Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Correlations between Olfactory Discrimination, Olfactory Receptor Neuron Responses and Chemotopy of Amino Acids in Fishes
Department of Biology, University of Ljubljana, Slovenia
Correspondence to be sent to: Tine Valentincic, e-mail: tine.valentincic{at}uni-lj.si
Key words: behavior, coding, conditioning, electroolfactogram, electrophysiology, fishes, mixtures
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Olfactory discrimination of amino acids in catfish and zebrafish |
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Catfishes (genera Ictalurus and Ameiurus) discriminate nearly every conditioned amino acid stimulus from every other amino acid stimulus (Valentinèiè and Caprio, 1994; Valentinèiè et al., 1994, 2000a). However, bullhead catfish were always unable to discriminate L-isoleucine (L-Ile) from L-valine (L-Val) and in some cases they were also unable to discriminate L-alanine (L-Ala) from L-serine (L-Ser) and glycine (Gly). We discovered that the olfactory discrimination capabilities of catfish and zebrafish (Danio rerio) are very similar. Knowledge of chemotopic projections of amino acid stimuli on the surface of the olfactory bulb studied with calcium labeling technique (Friedrich and Korsching, 1997
)
enabled us to predict which amino acids zebrafish can and cannot discriminate.
Differential bulbar activity patterns should facilitate olfactory discrimination, whereas
identical bulbar patterns should not enable olfactory discrimination. Largely different
bulbar activity patterns for short-chain neutral and long-chain neutral, acidic and basic
amino acids make their behavioral discrimination easy. Nearly identical bulbar activity
patterns that in most zebrafish occur after stimulation with L-Val and
L-Ile did not enable discrimination of these two amino acids irrespective of
the discrimination training applied. The bulbar activity patterns that occur after
stimulation with chemically similar amino acids, such as L-Ala and
L-Ser and L-arginine (L-Arg ) and L-lysine
(L-Lys) are not identical, their fractional differences allowed zebrafish to
discriminate these amino acid pairs. In conclusion, minor differences in bulbar activity
patterns enable olfactory discrimination of amino acids; the more similar these patterns
are, the less the chance of their olfactory discrimination. In individual zebrafish, the
bulbar activity patterns after stimulation with the same amino acid are slightly
different (Friedrich and Korsching,
1997), which makes discrimination capabilities of individual zebrafish
different. In bullhead catfish, the individual differences in L-Ala/
L-Ser discrimination capabilities depend on phenotypic expression rather than
genetic differences between individuals since the same catfish that did not discriminate
L-Ala from L-Ser before olfactory organ extirpation started to
discriminate these two amino acids subsequent to regeneration (Stenovec and
Valentinèiè, 2001). |
Olfactory discrimination of amino acid mixtures in bullhead catfish |
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Bullhead catfish initially detect binary and ternary mixtures of amino acids as their more stimulatory components; five to 12 successive comparisons of a mixture and its more stimulatory component alone enable the catfish to discriminate the more stimulatory amino acid from the mixture (Valentinèiè et al., 2000b). The more stimulatory components of binary mixtures were prepared using amino acid concentrations that were 3–30-fold higher than their equal stimulatory effectiveness concentrations determined by equal amplitude of electroolfactogram (EOG). Multimixtures composed of seven and 12 amino acids were studied at component concentrations that resulted in equal EOG amplitudes. Catfish discriminated a conditioned seven amino acid mixture from their six-, five- and four-component counterparts. The conditioned mixture of 12 amino acids was discriminated from its nine- and 10-component counterparts; however, irrespective of the missing component, they were unable to discriminate the 12-component mixture from its 11-component counterparts.
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Regenerated olfactory organs facilitate olfactory discrimination of amino acids |
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To study olfactory discrimination in catfish with regenerated olfactory organs, the fish were anesthetized with ethyl 3-aminobenzoate methanesulfonate salt (MS-222, dilution 1:8000) and olfactory organs were surgically excised (>95% of the olfactory organ was removed). The incompletely excised olfactory organs of bullhead catfish regenerated
3 months after surgery. The olfactory discrimination capabilities of
catfish were tested between 3 and 7 months post surgery. The regenerated olfactory roseta
was always several times smaller than the intact olfactory roseta; in some cases, only
few fingerlike lamellae had regrown. Olfactory discrimination capabilities of bullhead
catfish with the intact and regenerated olfactory organs were identical and in some cases
even better than the olfactory discrimination capabilities of the same catfish prior to
extirpation. At the end of the 5–6 months period after surgery even a tiny single
olfactory lamella enabled amino acid discrimination. We investigated the connectivity of
the olfactory receptor neurons (ORNs) with different areas of the olfactory bulb (OB) in
bullhead catfish with either intact or regenerated olfactory rosetae. In regenerated
olfactory rosetae, DiI crystals were introduced into either anterior ventral or lateral
ventral sites of the bulb. In both, intact and regenerated olfactory rosetae, the
insertion of the DiI into the anterior region of the ventral OB resulted in fluorescent
labeling of tall (ciliated) olfactory receptor neurons (Morita and Finger, 1998;
Hansen et al., 2003) whereas
lateral crystal insertion of the ventral OB resulted in labeling intermediate
(microvillus) ORNs. Both, the fine structure of the olfactory organ and the olfactory
discrimination abilities recovered during regeneration of the olfactory organs. |
Electroolfactogram and single olfactory receptor neurons responses to amino acid stimuli |
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The cellular olfactory code is provided by a layer of ORNs. A subpopulation of ORNs is spontaneously active and a second much larger subpopulation of ORNs is not spontaneously active (silent ORNs) prior to stimulation. For technical reasons, only the physiological responses of the spontaneously active ORNs were reported previously (Kang and Caprio, 1995
). The spontaneously active
ORNs predominantly responded to amino acid stimuli with suppression. However neurons that
code for amino acid odorants should respond to stimulation in a dose-dependent manner.
For the electrophysiological experiments, we anesthetized the catfish with the anesthetic
MS-222 and placed them into the recording chamber, perfused their gills with the
anesthetic and immobilized them with an intramuscular injection of gallamine triethiodide
(Flaxedil, 0.16 mg/100 g body wt). The concentration dependence of the suppressive
response to amino acid stimuli was at best ordinal (Stevens, 1946, 1951;
Velleman and Wilkinson, 1993). We found no correlation between the ability of catfish to discriminate amino acids and the relative number of suppressive responses of ORNs. Among several hundreds of spontaneously active ORNs tested, few (<3%) cells responded to stimulation with dose-dependent excitation. Spontaneous activity is a likely property of young olfactory receptor neurons establishing synaptic connections with the olfactory bulb glomeruli.
Olfactory organs of fishes are fully functional in freshwater that contain very small
ion concentrations. The physiological functions of the entire olfactory organ and of
individual ORNs were preserved for several hours even in highly purified water (HPW) that
contained 1000 times fewer ions than the artificial pond water (Figure
1). Due to the high resistance
(R > 18.2 M
cm) there was little shunting of the electrophysiological
signals in HPW. Responses to amino acid stimuli of numerous silent (non-spontaneously
active) ORNs could be observed in these conditions. The number of lamellar locations
where responses to amino acid stimuli were detected correlated highly with the amplitude
of EOGs to the same stimuli. These results corroborate the assumption that the summed
receptor potentials add up into the EOG amplitude. The silent ORNs responded to amino
acid stimuli repeatedly and the duration of their responses was dose-dependent. Most ORNs
(Figure
2) responded to one or two amino acid
stimuli; the most numerous were the neurons responding to L-methionine
(L-Met) and L-norvaline (L-nVal; 27% of all the
tested cells). Responses to stimuli that elicit very small magnitude EOGs, such as
L-proline (L-Pro), were also detected. At 23 recording locations,
responses to four to eight amino acids were observed; some of these activities originated
from single ORNs, whereas in other locations, single cell origin of action potentials
could not be confirmed in our extracellular recordings.
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Acknowledgements |
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Funded by Slovenian Ministry of Education and Science grant P0-0509-0487
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