the simultaneous activation
of relatively independent taste receptors by the components in the
mixture. The results of a subset of the present experiments (series B)
indicated further that the response enhancement observed with group I
binary mixtures was similar across widely differing stimulus
concentrations (µM to mM). This study also indicated that the MDIs of
group I and group II binary mixtures, respectively, calculated from
integrated multiunit and single fiber activity in the channel catfish,
were not significantly different. These latter results indicate that
the enhanced taste responses to group I binary mixtures were not
exclusively the result of the simultaneous activation of the different
types of taste fibers by the respective components in group I binary
mixtures. It is most likely that the enhancement observed with group I
binary mixtures occurred due to the simultaneous activation of
relatively independent taste receptor sites located on taste cells
innervated by a single taste fiber. The group I binary mixtures tested
were composed of the L-isomers of alanine, arginine, and
proline, all indicated to bind to independent receptors
(Kumazawa et al. 1998; Wegert and Caprio
1991
). Both electrophysiological (Kohbara et al.
1992
) and anatomic (Finger et al. 1996
) findings
are consistent in indicating that not only are the molecular receptors
for L-arginine and L-alanine different, but
that the majority of these receptors are expressed on different taste
cells. Because the majority of L-proline taste sensitivity
is correlated with a subset of L-arginine taste fibers
(Kohbara et al. 1992
), it is hypothesized that the proline receptors are localized on taste cells expressing few, if any,
alanine receptors. It is currently unknown, however, whether the
L-proline receptors are coexpressed on taste cells
possessing arginine receptors or are found on taste cells independent
of these other receptor sites, but innervated by a portion of the arginine fibers. Whether a single taste fiber innervating taste cells
coexpressing different molecular receptors would show a similar
enhancement of neural activity as a single taste fiber innervating
taste cells with nonoverlapping expression of molecular receptors
awaits future experiments. However, a single olfactory receptor neuron
that expresses different receptor sites for different odorants does
show enhanced activity to a binary mixture of these substances
(Cromarty and Derby 1997
; Kang and Caprio
1997
). Thus an explanation for the significantly greater
enhancement of taste activity to the group I binary mixtures (i.e., Ala + Arg; Ala + Pro; Arg + Pro) from the arginine-best compared with
alanine-best taste facial taste fibers in the present experiments is
currently unknown.
The present results also confirmed a recent finding that
L-glutamic acid, an acidic amino acid, acts as a neutral
amino acid with a short side-chain (i.e., SCN) in the facial taste
system of the sea catfish (Kohbara and Caprio 1996). For
both species of catfish, the taste MDI of a binary mixture of
L-glutamic acid and an SCN was not significantly different
from MDIs of group II binary mixtures and was significantly less than
those to group I binary mixtures. These results are also consistent
with previous electrophysiological experiments in the facial taste
system of the channel catfish that showed reciprocal cross-adaptation
between glutamic acid and SCNs (Wegert and Caprio 1991
).
It is rather interesting that in contrast to the gustatory system,
receptors for L-glutamic acid in the olfactory system of
the channel catfish are independent of those to neutral amino acids
(Bruch and Rulli 1988
; Caprio and Byrd
1984
; Caprio et al. 1989
; Kang and Caprio 1991
). Accordingly, a binary mixture of L-glutamic
acid and a neutral amino acid resulted in an olfactory receptor MDI
significantly greater than that determined from group II odorant
mixtures and equal to that for group I odorant mixtures (Caprio
et al. 1989
).
Simple chemical mixtures appear to be processed similarly by peripheral
olfactory and gustatory sensory nerves in vertebrates. Previous studies
indicated similar MDI values for group I ("across-group") (Caprio et al. 1989; Cromarty and Derby
1997
; Kang and Caprio 1991
; Kohbara and
Caprio 1996
) and group II ("within-group") binary stimulus mixtures, respectively, irrespective of the chemosensory system (olfaction or taste) tested (Table
3). A recent report of responses of
single olfactory receptor neurons of the spiny lobster Panulirus
argus also resulted in similar respective MDI values for group
I and group II mixtures for receptor neurons that responded excitedly
to the individual components of the tested mixtures (Cromarty
and Derby 1997
). In all these cases involving olfactory
receptor responses in the channel catfish and lobster and facial taste
responses in the channel and sea catfishes and hamster, MDI values for
binary group II mixtures, whose components bind to the same or highly
cross-reactive receptor sites, are similar to the responses to either
of the equipotent components (i.e., MDI averages ~1.06). MDI values
for group II mixtures are slightly >1.0, which most likely indicates
that the components of the group II binary mixtures tested
cross-reacted with some receptors in addition to their common receptor
sites (Cromarty and Derby 1997
; Kang and Caprio
1991
). MDI values ~1.0 also indicate that the components of
the tested group II mixtures interact by competitive agonism without
any evidence for mixture suppression (Caprio et al.
1989
; Cromarty and Derby 1997
; Kang and
Caprio 1991
; Kohbara and Caprio 1996
). However,
MDI values determined from responses of these same animals and systems
to binary mixtures (i.e., group I mixtures) whose components bind to
relatively independent receptor sites averaged 1.44, a 44% increase in
magnitude of the response over that to either component of the mixture
tested individually. It is currently unknown why in the present
experiments that, although the MDIs of group I mixtures were
significantly greater than those for group II, group I mixtures only
resulted in a 16% (integrated multiunit data) to 17% (single fibers)
enhancement of taste activity compared with the >40% enhancement for
olfactory receptor responses in the same animal (Caprio et al.
1989
) and for taste responses in the sea catfish Arius
felis (Kohbara and Caprio 1996
). One possible
explanation for the previous findings is that the amino acid receptors
for group I olfactory stimuli in the channel catfish and facial taste
stimuli in the sea catfish are more independent (i.e., a higher
percentage of receptor sites for the amino acids tested occurring on
independent receptor cells) than those within the facial taste system
of the channel catfish.
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ACKNOWLEDGMENTS |
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We thank R. Bouchard for computer graphics and Dr. E. Obata for partial financial support for K. Ogawa.
This research was supported by National Science Foundation Grant IBN-9221891 to J. Caprio.
Present address of K. Ogawa: Dept. of Otolaryngology, Kagoshima University Medical School, 8-35-1 Sakuragaoka, Kagoshima 890, Japan.
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FOOTNOTES |
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Address reprint requests to J. Caprio.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 January 1999; accepted in final form 27 April 1999.
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REFERENCES |
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