Department of Biological Sciences, Louisiana State University,
Baton Rouge, Louisiana 70803
 |
INTRODUCTION |
In recent studies, citric acid (or trisodium
citrate), a six-carbon tricarboxylic hydroxy acid (or salt), enhanced
taste responses to amino acids in the largemouth bass (Ogawa and
Caprio 1999
), catfish (Davis and Caprio 1996
),
and rat (Gilbertson et al. 1997
). Citric acid, which is
an additive to human food, acts as a preservative, a pH buffer, an
antioxidant, and a flavor enhancer (Gardner 1972
). Citrate is also used as an additive to increase the palatability of a
variety of animal foods, such as its combination with phosphoric acid
to enhance the flavor of cat food (Kealy 1975
).
Increased daily food intake of horses (Betz and Lantner
1980
) and the herbivorous fish Tilapia zillii
(Adams et al. 1988
) was also observed when citrate was
added to their respective foods. Behavioral tests in rodents revealed a
preference for stimulus mixtures of citrate with amino acids or sugars,
compounds known to depolarize taste receptors (Gilbertson et al.
1997
). Due to the electrophysiological response enhancement
seen to binary mixtures of citrate and amino acids in the taste system
of fish and mammals and the enhanced behavioral effects of citrate
addition in feeding studies of fish and mammals, the present study
tested whether the addition of citrate to an amino acid stimulus also
enhanced olfactory receptor responses in the channel catfish. The
results of the present study indicated that a binary mixture of citrate
and an amino acid enhanced asynchronous olfactory receptor activity and
also generated synchronized voltage oscillations [peripheral waves
(PWs)] within the sensory region of the olfactory epithelium. Although
a previous report documented the generation of PW activity in fish in
response to moderately high concentrations (1 mM) of amino acids
(Sutterlin and Sutterlin 1971
), the present study
indicates that lower concentrations (10 µM) of amino acids could
initiate PW activity when mixed with
0.5 mM citrate. Because the
enhancement of taste responses was recently suggested to be due to
citrate's ability to chelate calcium (Ogawa and Caprio
1999
), the present report tests this hypothesis on citrate's
enhancement of olfactory receptor responses.
 |
METHODS |
Experimental animals
Channel catfish, Ictalurus punctatus (25-50 g), were
obtained from three different sources. Most fish used in this study
were raised at the Louisiana State University Aquaculture Center and were maintained in floating cages held in ponds at the facility. Additional fish were obtained from Sander's Fish Farm, a local hatchery in Pride, Louisiana. A third source was the Louisiana State
University School of Veterinary Medicine. All experimental fish were
fed weekly with floating commercial fish chow. Each week catfish were
transferred to an aerated, 250-l polyethylene aquarium filled with
charcoal-filtered city tap water at the Louisiana State University
Animal Care Facility and maintained on a 12:12 light/dark regime. The
temperature was held above 27°C during the spring and summer and
below 20°C during the fall and winter to help avoid bacterial
(Edwardsiella ictaluri) infection (Morrison and Plumb
1994
). The fish were used experimentally within 1 wk of their
placement in the animal care facility and were not fed during this period.
Experimental procedures
The preparation of the animals was the same as that described by
Kang and Caprio (1991)
. Each catfish was initially immobilized with an
intramuscular injection of the neuromuscular blocking agent, Flaxedil
(gallamine triethiodide, 0.03 mg/100 g). During the experiments,
additional injections were applied as needed via a hypodermic needle
embedded in the flank musculature. The immobilized fish was wrapped in
a wet Kim-Wipe, placed into a Plexiglas container, and stabilized using
a pair of orbital ridge clamps. The gills were irrigated using an
orally inserted glass tube supplying a constant flow of aerated,
charcoal-filtered city tap water containing the anesthetic, 0.005%
(initial concentration) MS-222 (ethyl-m-aminobenzoate methane sulfonic
acid). Access to the olfactory organ was achieved by removing the skin
and connective tissue between the incurrent and excurrent nares,
superficial to the olfactory organ.
Stimuli
Representatives of four different classes of amino acids:
L-glutamic acid (acidic), L-arginine (basic),
L-methionine [neutral with a long side-chain (LCN)] and
L-alanine [neutral with a short side-chain (SCN)] were
presented individually and in binary mixtures with citrate (trisodium),
EGTA [ethylene
glycol-bis(aminoethylether)-N,N,N'N'-tetraacetic acid],
distilled water, calcium chloride, barium chloride, and strontium
chloride. All stimuli used in the study were prepared using
charcoal-filtered city tap water (containing 0.1-0.5 mM Ca2+), pH adjusted (8.5-9.0) to match the
control water bathing the olfactory organ and presented at
concentrations varying from 10
6 M to
10
2 M. Stock solutions at 10 mM were prepared
weekly, except for citrate, EGTA, CaCl2,
BaCl2, and SrCl2, which
were prepared daily. During the experiments, a series of four or five
consecutive odorants were preceded and followed by the presentation of
the standard (1 mM L-methionine). Data recorded in response
to these stimulus series were included in the study only if the initial
and final responses to the standard differed by <10%; if the
difference was more than a 10% change in response magnitude, the data
were excluded from the analysis. Stimulus delivery was via a
"gravity-feed" system employing a spring-loaded valve driven by a
pneumatic actuator at 40 psi. Stimulus solutions and charcoal-filtered
artesian water used to bathe the olfactory mucosa between stimuli were
delivered through separate Teflon tubes (0.8 mm diam) at a rate of 6-8
ml/min. The olfactory cavity was continuously perfused with
charcoal-filtered tap water to 1) facilitate stimulus
delivery, 2) protect the mucosa from desiccation,
3) avoid the introduction of mechanical artifacts associated
with stimulus presentation, and 4) thoroughly rinse the
olfactory cavity between stimuli (3-5 min interstimulus intervals). A
foot switch connected to an electronic timer triggered the valve to
introduce the odorants for a 5-s stimulus duration.
Recording technique and data analysis
In vivo recordings of multiunit olfactory neural activity
were made using metal-filled glass capillary electrodes plated with platinum (~15 µm ball; impedance, 10-40 k
) placed against the sensory face of an olfactory lamella (Caprio 1995
;
Gesteland et al. 1959
). The electrode was r.c.-coupled
(220 pF capacitor, 20 M
resistor) to a high-impedance probe at one
input with the other input grounded via a hypodermic needle embedded in
the flank musculature of the fish. The receptor neural activity was
amplified (band-pass 30-300 Hz), observed on an oscilloscope,
integrated (0.5 s rise time), and displayed on a pen recorder. These
amplified signals were stored on a hi-fi stereo VCR as an analogue
signal via one of the audio inputs or as a digitized video signal. All
recorded data were digitized at 32 kHz and analyzed off-line by
Discovery software (Brainwave Systems Discovery package Version 5.0, DataWave Technologies, Longmont, CO) and printed.
 |
RESULTS |
Citrate and EGTA enhance ORN activity and trigger PW activity
Citrate (0.1 and 0.5 mM) in binary mixtures with an amino acid
(0.1 mM) enhanced asynchronous olfactory receptor responses (Fig.
1, Table
1). In addition, single amino acids
(
0.1 mM) and binary mixtures of citrate and an amino acid selected
from any of four classes (acidic, basic, LCN and SCN) of amino acids generated PWs (Fig. 2). During the first
2 s of PW activity, the mean frequency displayed was 28 ± 5.6 Hz (mean ± SE, n = 25 fish, 283 trials);
however, at stimulus concentrations just above PW threshold, some
records show apparently lower frequency (e.g., Fig. 2, A and
B), but interspersed with the large waves are smaller ones
that increase in magnitude with increasing stimulus potency. Frequencies sampled from the responses of nine fish during the 5th s of
PW activity in response to a 5-s stimulus decreased by 7 ± 3.7 Hz
(n = 84) from those measured during the 1st s of
synchronous activity. No specific frequency or range of frequencies was
associated with a particular stimulus or fish preparation (Table
2). Components of binary mixtures that
triggered PW activity included
0.5 mM amino acid and
0.1 mM
citrate. A lower concentration of an amino acid (10 µM) resulted in
PWs if the concentration of citrate was raised to
0.5 mM. Individual
presentations of
0.1 mM EGTA or
3 mM trisodium citrate alone
triggered PWs (Fig. 3). PW activity is
stimulus driven, because intrinsic oscillatory activity was never
observed.

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Fig. 1.
Citrate enhances olfactory receptor responses to amino acids. Absence
of response to control water (A1 and A2)
and responses to 0.1 mM L-methionine (L-Met;
B1 and B2), 0.1 mM citrate
(C1 and C2) and enhanced response to the
binary mixture 0.1 mM L-Met and 0.1 mM citrate
(D1 and D2). Labels with the number 1 are
raw data, and those with 2 are integrated (0.5 s). In this and
succeeding figures, stimulus duration was 5 s, and arrows indicate
stimulus onset.
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Fig. 2.
Peripheral waves (PWs) are generated in response to mixtures of citrate
and a representative from each of 4 classes of amino acids. PW
responses to binary mixtures of 1 mM trisodium citrate and 1 mM
L-Met [neutral with long side chain (LCN);
A], 1 mM L-alanine [L-Ala;
neutral with short side chain (SCN); B], 1 mM
L-arginine (L-Arg; basic; C),
and 2 mM L-glutamic acid (L-Glu; acidic;
D).
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Fig. 3.
Calcium chelators trigger PW activity. Trisodium citrate (2 mM;
A) and 10 µM EGTA (C) were insufficient
to trigger PWs; however, 3 mM citrate (B) and 0.1 mM
EGTA (D) caused PW activity.
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Divalent cations abolish both the enhancement of asynchronous
activity and PWs
The possibility that the enhancement of olfactory receptor neural
activity was due to citrate's ability to chelate calcium prompted the
testing of EGTA, a classical calcium chelator, to see whether it also
caused an increase in responsiveness. EGTA (30 µM) caused a similar
enhancement of asynchronous responses (Fig.
4) as was seen with citrate (0.1 mM). The
addition of divalent cations (1 mM CaCl2,
BaCl2, or SrCl2) to binary
mixtures of an amino acid and citrate (Fig.
5, A-C) or EGTA (Fig.
5D) eliminated the enhancement of asynchronous olfactory
receptor activity to a response similar to that to the single amino
acid alone. PWs triggered by binary mixtures of citrate and an amino
acid were also abolished in 100% of 42 trials by the addition of
CaCl2, BaCl2, or
SrCl2, but the magnitude of responses to single
amino acids in the presence of divalent cations was not appreciably reduced (Fig. 6). PWs in response to
calcium chelators (citrate and EGTA) alone were abolished in 100% of
16 trials by the addition of CaCl2 or
BaCl2 (Fig. 7).

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Fig. 4.
EGTA enhances asynchronous olfactory receptor responses to amino acids.
The integrated (0.5 s) response to the binary mixture 0.1 mM
L-Met +30 µM EGTA is enhanced when compared with the
responses to the individual components.
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Fig. 5.
Enhancement of the integrated (0.5 s) response to L-Met + calcium chelator [citrate (0.1 mM; A-C) or EGTA (30 µM; D)] is abolished by divalent cations [1 mM
CaCl2 (A and D),
BaCl2 (B and D) and
SrCl2 (C and D)], which do
not affect the response to L-Met alone
(A-C).
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Fig. 6.
Addition of calcium chloride to a binary mixture of an amino acid and
citrate abolishes PW activity. PW activity is generated to the mixture
of 1 mM L-Met +1 mM citrate (A), but not to
the binary mixture in the presence of 1 mM calcium chloride
(B). The response to the binary mixture in the presence
of calcium chloride is similar to that seen to L-Met alone
(C). D: lack of response to a water
control.
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Fig. 7.
Addition of calcium chloride abolished PW activity to calcium-chelating
stimuli. PWs generated in response to 5 mM citrate (A)
and 0.1 mM EGTA (C) were abolished by the addition of 1 mM calcium chloride (B and D).
E: lack of response to a water control.
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Effects of distilled water
Single amino acid stimuli presented in four fish
preparations (5-s duration) at concentrations normally insufficient to
produce PW activity generated PWs when prepared in distilled water. The addition of 1 mM CaCl2, however, prevented PW
activity (Fig. 8).

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Fig. 8.
Amino acid dissolved in distilled water triggers PWs. PWs were
generated by 1 mM L-Met in distilled water
(A). The same amino acid dissolved in charcoal-filtered
tap water did not initiate PWs (B). Calcium chloride (1 mM) abolished PW activity observed in A
(C).
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DISCUSSION |
Prior studies reported enhancements of taste responses to
binary mixtures of citrate and amino acids (Davis and Caprio
1996
; Gilbertson et al. 1997
; Ogawa and
Caprio 1999
). Because both olfactory and taste systems of
channel catfish are highly sensitive to amino acids (Caprio
1978
), it was hypothesized that a similar enhancement by
citrate as seen in gustation might also occur in olfaction. This study
shows that trisodium citrate also enhances olfactory receptor responses
in the channel catfish to amino acids (Fig. 1) and can trigger voltage
oscillations known as PWs (Fig. 2). Olfactory receptor response
enhancement was observed to binary mixtures of citrate and a
representative of each of four classes of amino acids: neutral amino
acid with a long, linear side-chain (L-methionine), neutral
amino acid with a short side-chain (L-alanine), acidic
amino acid (L-glutamate), and basic amino acid
(L-arginine).
The generation of PW activity depends on surpassing a threshold level
of depolarization required to initiate a synchronization of ORN
responses. The coordinated activity of multiple ORNs responding to an
odorant generates large PWs, which have been observed in representatives of every class of vertebrate [fish (Sutterlin and Sutterlin 1971
), amphibian (Ottoson 1959
;
Takagi and Shibuya 1961
), bird (Tucker
1975
), and mammal (Adrian 1956
)]. PWs
1) are odorant-driven (i.e., do not occur intrinsically),
and in most species studied have a frequency that is significantly
higher than olfactory bulbar waves; 2) are generated within
the olfactory sensory epithelium (Parker et al. 1997
;
Sutterlin and Sutterlin 1971
; Takagi and Shibuya
1961
); 3) are not derived from elements other than
ORNs (Takagi and Shibuya 1961
); 4) are not
influenced by bulbar electroencephalographic (EEG) waves, because
transection of the olfactory nerves have no effect on either their
magnitude or frequency (Sutterlin and Sutterlin 1971
);
and 5) possibly function as a logic gate to facilitate the
release of glutamate onto mitral/tufted cells possessing
N-methyl-D-aspartate (NMDA) receptors
(Ennis et al. 1996
), resulting in long-term potentiation
associated with learning and memory (Brunjes 2000
;
Parker et al. 1999
).
The present report clearly indicates that response enhancement by
citrate is due to its ability to chelate calcium. The addition of
calcium, barium, or strontium chloride to the binary mixture of an
amino acid and citrate abolished this enhancement by inactivating the
calcium chelating ability of citrate, thus confirming that it is the
reduction of divalent cations that is responsible for the hyperexcited
state of the ORNs. Lowering the extracellular calcium concentration
with a conventional calcium chelator (EGTA) in a binary mixture with an
amino acid produced a similar enhancement to that seen with citrate.
This enhancement was also eliminated by the addition of divalent
cations (1 mM CaCl2, BaCl2,
or SrCl2) to the stimulus mixture, which did not
affect olfactory responses to single amino acids (Fig. 5). PW activity
triggered by the presence of a calcium chelator in a binary mixture
with an amino acid was also abolished by the addition of divalent
cations (Fig. 6). One millimolar calcium is a concentration similar to
that reported in the mucus surrounding the cilia of ORNs in the frog
(Joshi et al. 1987
). However, due to the greater potency
for calcium chelation by EGTA, lower concentrations of EGTA (30 µM)
achieved a similar enhancement of asynchronous olfactory activity as
that observed with citrate (0.1 mM). In addition, PWs were observed to
amino acid stimuli that by themselves were not sufficiently potent to
generate synchronized voltage oscillations, but did so dissolved in
distilled water. Peripheral waves were abolished with the addition of
calcium chloride or barium chloride, which resulted in a response
similar to that to the amino acid component alone (Fig. 8).
A reduced extracellular calcium ion concentration can result
theoretically in the increased excitability of vertebrate ORNs due
either to 1) lessening the calcium block on olfactory
receptor cyclic nucleotide-gated (CNG) channels (Frings et al.
1995
; Kleene 1995
; Kleene and Pun
1996
; Zufall and Firestein 1993
) and/or
2) by lowering of the surface potential of neurons, shifting
ion channel activation to a more negative potential (Hille
1992
). Although CNG channels are involved in the transduction
of olfactory information in tetrapods (Belluscio et al.
1998
; Brunet et al. 1996
; Firestein et
al. 1991
; Kurahashi 1990
; Nakamura and
Gold 1987
), and in situ hybridization studies demonstrated the
extensive presence of CNG channels in the olfactory epithelium of the
channel catfish (Ngai et al. 1993
), there is no evidence
linking this specific channel type to olfactory responses to amino
acids in this species (Bruch and Teeter 1990
). Current
data clearly show that IP3-gated ion channels
mediate olfactory responses to amino acids in the channel catfish
(Bruch 1996
; Restrepo et al. 1993
) and
other teleosts (Speca et al. 1999
). Thus the mechanism
for the presently described enhancement of ORN activity is consistent with being an effect on the divalent field charge at the membrane of
ORNs. Under conditions of a lowered divalent cation concentration as
occurring in the presence of citrate and EGTA, sodium ions entering through ciliary and microvillous IP3
channels are sufficient to activate the ORNs (Schild and
Restrepo 1998
).
Binary mixtures of citrate and amino acids are most likely
similar to chemical stimuli the catfish would normally be exposed to
during feeding in its natural environment. Amino acids are present in
high (up to a few hundred millimoles/liter) concentration in all living
tissue (Carr 1988
; Carr et al. 1996
) and
are naturally released into the aqueous environment. Citrate is present
in all living cells that undergo aerobic respiration via the citric
acid (i.e., Krebs) cycle and is also released in high (>30 mM)
concentrations by common food sources for the channel catfish (e.g.,
insects) (Wyatt 1961
). The use in the present study of
naturally occurring and physiologically relevant concentrations of
stimuli suggests that PWs occur in the animal's natural environment.
The enhancement of taste responses with citrate in rodents occurred
only in response to stimuli that caused depolarization of the taste
receptor cells (Gilbertson et al. 1997
). This finding correlates with the proposed mechanism in the present experiments for
the enhancement of olfactory receptor responses, i.e., the ability of
citrate to chelate calcium. With respect to citrate's flavor-enhancing
ability in human food (Gardner 1972
), citrate may not
add only a sour component, but is likely to enhance the taste of
gustatory stimuli whose normal action is to depolarize taste cells.
This research was supported by National Science Foundation Grant
IBN-9221891 and National Institute on Deafness and Other Communication
Disorders Grant DC-03792 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.