Associative learning of plant odorants activating the same or different receptor neurones in the moth Heliothis virescens
1 Neuroscience Unit, Department of Biology, Norwegian University of Science
and Technology, Olav Kyrres gate 3, NO-7489 Trondheim, Norway,
2 Research Centre on Animal Cognition, Paul Sabatier University, 118 Route
de Narbonne, FR-31062 Toulouse cedex 4, France,
3 Institut für Biologie-Neurobiologie, Freie Universität Berlin,
Königin Luise Straße 28-30, DE-14195 Berlin, Germany
4 Centre for the Biology of Memory, Norwegian University of Science and
Technology, Olav Kyrres gate 3, NO-7489 Trondheim, Norway
* Author for correspondence (e-mail: hanna.mustaparta{at}bio.ntnu.no)
Accepted 6 December 2004
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Summary |
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Key words: olfactory learning, linalool, ocimene, myrcene, proboscis extension response, differential conditioning, primary odorant, recordings
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Introduction |
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A range of neurophysiological methods in the honeybee and in the fruitfly
Drosophila melanogaster have allowed characterisation of the brain
structures involved in olfactory learning and memory
(Heisenberg, 2003;
Menzel, 2001
). In the
honeybee, multiple convergence sites between the olfactory (CS) and the taste
(US) pathway have been identified. Thus, a ventral unpaired median neurone
(VUMmx1) was found, the depolarisation of which was shown to substitute for
the rewarding part of the US in associative learning experiment
(Hammer, 1993
). This neurone
has dendrites and its cell soma in the taste centre, the suboesophageal
ganglion, and has extensive arborisations in three olfactory areas: the
antennal lobe, the lateral protocerebrum and the calyces of the mushroom
bodies. Several studies using optical or intracellular recordings have shown
changes of odour responses in the antennal lobe and in the mushroom bodies
after olfactory learning (Faber et al.,
1999
; Faber and Menzel,
2001
; Mauelshagen,
1993
; Sandoz et al.,
2003
; Daly et al.,
2004
; Yu et al.,
2004
). At the peripheral level, possible changes of odour
responses by antennal receptor neurones (RNs) have been discussed in studies
that have given contradictory results (De
Jong and Pham-Delègue, 1991
;
Pham-Delègue et al.,
1997
; Sandoz et al.,
2001
; Vet et al.,
1990
; Wadhams et al.,
1994
). In most of these studies techniques have been used that
recorded summated peripheral responses or processed responses in higher-order
neurones, making it difficult to understand where the changes occur.
Heliothine moths, for which the olfactory RNs have been well described,
represent an interesting model for evaluating possible changes in odorant
responses through learning. However, before the search for learning-induced
changes can begin, a particular effort must first be made to describe better
the learning mechanisms in these species. The present study represents such an
effort in H. virescens, focusing on three plant odorants with a
strong biological relevance for this moth.
By the use of gas chromatography linked to electrophysiological recordings
from single RNs, 19 types of plant odour RNs have been classified in this
species (Mustaparta, 2002;
Røstelien et al.,
2000a
,b
;
Stranden et al.,
2003a
,b
;
T. Røstelien, M.S., A.-K. Borg-Karlson and H.M., unpublished). The RNs
are characterised by strong responses to one odorant (primary odorant) and
weak responses to a few others with related molecular structures (secondary
odorants). Among them are three frequently occurring RN types, tuned to
E-ß-ocimene (3E-3,7-dimethyl-1,3,6-octatriene),
geraniol (2E-3,7-dimethyl-2,6-octadien-1-ol) and
(S)-(+)-linalool (3,7-dimethyl-1,6-octadien-3-ol), respectively.
ß-Myrcene (7-methyl-3-methylene-1,6-octadiene) and
Z-ß-ocimene (3Z-3,7-dimethyl-1,3,6-octatriene) are
secondary odorants of the E-ß-ocimene RN type, (R)-(-)-linalool
of the (S)-(+)-linalool type, and both enantiomers of linalool are
secondary odorants of the geraniol type.
In the first part of this study, parameters of the learning procedure affecting the acquisition of the CS-US association were tested. Male and female moths were subjected to acquisition procedures with increasing concentrations of racemic linalool, a floral volatile compound activating at least two RN types. As the time parameters of CS and US presentations are critical for learning, we also studied the influence on acquisition of the interval between CS and US onset, i.e. the inter-stimulus interval (ISI). We then asked whether odorants with differential activation of RN types cause different learning rates, a fact that would point to differences in their salience. As ß-myrcene activates the same RN type as E-ß-ocimene, but with a lower efficacy, we would expect E-ß-ocimene to be efficiently learned at lower concentrations than ß-myrcene. Likewise, since racemic linalool would activate at least two RN types (one with high and the other with low efficacy), we asked whether it might be learnt at lower concentrations than the two other odorants. Thus, the learning rate of the three odorants at different concentrations was compared. To clarify the relationship between RN activation and the strength of olfactory input to the brain, electroantennograms (EAG) were recorded as responses to the same concentrations of the three odorants. Finally, we asked whether the moths have a higher ability to discriminate odorants that activate different RN types than odorants activating the same type. Choosing concentrations that gave a similar learning rate in the previous experiment, differential conditioning experiments with all six odour pairs were carried out. The hypothesis was that moths would more easily discriminate racemic linalool from ß-ocimene/ß-myrcene than ß-ocimene from ß-myrcene, since the two latter odorants activate the same RN type.
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Materials and methods |
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Test compounds
The odorants used as CS were the plant odorants ß-ocimene (21%
Z-ß-ocimene and 58% E-ß-ocimene), ß-myrcene
(75%), and racemic linalool (Experiment 1 and 2, 87% racemic
linalool; or Experiment 3, 4 and 5, 82% racemic linalool). We chose
to use the commercially available racemate of linalool, since both enantiomers
activated the two RN types. The given purities of the odorants were determined
by analyses of injections in a gas chromatograph with DB-wax column (J&W
Scientific, Agilent Technologies, Palo Alto, CA, USA; 30 m, i.d. 0.25 mm, film
thickness 0.25 µm). Separations in the column were performed from the
initial temperature 80°C with an increase rate of 6°C min-1
to 180°C, and a further increase rate of 15°C min-1 to
220°C. Except for the racemic linalool (Sigma-Aldrich, Steinheim,
Switzerland) used in Experiment 1 and 2, all odorants originated from Fluka
Chemika (Sigma-Aldrich, Steinheim, Switzerland). For experiment 1, we applied
10, 100 and 1000 mg of undiluted racemic linalool onto pieces of
filter paper inserted into glass cartridges. The lowest amount (1 mg) of
racemic linalool was obtained from a dilution in n-hexane (>99%).
In Experiments 2, 3, 4 and 5 the compounds were first diluted in hexane, and
from each dilution 100 µl were evaporated onto a piece of filter paper
placed in a weak stream of pure nitrogen (99.99%). Each filter paper was
placed in a glass cartridge sealed with teflon caps. Using these cartridges,
we stimulated the animals with the different concentrations of the three
odorants. Sucrose (1 mol l-1) was used as the US stimulus.
Experiment 1. Effect of CS concentration on acquisition and retention
As a first approach to conditioning experiments in moths we wanted to
investigate the effect of CS concentration on acquisition and retention. For
each conditioning trial the insects were placed one at a time into a purified
air stream (240 ml min-1). The antennae were stimulated by
blowing an air pulse (
50 ml min-1, 5 s) through the test
cartridge into the continuous air stream. After 2.5 s of the onset of the CS,
the US was given for
4 s. The US was always a compound US, and was given
first to the antennae and then to the extended proboscis, which allowed an
uptake of sucrose solution. If the moth did not show a response to the sucrose
stimulation of the antenna during the first conditioning trial, extension of
the proboscis was forced and was stimulated with the sucrose solution. The
inter-trial interval (ITI) was set to 7 min, and 12 trials were performed.
Insects that did not respond to the US in three subsequent training trials
were considered to be not properly motivated, and were excluded from the
analysis. The four concentrations of racemic linalool were tested in
separate experiments for each sex. The results obtained for all concentrations
were plotted as the percentage of animals showing the CR as a function of the
number of conditioning trials (acquisition curves). Extinction tests were
performed 15 and 120 min after the end of training.
Experiment 2. The effect of different inter-stimulus intervals (ISIs) on learning
One important parameter in learning experiments is the interval between CS
and US. We therefore tested five groups for different ISIs to determine the
optimal delay between onset of CS and US. The ISIs -1 (backward pairing), 0,
1, 2 and 3 s were chosen for this experiment according to previous results
from the moth S. littoralis (Fan
et al., 1997). Racemic linalool was used as the CS in a
concentration of 100 mg on filter paper, which produced good acquisition in
Experiment 1. Both males and females were used, but in separate test series.
To adapt the insects to the test conditions, they were placed in the constant
air stream (
400 ml min-1) 15 s prior to the odour stimulation
(
100 ml min-1, 5 s) and left in this position until 5 s after
the end of the US stimulation. The sucrose stimulus (US) was given for 5 s,
first to the antennae and then to the extended proboscis. All ISIs were tested
in all experiments. Each individual was given eight conditioning trials.
Precise timing of the CS and US stimulation was ensured by an auditory signal
given every second. The ITI was set to 15 min. Acquisition curves were made
for the forward-paired groups (i.e. groups trained with the ISIs 1, 2 and 3
s). An extinction test was performed 60 min after the last conditioning trial.
The results were plotted as the percentage of animals showing the CR in the
extinction test for the different ISIs. Exclusion of non-motivated animals was
carried out as in Experiment 1.
Experiment 3. Dose-response relationships of learning rate with racemic linalool, ß-ocimene and ß-myrcene
The effect of different concentrations of odorants used as CS on the
learning rate was compared between the three odorants racemic
linalool, ß-myrcene and ß-ocimene. Female moths were subjected to
conditioning procedures with the three odorants at four different
concentrations (0.1, 1.0, 10 and 100 mg of the component on filter paper). Ten
conditioning trials with 1 s ISI and 15 min ITIs were performed. Extinction
tests were performed 15 min after the last conditioning trial. Otherwise the
experimental procedure was identical to that in Experiment 2. Since all four
concentrations of linalool showed a high percentage of CR in the test,
additional conditioning experiments were performed with a lower amount of this
odorant (0.001 mg). The results obtained for the three odorants were plotted
as the percentage of animals showing CR in the extinction test as a function
of odorant concentrations.
Experiment 4. Electroantennogram dose-response relationships for racemic linalool, ß-ocimene and ß-myrcene
Since the three odorants caused different dose-response relationships for
the learning rates, EAG recordings were performed to find out whether these
differences could be correlated with different antennal sensitivity to the
odorants. Moths were immobilised with the antenna fastened at the base and the
tip by tungsten hooks to a dental-wax layer on the top of the plexiglass
holder. Glass capillary microelectrodes filled with Ringer's solution (150 mM
NaCl, 3 mM CaCl2, 3 mM KCl, 10 mM TES buffer, pH 6.9) were used as
recording electrodes. The reference electrode was placed into the haemolymph
of a proximal antennal segment or into the eye, and the recording electrode
into the cut tip of the antennae. The three odorants racemic
linalool, ß-myrcene and ß-ocimene were tested in five concentrations
(from 0.01 to 100 mg) in two parallel series, starting with the lowest
concentrations. Antennae of five females were tested, and in two of these a
lower concentration of the three odorants (0.001 mg) was included. The glass
cartridges used for a maximum of 2 days were stored at -20°C. A double set
of control cartridges, each with hexane or pure air, were used as controls
before each test series and between each concentration step. The preparation
was placed in a constant clean air stream (250 ml min-1),
which was turned off during stimulation. Stimulation was performed
via a parallel air stream through the odour cartridges (
500 ml
min-1) onto the antenna. The time between each odour stimulus
increased from 1 min for the lowest to 8 min for the highest concentrations to
avoid adaptation. The signals were amplified 1000 x and visualised and
analysed using the EAG software (version 2.6d, Syntech, Hilversum, The
Netherlands). The mean response to the pure air stimulus was subtracted from
all odorant responses and the results were given as the percentage of the
response to 10 mg racemic linalool, which elicited a high response in
all animals. The results are given as dose-response curves of the average EAG
response of the two parallel test series to each odorant concentration in all
individuals tested. Finally, we plotted the learning rate for each
concentration of each odorant (% proboscis extension in the extinction test in
Experiment 3) as a function of the EAG response (% of the 10mg
racemic linalool standard) and calculated the Pearson R
correlation coefficient.
Experiment 5. Discrimination between racemic linalool, ß-ocimene and ß-myrcene
We investigated the ability of female moths to discriminate the three
odorants, and evaluated whether there was a difference in discrimination
between odorants activating the same RN type and odorants activating different
RN types. Concentrations of the three odorants giving similar learning levels
in Experiment 3 were chosen for this experiment: 0.1 mg for racemic
linalool, 10 mg for ß-ocimene and 100 mg for ß-myrcene. Each animal
was trained with two odorants, one rewarded (CS+) and one explicitly presented
without a reward (CS-). Rewarded and unrewarded trials were performed six
times each in a pseudo-randomised order (CS+, CS-, CS-, CS+, CS-, CS+, CS+,
CS- or CS-, CS+, CS+, CS-, CS+, CS-, CS-, CS+). The ISI was set to 1 s and the
ITI to 15 min. Fifteen minutes after the last conditioning trial, both
odorants were presented in extinction tests in a balanced order.
Statistics
In all conditioning experiments, dichotomous data were recorded, the moths
responding or not to the CS at each trial. We compared responses either within
each group, to see the development of responses throughout the experimental
procedures, or among groups, to compare moths' performance in different
conditions.
Within-group comparisons
To see whether acquisition took place, i.e. if the responses within each
group increased significantly throughout the conditioning procedure, we used
Cochran's Q test (Experiments 1, 2 and 5). When testing for differences in the
proportion of responding individuals within the same group, at two points in
time or to two different stimuli, we used the exact McNemar test (Experiments
1 and 5).
Among-group comparisons
To compare the overall learning rate among groups, we counted the number of
responses given by each moth during the whole conditioning procedure and used
the Kruskal-Wallis test (Experiment 1). This test was also used in Experiment
3 to test for significant differences between concentrations of the three
odorants giving the same degree of CR. When only two groups were involved,
i.e. when comparing sexes in Experiment 1, the exact Mann-Whitney test was
used. Moreover, to compare moths' responses in the test phase, a Fisher's
exact test (Nd.f.) was used on the proportion of responding insects
in the different groups (Experiments 1, 2, 3 and 5). In Experiment 2, we also
carried out pair-wise comparisons between groups conditioned with different
ISIs, using Fisher's exact tests with Bonferroni correction. With an overall
significance level of 5% and 10 combinations of groups being tested, the new
threshold for each test was set to 0.5% (5%/10).
To compare EAG responses to the three odorants at the five highest odorant concentrations (Experiment 4) a General Linear Model with repeated measurements test was used (sphericity assumed) with a Tukey (honestly significant difference) correction (threshold 5%), SPSS software (versions 11.0 and 12.0) was used for all the statistical analyses. The correlation between the learning rate (% proboscis extension in the extinction test in Experiment 3) and the EAG response (% of the 10 mg racemic linalool standard) for each odorant concentration was estimated by the Pearson R linear correlation coefficient.
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Results |
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Extinction trials were performed for all individuals 15 and 120 min after the last conditioning trial (Fig. 1C,D). The group conditioned to the highest odorant concentration (1000 mg) showed the highest response level after 15 min (60% of the females and 53% of the males). A significant decrease of the response level with decreased odorant concentration at the 15 min test was found for both sexes (Fisher's exact test, P<0.05, 3 d.f.). However after 120 min a tendency for differences was seen only among the male groups; no significant differences were observed among the female groups (Fisher's exact test, P=0.055 and P>0.05, 3 d.f., respectively). By comparing the response level between the sexes at the 15 min and the 120 min tests, no significant differences were found at any concentration (Fisher's exact test, P>0.05, 3 d.f.). A general decrease of the response level was found during the period of 15 to 120 min after the last conditioning trial. However, the decrease was only significant for one trained concentration in the females (1000 mg) and another one in the males (10 mg) (exact McNemar test, P<0.05).
Experiment 2. The effect of different inter-stimulus intervals (ISI) on learning
We found in Experiment 1 that PER increased with repeated presentations of
CS and US paired with an ISI of 2.5 s. We wanted to check the associative
nature of this conditioning, and find the optimal ISI for building the CS-US
association. In each sex, five groups of moths were subjected to eight
conditioning trials with different ISIs (-1, 0, 1, 2 and 3 s). Owing to the
different durations of CS-alone presentation in each trial, it was not
possible to compare acquisition curves. For 1 and 2 s ISIs, responses
increased significantly during training (data not shown, Cochran's Q tests,
P<0.05 in both sexes). For an ISI of 3 s there were no significant
performance increases in females, and only a trend in males
(P>0.05 and P=0.06, respectively). Acquisition could not
be recorded in the groups with -1, 0 and 1 s ISI, because the odorant never
appeared before the US.
Responses of the different groups in the extinction test could be compared directly because all groups received the same odorant stimulation (Fig. 2A,B). In both sexes, a clear heterogeneity appeared among the different ISI groups (Fisher's exact test, P<0.05, 4 d.f.). The presentation of the US one second before the CS (backward pairing) resulted in the lowest percentage of responses, whereas the CS presented one second before the onset of the US gave the highest number of responses. Longer intervals of 2 and 3 s showed a decreased percentage of responding females, whereas in males no change was seen in responding individuals for these intervals. In both sexes, pair-wise comparisons between ISI groups showed a clear significant difference between the group of 1 s ISI and the group -1 s ISI (Fisher's exact test with Bonferroni correction, P<0.005 in both sexes). This difference between responses in the forward- and in the backward-pairing groups was a critical control for showing that PER performance in our experiments was due to the formation of a CS-US association, i.e. to associative learning. Based on these results, the ISI of 1 s was chosen for Experiments 3 and 5. No significant differences were found between the results obtained for each ISI in the two sexes (Fisher's exact test, P>0.05).
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Experiment 3. Dose-response relationships of learning rate with racemic linalool, ß-ocimene and ß-myrcene
As in Experiment 1, where we found that the learning rate increased with
increasing concentrations of racemic linalool, we compared in this
experiment dose-response relationships for the three odorants,
racemic linalool, ß-ocimene and ß-myrcene. In these
experiments, no spontaneous responses to any of the odorants were ever
observed. Moreover, due to the fact that moths are slow to extend the
proboscis, and that the ISI was 1 s, very few moths showed CR during the
conditioning procedure. Thus, only the results of the extinction tests (5 s
odour presentations, allowing the moths to respond) performed 15 min after the
conditioning trials, were used and are shown in
Fig. 3A. The results of the
extinction tests generally showed an increase of responses to the CS with
increasing concentrations of the three odorants. This dose-response effect was
significant for ß-myrcene (Fisher's exact test, P<0.05), but
not for the two other odorants (Fisher's exact test, P>0.05).
However, the concentration-dependent PER curves differed between the odorants.
A much lower concentration threshold (0.001 mg) was found for
racemic linalool than for ß-ocimene (
0.1 mg) and
ß-myrcene (
1.0 mg). The CR to racemic linalool reached
maximum already at the concentration of 0.1 mg. Higher concentrations of
ß-ocimene and ß-myrcene were required for learning; ß-ocimene
showing qualitatively higher percentages of CR than ß-myrcene at the
three concentrations of 0.1, 1.0 and 10.0 mg.
|
Experiment 4. Electroantennogram dose-response relationships for racemic linalool, ß-ocimene and ß-myrcene
Since we found dose-response relationships for the three odorants in the
conditioning experiments, we evaluated antennal sensitivity to the same
stimuli using EAG recordings. Fig.
3B shows the dose-response curves obtained for the three odorants
racemic linalool, ß-ocimene and ß-myrcene. The results
showed that the moths were more sensitive to racemic linalool than to
the two other odorants, with the strongest response increase from 0.1 mg to 1
mg. The other two odorants elicited increased responses from the concentration
of 1 mg, reaching maximum responses at 10 mg. At the five highest
concentrations (0.01 mg to 100 mg) a stronger response was obtained to
racemic linalool than to the two other odorants. The differences were
significant between the responses to racemic linalool and
ß-myrcene [General Linear Model with repeated measurements test and a
Tukey (honestly significant difference) correction, (P<0.05)] and
showed a trend toward significant difference (P=0.07) between the
responses to racemic linalool and ß-ocimene. No significant
difference was found between the ß-ocimene and ß-myrcene responses
(P>0.05). The average response to evaporated hexane on filter
paper is also indicated in Fig.
3B, being slightly higher than the responses to the air controls.
The same trend toward sensitivity was found for the EAG recordings and
conditioning of the moths, being more sensitive to racemic linalool,
than to ß-ocimene, and, finally, least sensitive to ß-myrcene. When
representing conditioning performance relative to EAG responses
(Fig. 3C), a clear and
significant correlation (the Pearson R correlation coefficient,
R2=0.46, P<0.05) was found.
Experiment 5. Discrimination between racemic linalool, ß-ocimene and ß-myrcene
We compared how the three odorants were differentiated from each other
using differential conditioning experiments in which one odorant was rewarded
(CS+) and a second one was explicitly non-rewarded (CS-). To be able to
compare differentiation between odorants controlling for different odour
saliencies, we chose three concentrations of the odorants inducing similar
learning levels in Experiment 3: moths trained with concentrations of 0.1 mg
of racemic linalool, 10 mg of ß-ocimene and 100 mg of
ß-myrcene, showed the same level of CR (26%, 27% and 34%, respectively,
exact Kruskal-Wallis test, P>0.05, 2 d.f.). Thus, these
concentrations of the three compounds were chosen for this experiment. During
the differential conditioning procedure the moths showed only minimal
responses to the presentations of the CS+ or of the CS-(data not shown,
Cochran's Q test in all cases non-significant). This low response level to the
CS+ was probably due to the short ISI (1 s, as in Experiment 3) in addition to
the higher complexity of this differential conditioning procedure for moths.
The results of the extinction tests performed 15 min after differential
conditioning are shown in Fig.
4. Pooling the responses to CS+ and CS- in each of the three
odorant pairs, irrespective of which odorant was the CS+, showed that the
moths responded significantly more to the CS+ than to the CS-
(Fig. 4A, exact McNemar test,
P<0.05). When separating the responses within each odorant pair,
we could evaluate possible asymmetries in response differentiation when each
odorant was a CS+ or a CS- (Fig.
4B). When comparing CS+ and CS- responses in the six different
odorant combinations, we only found significant differences in three cases:
CS+myrcene/CS-linalool,
CS+myrcene/CS-ocimene,
CS+ocimene/CS-myrcene (exact McNemar test,
P<0.05). Comparing responses with the CS+ in the two combinations
of each odour pair, we found a significant difference for the linalool/myrcene
pair. These findings point to an asymmetry between linalool and myrcene,
although we corrected for potential concentration effects.
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Discussion |
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In Experiments 3 and 4, we used an ISI of 1 s between CS and US, as it gave the best learning performance. However, it appeared to be an important drawback, because it did not allow us to record moths' acquisition performance. During conditioning, we usually measure learning performance at each trial, when presenting the odour CS alone, before the US. In moths, which have a long proboscis and tend to respond slowly, CS-induced responses could usually not be recorded within 1 s, producing very low acquisition curves. In contrast, moths conditioned with 1 s ISIs showed response levels as high as 35-45% in the test trials, in which the odorant was presented for 5 s (Fig. 2A,B). We thus believe that such short ISIs are rather impracticable with moths, and future experiments should use a somewhat longer ISI (e.g. ISI of 2 to 3 s), which also support good learning performance.
Another important parameter in olfactory learning is its dependency on CS
concentration. The present study showed that increased odorant concentrations
resulted in a higher percentage of moths showing the CR (Figs
1A,B,
3A). Higher odour
concentrations have also been shown to increase the percentage of moths
showing the CR (increased cibarial pump response) in M. sexta
(Daly et al., 2001) and to
increase discrimination and induce better memory consolidation in PER
conditioning in A. mellifera
(Bhagavan and Smith, 1997
;
Pelz et al., 1997
;
Wright and Smith, 2004
). In
the present study, moths showed the ability to learn racemic linalool
at a much lower concentration than the other two odorants. Already at the
concentration of 0.001 mg, conditioning to racemic linalool took
place and maximum proportions of CR were reached after training with
concentrations of 0.1 mg, as shown by Experiment 3
(Fig. 3A). In comparison, the
highest proportion of CR to ß-ocimene and ß-myrcene was reached at
the 10 mg and 100 mg concentrations, respectively. These concentration
differences in learning performance may be partly due to a higher sensitivity
of the moth olfactory system to linalool than to the other two odorants, as
was indicated by the significant correlation of learning rate with the
amplitude of EAGs recorded in Experiment 4
(Fig. 3). This finding is also
in accordance with the increased responses to higher odorant concentrations
obtained from single RNs measured by electrophysiological recordings
(Stranden et al., 2003b
; T.
Røstelien, M.S., A.-K. Borg-Karlson and H.M., unpublished) and with
increased calcium responses obtained in the antennal lobe in optical imaging
recordings (Skiri et al.,
2004
; Stranden et al.,
2003b
). Since single-cell recordings have shown similar
sensitivities of the RN types to their primary odorants
(Stranden et al., 2003b
), the
present EAG recordings may indicate that H. virescens has a higher
number of RNs responding to linalool than to the other two odorants. In
addition, the EAG and calcium imaging experiments showed somewhat stronger
responses to ß-ocimene than to ß-myrcene, the two compounds known
from electrophysiological recordings to activate the same RN type,
ß-ocimene being the primary and ß-myrcene a secondary odorant
(Røstelien et al.,
2000b
; Stranden et al.,
2003b
). Thus, the results of the present study may indicate that
learning performance with the three odorants increases with a larger number of
RNs responding and/or with a higher firing rate of the RNs.
Electrophysiological studies have previously shown that ß-ocimene and
ß-myrcene activate the same RN type, whereas linalool activates two other
types; linalool being the primary odorant of one and a secondary odorant of
the other (Røstelien et al.,
2000b; Stranden et al.,
2003b
; T. Røstelien, M.S., A.-K. Borg-Karlson and H.M.,
unpublished). One would therefore expect the moths to discriminate between
linalool and the two other compounds more easily than between ß-ocimene
and ß-myrcene. Our conditioning experiments indicated that the moths do
indeed have the ability to discriminate between all three odorants (although
not as well in all cases), including between ß-ocimene and ß-myrcene
(Fig. 4). This is surprising,
since the concentrations of ß-ocimene and ß-myrcene used should
induce the same spiking rate in the RNs (i.e. we used 10x more ß-myrcene
than ß-ocimene). It is likely that discrimination between these two
odorants is due to the activation of other RN types by only one of the
compounds, although this has not yet been found in electrophysiological
recordings. In fact, in a calcium imaging study, one or two glomeruli in males
were activated by ß-ocimene but not by ß-myrcene
(Skiri et al., 2004
).
Considering the number of ordinary glomeruli (61-63) in the antennal lobe of
the heliothine species that are believed to receive plant odour information
(Berg et al., 2002
; H.T.S., B.
G. Berg and H.M., unpublished) and the 19 RN types identified so far (T.
Røstelien, M.S., A.-K. Borg-Karlson and H.M., unpublished), we think
that not all RN types have yet been described. Future work will attempt to
broaden our knowledge of RN types on H. virescens antennae.
Alternatively, since we used commercially available chemicals, we cannot
exclude that impurities in the high concentrations tested may have contributed
to the discrimination between the two test stimuli ß-ocimene and
ß-myrcene.
A significant discrimination asymmetry was found in Experiment 5. Moths
learned to discriminate the pair CS+myrcene/CS-linalool
but not the pair CS+linalool/CS-myrcene, whereas they
learned to discriminate equally well the pair ß-myrcene/ß-ocimene,
irrespective of which odorant was trained
(Fig. 4B). Since the odorant
concentrations showing equal acquisition were chosen, the asymmetry in the
ß-myrcene/racemic linalool discrimination tests cannot be
explained on the basis of different sensitivities to these odorants. Sensory
similarity (or difference) should be indicated by the amount of differential
responding, and should be independent of which element of an odour pair is
being used as the CS+ or CS-. Discrimination values depend on the stimulus
trained, and here stimulus strength can be excluded as a parameter. The
better-learned stimulus differs qualitatively in its potential to gain the
properties of a learned odour. This potential is called the `salience' of a
stimulus in learning theory (Rescorla and
Wagner, 1972). Salience differences become apparent when the
pair-wise comparison between CS+ and CS-responses of the two odorants
indicates an asymmetry, assigning a higher salience to the odorant that is
responded to with higher probability when it is used as a CS+ than as a CS-.
It is not surprising that a rank order of salience is not simply additive, as
found in Experiment 5 (ß-myrcene=ß-ocimene,
ß-myrcene>racemic linalool but not
ß-ocimene>racemic linalool). This means that salience is not
an isolated parameter of a stimulus, but depends on the conditions under which
the stimulus is learned, here the not rewarded odorant in a differential
conditioning paradigm.
In conclusion, the present study has shown that performance in PER
conditioning in moths depends on the concentration of the odorant CS as well
as on the precise timing between CS and US presentations, and has identified
conditions in which PER conditioning is relatively efficient. Also, we have
shown that three different odorants, inducing quite different dose-responses
in EAG experiments (this study) and in calcium imaging experiments
(Skiri et al., 2004) also had
different thresholds in the learning experiments, in such a way that the EAG
dose-response relationship could predict the learning rate. By using a
differential conditioning procedure, we found that moths could discriminate
all three odorants, which was surprising given the fact that two odorants,
ß-ocimene and ß-myrcene, activate the same RNs. Furthermore, we
found that differential conditioning of odorant pairs leads to discrimination
values that are biased by differences in the salience of the stimuli, even if
the stimuli are made subjectively equally strong. The present results thus
show that moths can be used to answer specific questions about how olfactory
learning performance in insects relates to odorant detection, processing and
perception. The development of coupled electrophysiological recordings and
behavioural experiments on moths would be critical in this endeavour.
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