Download Appetitive and aversive olfactory learning induce similar

Anim Cogn (2014) 17:399–406
DOI 10.1007/s10071-013-0671-6
ORIGINAL PAPER
Appetitive and aversive olfactory learning induce similar
generalization rates in the honey bee
Nick Bos • Edith Roussel • Martin Giurfa
Patrizia d’Ettorre
•
Received: 26 December 2012 / Revised: 31 July 2013 / Accepted: 6 August 2013 / Published online: 20 August 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Appetitive and aversive learning drive an animal toward or away from stimuli predicting reinforcement,
respectively. The specificity of these memories may vary
due to differences in cost–benefit relationships associated
with appetitive and aversive contexts. As a consequence,
generalization performances may differ after appetitive and
aversive training. Here, we determined whether honey bees
show different rates of olfactory generalization following
appetitive olfactory conditioning of the proboscis extension
response, or aversive olfactory conditioning of the sting
extension response. In both cases, we performed differential conditioning, which improves discrimination learning
between a reinforced odor (CS?) and a non-reinforced
odor (CS-) and evaluated generalization to two novel
odors whose similarity to the CS? and the CS- was different. We show, given the same level of discriminatory
Martin Giurfa and Patrizia d’Ettorre shared senior authorship.
Electronic supplementary material The online version of this
article (doi:10.1007/s10071-013-0671-6) contains supplementary
material, which is available to authorized users.
N. Bos (&) P. d’Ettorre
Department of Biology, Centre for Social Evolution,
University of Copenhagen, Copenhagen, Denmark
e-mail: [email protected]
E. Roussel M. Giurfa
Research Center on Animal Cognition, University of Toulouse,
118 Route de Narbonne, 31062 Toulouse Cedex 9, France
E. Roussel M. Giurfa
Research Center on Animal Cognition, CNRS, 31062 Toulouse
Cedex 9, France
P. d’Ettorre
Laboratoire d’Ethologie Expérimentale et Comparée (LEEC),
Université Paris 13, Sorbonne Paris Cité, Villetaneuse, France
performance, that rates of generalization are similar
between the two conditioning protocols and discuss the
possible causes for this phenomenon.
Keywords Appetitive learning Aversive learning Olfactory PER conditioning Olfactory SER
conditioning Generalization Honey bee
Introduction
Associative learning allows animals to make predictions
about the outcome of events in their environment. Several
forms of associative learning can be distinguished
depending on the nature of the events associated and on the
valence of reinforcements involved (Staddon 1983). Taking into account reinforcement valence, associative learning can be classified as either appetitive or aversive. In the
former case, appetitive reinforcements are used, which
drive the animal toward reinforcement. In the latter case,
aversive reinforcements are used, which move the animal
away from reinforcement (Schull 1979; Mackintosh 1983).
These learning forms imply different motor responses
and are mediated by distinct neural circuits mediating
reinforcements with opposed valences (Rudy 2008). Predominance of one form over the other is difficult to predict.
On one hand, fleeing from potential enemies or noxious
stimuli may be seen as crucial for survival so that, from this
perspective, aversive learning would in some sense dominate over appetitive learning. On the other hand, appetitive
behavior is also fundamental for an organism’s sustainment
so that it may dominate over aversive learning.
An animal in which a comparative analysis of appetitive
and aversive learning is possible is the honey bee, Apis
mellifera. Honey bees are well known for their impressive
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learning capacities in various sensory domains such as
vision and olfaction (Giurfa 2007; Avarguès-Weber et al.
2011; Giurfa and Sandoz 2012). Such learning is amenable
to laboratory investigation as operant and Pavlovian
(classical) conditioning protocols have been developed to
study learning and memory in honey bees (Giurfa 2007). In
the former, bees learn the association between a specific
behavior and a reinforcement (Skinner 1938); in the latter,
they learn to associate a conditioned (originally neutral)
stimulus (CS) with an unconditioned (biologically relevant) stimulus (US) (Pavlov 1927). The main learning
protocols established for harnessed bees are Pavlovian. The
most traditional one is the appetitive olfactory conditioning
of the proboscis extension response (PER, Takeda 1961;
Bitterman et al. 1983; Giurfa and Sandoz 2012). In this
Pavlovian protocol, individually restrained hungry bees,
which exhibit reflexive PER to sucrose solution delivered
to their antennae, learn an association between an odor (the
CS) and sucrose reward (the US). As a consequence, they
release PER upon subsequent presentations of the olfactory
CS, even in the absence of sucrose. This protocol is clearly
appetitive as bees trained in this way, and if afterward
released into a Y-maze where they can freely chose
between the conditioned odor and a different odor, they
significantly prefer the odor previously paired with sucrose
(Carcaud et al. 2009).
An aversive Pavlovian protocol is also available for
honey bees (Vergoz et al. 2007; Giurfa et al. 2009). In this
case, individually restrained bees, which exhibit a reflexive
sting extension response (SER) upon stimulation with
noxious stimuli, learn to associate an odor (the conditioned
stimulus or CS) and an electric shock (the aversive
unconditioned stimulus or US). As a consequence, they
extend their sting to the mere presentation of the olfactory
CS, even in the absence of shock. This protocol is clearly
aversive as bees trained in this way, and afterward released
in a Y-maze, they significantly avoid the odor previously
associated with the electric shock (Carcaud et al. 2009).
Are memories induced by appetitive and aversive
olfactory conditioning equally robust and specific? Or does
one learning form dominate over the other? A possible way
to address this question is to perform generalization tests
following olfactory conditioning (Guerrieri et al. 2005).
Generalization is defined as the capacity that allows
responding to occur to stimuli that are perceptually similar
to a CS used in original training (Spence 1937; Shepard
1987; Ghirlanda and Enquist 2003). While responses to the
learned CS are generally maximal, generalized responses
decrease with decreasing similarity along a perceptual
dimension, thus establishing generalization gradients
(Spence 1937; Shepard 1987; Ghirlanda and Enquist 2003).
Generalization gradients depend not only on similarity, but
also on prior experience with stimuli, as the steepness of
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Anim Cogn (2014) 17:399–406
the generalization gradient is an indicator of how much
experience an organism has had with the stimuli (Lashley
and Wade 1946). Thus, when comparing generalization
across conditioning protocols, caution is needed to ensure
similar levels of CS learning. Generalization has been
studied in a variety of modalities (e.g., visual and olfactory) in many different organisms (e.g., rhesus monkeys:
Hearst 1960, 1962; moths: Daly et al. 2001; rabbits: Coureaud et al. 2009; honey bees: Guerrieri et al. 2005; ants:
Bos et al. 2011; and cuttlefish: Guibé et al. 2012).
Two main alternatives can be conceived: on one hand, if
the amount of generalization were determined entirely by
the sensory features of the conditioned odors, one would
expect that generalization would be the same after appetitive and aversive conditioning if the same odors are used
across protocols; on the other hand, if associative learning
and US valence themselves contribute to the amount of
generalization, then, different generalization gradients
could be found after appetitive and aversive conditioning,
even if the same odors are used across protocols, and the
same level of discriminatory performance is reached at the
end of conditioning.
Here, we investigated whether the rate of generalization
between odorants differs between aversive (SER) and
appetitive (PER) conditioning in honey bees. In both cases,
we performed differential conditioning, in which bees learn
to discriminate a reinforced odor (CS?) from a non-reinforced odor (CS-) and evaluated generalization to two
novel odors whose similarity to the CS? and the CS- was
different. Differential conditioning was chosen because it
improves reinforced stimulus learning with respect to
absolute conditioning (training of a single reinforced
stimulus) at least in the visual domain (Dyer and Chittka
2004; Giurfa 2004). In this way, responses to novel stimuli
evaluated after conditioning would reflect true generalization rather than absence of discrimination resulting from
potential lower acquisition in absolute conditioning.
Materials and methods
Choice of stimuli
Four odorants were used in our experiments: 1-nonanol;
2-hexanone; 1-octanol and hexanal (all Sigma Aldrich,
Saint-Quentin Fallavier, France). Odors were chosen based
on the similarity of the neural activation patterns they
evoke in naive bees at the level of the antennal lobe, the
primary olfactory neuropile of the bee brain (Joerges et al.
1997). This choice is justified as behavioral measures of
odor similarity have been obtained using exclusively
appetitive absolute conditioning (Guerrieri et al. 2005) and
may therefore not be fully valid in the case of differential
Anim Cogn (2014) 17:399–406
401
conditioning. Using activation patterns provided by Sachse
et al. (1999), we calculated the Euclidian distance between
the four odors, as this measure provides a good estimation
of perceptual similarity (Deisig et al. 2006; Deisig et al.
2010). Odors with a low Euclidian distance are perceptually similar and promote generalization while those with a
high Euclidian distance are perceptually different and
promote discrimination (Deisig et al. 2002). The highest
distance (Table 1) found was between 1-nonanol and
2-hexanone, which were assigned either as CS? or CS- in
a balanced way; 1-octanol and hexanal were used as novel
test odors to assess generalization after conditioning;
1-octanol was very similar to the conditioned stimulus
1-nonanol (and thus different from the other conditioned
stimulus 2-hexanone) while hexanal was equally similar to
both conditioned stimuli 1-nonanol and 2-hexanone.
Preparation
Honey bees of four different colonies were collected near
the entrance of outdoor hives, located in the campus of the
University of Toulouse (France) and chilled on ice until
they stopped moving. Bees were individually fixed on a
metal holder used in olfactory SER conditioning and
allowing stimulation with an electric shock (Vergoz et al.
2007). For olfactory PER conditioning, bees were harnessed in the same holders and were stimulated with
sucrose solution. Sucrose responsiveness is maintained in
this preparation, and bees exhibit a high level of motivation
for the appetitive US despite not being in conventional
PER holders (Roussel et al. 2009). Once harnessed, bees
received a droplet of sucrose solution (50 % w/w) and were
left to habituate to the experimental conditions for 2 h.
Five microliters of the four pure odorants were applied
to individual 1-cm2 filter paper pieces and transferred to
20-ml syringes which were used to stimulate the bees’
antennae. An air extractor behind the bee impeded accumulation of residual odors after odor delivery. For PER
conditioning, the US was 50 % (w/w) sucrose solution. For
SER, the US was a 7.5 V electric shock. In both appetitive
and aversive conditioning procedures, the CS? was paired
with its corresponding US, while the CS- was presented
alone.
Olfactory conditioning
Both appetitive conditioning (PER) and aversive conditioning (SER) were accomplished using a differential
conditioning protocol, where one stimulus is reinforced
(the CS?), while the other stimulus in not reinforced (the
CS-). Conditioning consisted of 12 trials in total for each
individual (six CS? and six CS- trials). Trials were
pseudo-randomized so that the same CS was never presented more than twice in a row. The intertrial interval
(ITI) was 10 min.
Each conditioning trial lasted 1 min. The bee was placed
in front of the air extractor and left for 20 s before being
exposed to the CS for 5 s. In CS? trials, the unconditioned
stimulus (US) was presented for 2–3 s after CS onset. In
CS- trials, no US was delivered. Response to the CS? and
to the CS- was measured during the 3 s prior to the
administration of the US. Afterward, the bee was left in the
setup for 35 s and then removed. In total, 164 individual
honey bees were conditioned, either in appetitive PER
(n = 75) or in aversive SER conditioning (n = 99). In both
PER and SER experiments, for half of the bees, 1-nonanol
(A) was the CS? and 2-hexanone (B) the CS-, while for
the other half, 2-hexanone was the CS? and 1-nonanol the
CS-. For establishing acquisition curves, conditioned
responses (i.e., responses to the CS prior to US delivery)
were quantified for each CS and represented as a function
of trial number.
Tests
Tests were performed 10 min after conditioning. The
procedure for the tests was similar to that of the conditioning trials, except that, no US was delivered following
odor stimulation. Each bee was presented with both CS?
and CS-, as well as with the two novel test odors. The
sequence of odor presentation was pseudo-randomized as
the CS? was always presented last in order to observe
whether extinction had occured; only individuals that
reacted to the CS? and not to the CS- were used in the
analyses (66 % for appetitive PER learning, 46 % for SER
aversive learning). After the four tests, PER/SER to the US
was checked once again. Individuals not exhibiting
Table 1 Properties of chemical substances used chemical structure, as well as the chemical distance (as measured by Euclidean distance) of the
test stimuli (TS) to the conditioned stimuli (CS)
A (1-nonanol) CS?/CS-
B (2-hexanone) CS?/CS-
C (1-octanol) TS
D (hexanal) TS
Chemical distance from A
–
Chemical distance from B
82
82
16
57
–
77
48
Chemical structure
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402
unconditioned responses to the US were not considered for
analysis (10 % for PER, 13 % for SER).
Anim Cogn (2014) 17:399–406
2011) using the function lmer of the R-package lme4
(Maechler and Bates 2010).
Statistical analysis
Results
To analyze the variation of performance during conditioning trials, we used a Generalized Linear Mixed Model
(GLMM, Baayen et al. 2008) with binomial error structure
and logit link function, including trial number, conditioning protocol (PER or SER), conditioned stimulus (1-nonanol or 2-hexanone) and stimulus type (CS? or CS-) as
fixed factors. Also, the interaction between protocol, trial
number and conditioned stimulus, as well as the interaction
between conditioned stimulus and trial number were
included. The effect of trial number might, to some extent,
be individually specific. Therefore, we included a randomeffect structure allowing for random intercepts and random
slopes, as well as a correlation between them.
For both PER and SER, differences in acquisition level
between CS? and CS- on trial 6 were analyzed using
McNemar’s test.
To investigate how perceptual similarity between test
and conditioned stimuli affected generalization performances depending on the conditioning protocol (PER or
SER conditioning), we used a GLMM, with binomial error
structure and logit link function, including protocol and
Euclidean distance between odorants as fixed effects and
subject (individual bee) as a random effect. To account for
the possibility that the different protocols lead to different
changes in the probability of responding to novel stimuli,
we also included the interaction between protocol and
chemical distance as a fixed effect into the model. However, this interaction was not significant (likelihood ratio
test: v2 = 0.11, df = 1, P = 0.74) and was, therefore,
removed from the model. The effect of Euclidean distance
between odorants might, to some extent, be individual
specific; we included, therefore, a random-effect structure
allowing to account for random intercepts, random slopes,
as well as a correlation between them. In this model, we
only included bees that effectively learned the discrimination task, i.e., bees which during the test responded to the
CS?, but not to the CS-. For these bees, we analyzed
generalization responses to novel test stimuli, focusing on a
possible correlation between chemical distance and
generalization.
We checked stability of both models by excluding data
points one by one from the data and comparing the estimates derived with those obtained for the full model, which
indicated that no influential cases existed. Variance Inflation Factors (VIF, Field 2005) were derived using the
function vif of the R-package car (Fox and Weisberg 2011)
applied to a standard linear model excluding the random
effect. Models were fitted in R (R Development Core Team
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Acquisition
In both conditioning protocols, appetitive PER conditioning and aversive SER conditioning, bees learned the
olfactory discrimination, irrespective of whether 1-nonanol
(A) or 2-hexanone (B) was used as CS? or CS-. There
were no significant differences in acquisition performance
with respect to odorant identity (GLMM, z = -1.26,
P = 0.21).
Figure 1 shows the acquisition curves of bees that successfully learned to discriminate between the CS? and
CS- during the test (for acquisition curves of all bees,
including those that did not learn correctly, see supplementary Fig. 1). Comparison between acquisition levels
reached in each protocol showed that there was a clear
effect of the protocol on the performance of bees (GLMM,
Trial*Protocol, z = -5.96, P \ 0.01). During the first
trial, there was a higher level of spontaneous responses in
the SER than in the PER protocol, a fact that is commonly
observed (e.g., Carcaud et al. 2009; Vergoz et al. 2007) and
is probably related to the unnatural position of bees in the
SER protocol (they are upside-down), which drives aversive responding to the first stimulus delivered. Despite this
difference, in both protocols, bees learned the discrimination, but, as previously shown (Carcaud et al. 2009; Vergoz
et al. 2007), the proportion of learners was higher in
appetitive PER conditioning than in SER conditioning.
When bees that did not learn successfully are included, the
maximum probability of responding to the CS? in the last
trial of SER conditioning was 40 %, while it was 80 % in
appetitive PER conditioning (supplementary figure. 1).
Nevertheless, both conditioning protocols induced successful learning, as evinced by a significant higher amount
of individuals responding to the CS? than to the CS- in
the sixth trial (McNemar’s test, P \ 0.01 for both PER and
SER).
Generalization tests
For analyzing generalization responses to the novel odors
1-octanol and hexanal after PER/SER conditioning, only
bees that learned the discrimination between 1-nonanol and
2-hexanone (i.e., that responded to the CS? and not to the
CS- in the test) were considered in order to evaluate
generalization in bees that reached the same level of discriminatory performance. In this case, given the variable
similarity between the novel odors and the conditioned
Anim Cogn (2014) 17:399–406
403
We also analyzed the impact of chemical distance
between the CS? and the novel odorant on the probability
of generalizing. We found that the probability of generalizing decreased with chemical distance (GLMM, z =
-5.49, P \ 0.001; estimate ±SE = -2.29 ± 0.42, see
Fig. 3).
Discussion
Fig. 1 Learning acquisition curves for both appetitive (PER, n = 50)
and aversive (SER, n = 41) differential conditioning paradigms. As
there was no significant difference in acquisition depending on
whether A or B was CS?, data are pooled here
odors [see Table 1: 1-octanol (C) was very similar to the
conditioned stimulus 1-nonanol (A) and thus different from
the other conditioning stimulus 2-hexanone (B), while
hexanal (D) was equally similar to both conditioned stimuli
1-nonanol and 2-hexanone], the analysis of generalization
performances has to take into account the specific odors
used as CS? and the CS-.
Figure 2a shows the generalization performances after
PER conditioning when 1-nonanol and 2-hexanone were
assigned as CS? and CS-, respectively (left panel) and
when their roles were reversed (right panel). Figure 2b
shows the same information in the case of SER conditioning. No significant differences were found between
generalization performances after PER and SER conditioning, irrespective of whether 1-nonanol or 2-hexanone
were used as CS? (GLMM, z = 0.58, P = 0.56). Thus,
given the same level of discriminatory performance,
appetitive reinforcement yielded the same generalization
profile as aversive reinforcement, showing there were no
class-wide differences between the generalization responses induced by these two kinds of reinforcements.
Our study evaluated the impact of reinforcement valence
on post-training stimulus generalization. As bees can be
trained to associate odors either with an appetitive reinforcement of sucrose solution (Takeda 1961; Bitterman
et al. 1983; Giurfa and Sandoz 2012) or with an aversive
reinforcement of electric shock (Vergoz et al. 2007; Carcaud et al. 2009; Giurfa et al. 2009), we studied olfactory
generalization after differential conditioning in bees that
effectively learned the discrimination between a reinforced
odor (CS?) and a non-reinforced odor (CS-). In this way,
generalization responses were determined in animals that
reached the same level of discriminatory performance both
in aversive and appetitive conditioning.
Honey bees possess the ability to generalize between
odorants after being trained in appetitive PER conditioning
(Vareschi 1971; Guerrieri et al. 2005). This capacity has
been considered adaptive in a foraging context, where
different flowers can give similar rewards in the form of
nectar and pollen (Waser et al. 1996). Until now, generalization performances after aversive SER conditioning
had not been studied, despite the importance of aversive
stimuli in the life of a bee. Generalization in this context is
expected as colonies are under constant threat of being
attacked, parasitized and robbed by a series of different
natural enemies (reviewed in Breed et al. 2004) so that
similarity between potential noxious stimuli may trigger
common defensive responses.
Two main outcomes were predicted concerning olfactory generalization after aversive and appetitive SER and
PER conditioning, respectively: (1) equivalent rates of
generalization, if generalization entirely depends on the
sensory features of the odors conditioned (which were the
same in both protocols), and thus on general properties of
nervous systems (Ghirlanda and Enquist 2003) or (2) different rates of generalization, if generalization is affected
by associative learning and the nature of the reinforcement.
In our results, olfactory generalization was similar after
appetitive PER and aversive SER conditioning. This suggests that odorant features and their processing in the
nervous system are the main factor driving generalization
in bees. The effect of a variable US, with different hedonic
values (sucrose vs. electric shock), appears to be negligible.
However, the fact that we evaluated generalization only in
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404
a
Anim Cogn (2014) 17:399–406
CS+
1,0
n=24
TS
n=26
Proportion PER
0,8
0,6
0,4
0,2
0,0
b
1,0
n= 2020
nnn=21
Proportion SER
0,8
0,6
0,4
0,2
0,0
A)
)
l(
o
an
e
n
no
n
no
1-
xa
he
2-
(B
C)
t
oc
1-
Perceptual distance of TS from CS+
D)
a
an
x
he
16
A)
)
l(
l(
l(
o
an
o
an
e
n
no
n
no
1-
57
xa
he
2-
(B
C)
t
oc
1-
D)
l(
l(
o
an
a
an
x
he
77
48
Fig. 2 Generalization toward novel stimuli generalization toward
novel stimuli of bees that learned to correctly discriminate between
a and b in appetitive (a) and aversive (b) conditioning. Generalization
toward novel stimuli does not depend on conditioning paradigm, but
only on the conditioned stimulus, and thus, the chemical distance of
the conditioned stimulus to the novel, test stimulus (see also Table 1).
Data shown as mean ? 95 % confidence intervals. The number of
bees that responded to the CS? and not to the CS- in the test is
shown in the CS? bar
those bees that learned perfectly the discrimination in both
protocols could also explain the absence of differences in
generalization rates after aversive and appetitive training.
Given that the associative strength reached by the CS was
highest in both protocols, then generalization profiles were
also identical in both cases. Our results are different from
those found for instance in rhesus monkeys (Hearst 1960,
1962), where the generalization gradient after aversive
conditioning was broader than that induced by appetitive
conditioning. These results were interpreted as being
determined by differential response rates instead of motivational or reinforcement factors, as the response rate to
food reward was lower than that of avoidance response.
Response rates were not relevant in our study as both in
appetitive PER and aversive SER conditioning, responses
quantified were all-or-none and unique by trial/test. Further
factors may be influential for determining variation (or lack
of it) in generalization performances following appetitive
and aversive conditioning. Differences in species-specific
sensitivity toward different sensory modalities (either at the
CS or the US level), variable reinforcement intensities and
levels of acquisition reached before testing generalization,
among others, may affect post-conditioning generalization
gradients dramatically.
The notion that chemical identity was the leading criterion in olfactory generalization in bees is supported by
our similarity analyses. We showed that the function
relating the probability of generalizing and the chemical
distance between odorants was the same irrespective of the
conditioning protocol (Fig. 3). Previous work showed that
chemical features of aliphatic odorants such as carbon
chain length and functional group are odorant dimensions
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Anim Cogn (2014) 17:399–406
Fig. 3 Effect of chemical distance on generalization fitted values of
the probability of response according to the chemical distance of the
novel stimulus to the conditioned stimulus. Data points are scattered
in order to increase clarity
used by bees and also other social insects to evaluate
odorant similarity (Guerrieri et al. 2005; Bos et al. 2013).
The study by Guerrieri and co-workers also showed that
generalization performances between odorants after absolute appetitive PER conditioning (a single CS reinforced)
could be predicted based on similarity between glomerular
activation maps induced by these odorants in the antennal
lobe of naı̈ve (non-conditioned) bees. In the present study,
we performed differential conditioning with one odor
reinforced and the other not. We did not use the behavioral
measures of Guerrieri et al. (2005) to choose our odors
based on their perceptual similarity because of the use of a
different conditioning protocol. Instead, we used glomerular activation maps at the level of the antennal lobe of
non-conditioned bees to predict odor similarity. Our results
show that it was possible to predict similarity using these
activation maps, and we also found a correlation between
neural similarity as evaluated in non-conditioned bees and
perceptual generalization as measured in conditioned bees.
Thus, neural similarity between odorants predicts generalization irrespective of conditioning procedure and hedonic value of reinforcements used. This finding concurs with
405
the interpretation that generalization depends solely on
chemical features of odorants and on the general properties
of the olfactory circuit processing them (Ghirlanda and
Enquist 2003). A systematic study comparing absolute and
differential conditioning both in the aversive and the
appetitive modality, using more odorants systematically
varying in carbon chain length and functional group, would
be needed to confirm this conclusion.
Another point to take into account is the moment in
which generalization is evaluated. In our case, generalization tests were performed shortly (10 min) after the last
conditioning trial so that memories addressed corresponded
to the initial phases of the mid-term memory (Menzel
1999). An interesting question would be whether generalization profiles remain the same when olfactory memories
are consolidated into long-term memories retrievable
3 days after training or whether appetitive and aversive
conditioning result in different generalization profiles
pointing toward different consolidation processes.
Finally, our data provide support for the inclusion criterion hypothesis (e.g., Guerrieri et al. 2009; Bos et al.
2011) stating that generalization mostly occurs when the
test stimuli have a shorter carbon chain than the conditioned stimulus. Generalization occurs less frequently
when the test stimulus is of longer chain length than the
conditioned stimulus. In our study, honey bees generalized
to hexanal and 1-octanol, when 1-nonanol was the CS?;
however, when 2-hexanone was the CS?, bees only generalized to hexanal. The proximate mechanisms of this
inclusion criterion remain to be elucidated.
Our work represents the first direct comparative analysis
of behavioral performances between appetitive and aversive olfactory conditioning in honey bees. We studied
olfactory generalization following differential conditioning
and found that irrespective of the different US nature,
appetitive and aversive learning led to same generalization
profiles and that odorant generalization was governed by
chemical similarity, which in turn directly relates to neural
similarity. Further studies should determine whether
behavioral and neural processes underlying appetitive and
aversive learning are similar and if not, where differences
reside.
Acknowledgments M.G. acknowledges the support of the French
Research Council, the University Paul Sabatier and of the Institut
Universitaire de France. NB and PdE were supported by a grant of the
Faculty of Science, University of Copenhagen and by the Danish
National Research Foundation (Centre for Social Evolution). Thanks
to the Centre of Excellence in Biological Interactions for allowing
allocation of working time and salary (provided by project grant
1251337) on the writing of our manuscript. We thank the three
anonymous referees for their help in improving the manuscript. The
authors declare that they have no conflict of interest. Treatment of the
experimental animals complied with European laws on animal care
and experimentation.
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References
Avarguès-Weber A, Deisig N, Giurfa M (2011) Visual cognition in
social insects. Annu Rev Entomol 56:423–443
Baayen RH, Davidson DJ, Bates DM (2008) Mixed-effects modeling
with crossed random effects for subjects and items. J Mem Lang
59(4):390–412
Bitterman ME, Menzel R, Fietz A, Schäfer S (1983) Classical
conditioning of proboscis extension in honeybees (Apis mellifera). J Comp Psychol 97(2):107–119
Bos N, Dreier S, Jørgensen CG, Nielsen J, Guerrieri FJ, d’Ettorre P
(2011) Learning and perceptual similarity among cuticular
hydrocarbons in ants. J Insect Physio 58(1):138–148
Bos N, d’Ettorre P, Guerrieri FJ (2013) Chemical structure of
odorants and perceptual similarity in ants. J Exp Biol
216:3314–3320
Breed MD, Guzman-Novoa E, Hunt GJ (2004) Defensive behavior of
honey bees: organization, genetics, and comparisons with other
bees. Annu Rev Entomol 49:271–298
Carcaud J, Roussel E, Giurfa M, Sandoz JC (2009) Odour aversion
after olfactory conditioning of the sting extension reflex in
honeybees. J Exp Biol 212(Pt 5):620–626
Coureaud G, Hamdani Y, Schaal B, Thomas-Danguin T (2009)
Elemental and configural processing of odour mixtures in the
newborn rabbit. J Exp Biol 212:2525–2531
Daly KC, Chandra S, Durtschi ML, Smith B (2001) The generalization of an olfactory-based conditioned response reveals unique
but overlapping odour representations in the moth Manduca
sexta. J Exp Biol 204:3085–3095
Deisig N, Lachnit H, Giurfa M (2002) The effect of similarity
between elemental stimuli and compounds in olfactory patterning discriminations. Learn Mem 9(3):112–121
Deisig N, Giurfa M, Lachnit H, Sandoz JC (2006) Neural representation of olfactory mixtures in the honeybee antennal lobe. Eur J
Neurosci 24(4):1161–1174
Deisig N, Giurfa M, Sandoz JC (2010) Antennal lobe processing
increases separability of odor mixture representations in the
honeybee. J Neurophysiol 103(4):2185–2194
Dyer AG, Chittka L (2004) Fine colour discrimination requires
differential conditioning in bumblebees. Naturwissenschaften
91(5):224–227
Field A (2005) Discovering statistics using SPSS. Sage Publications,
London
Fox J, Weisberg S (2011) An R companion to applied regression, 2nd
edn. Sage Publications, London
Ghirlanda S, Enquist M (2003) A century of generalization. Anim
Behav 66:15–36
Giurfa M (2004) Conditioning procedure and color discrimination in
the honeybee Apis mellifera. Naturwissenschaften 91(5):228–231
Giurfa M (2007) Behavioral and neural analysis of associative
learning in the honeybee: a taste from the magic well. J Comp
Physiol A 193(8):801–824
Giurfa M, Sandoz JC (2012) Invertebrate learning and memory:
50 years of olfactory conditioning of the proboscis extension
response in honeybees. Learn Mem 19(2):54–66
Giurfa M, Fabre E, Flaven-Pouchon J, Groll H, Oberwallner B,
Vergoz V, Roussel E, Sandoz JC (2009) Olfactory conditioning
of the sting extension reflex in honeybees: memory dependence
on trial number, interstimulus interval, intertrial interval, and
protein synthesis. Learn Mem 16(12):761–765
123
Anim Cogn (2014) 17:399–406
Guerrieri FJ, Schubert M, Sandoz JC, Giurfa M (2005) Perceptual and
neural olfactory similarity in honeybees. PLoS Biol 3(4):e60
Guerrieri FJ, Nehring V, Jørgensen C, Nielsen J, Galizia G, d’Ettorre
P (2009) Ants recognize foes and not friends. Proc R Soc Lond B
Biol Sci 276(1666):2461–2468
Guibé M, Poirel N, Houdé O, Dickel L (2012) Food imprinting and
visual generalization in embryos and newly hatched cuttlefish,
Sepia officinalis. Anim Behav 84(1):213–217
Hearst E (1960) Simultaneous generalization gradients for appetitive
and aversive behavior. Science 132(3441):1769–1770
Hearst E (1962) Concurrent generalization gradients for foodcontrolled and shock-controlled behavior. J Exp Anal Behav
5(1):19–31
Joerges J, Kuettner A, Galizia G, Menzel R (1997) Representations of
odours and odour mixtures visualized in the honeybee brain.
Nature 387:285–288
Lashley KS, Wade M (1946) The Pavlovian theory of generalization.
Psychol Rev 53:72–87
Mackintosh NJ (1983) Conditioning and associative learning. Oxford
psychology series, vol 3. Oxford University Press, New York
Maechler M, Bates D (2010) lme4: linear mixed-effects models using
S4 classes. R package version 099937–099935
Menzel R (1999) Memory dynamics in the honeybee. J Comp Physiol
A 185:323–340
Pavlov IP (1927) Conditioned reflexes: an investigation of the
physiological activity of the cerebral cortex. Oxford University
Press, London
R Development Core Team (2011) R: a language and environment for
statistical computing. R Foundation for Statistical Computing,
Vienna
Roussel E, Carcaud J, Sandoz JC, Giurfa M (2009) Reappraising
social insect behavior through aversive responsiveness and
learning. PLoS ONE 4:e4197
Rudy JW (2008) The neurobiology of learning and memory. Sinauer
Ass, Sunderland
Sachse S, Rappert A, Galizia CG (1999) The spatial representation of
chemical structures in the antennal lobe of honeybees: steps
towards the olfactory code. Eur J Neurosci 11(11):3970–3982
Schull J (1979) A conditioned opponent theory of Pavlovian
conditioning and habituation. Psychol Learn Motiv 13:57–90
Shepard RN (1987) Toward a universal law of generalization for
psychological science. Science 237:1317–1323
Skinner BF (1938) The behavior of organisms: an experimental
analysis. The century psychology series. D. Appleton-Century
Company, New York
Spence KW (1937) The differential response in animals to stimuli
varying within a single dimension. Psychol Rev 44:430–444
Staddon JE (1983) Adaptive behavior and learning. Cambridge
University Press, New York, pp 121–161
Takeda K (1961) Classical conditioned response in the honey bee.
J Insect Physiol 6:168–179
Vareschi E (1971) Duftunterscheidung bei der Honigbiene—Einzelzell-Ableitungen und Verhaltensreaktionen. Z vergl Physiol
75:143–173
Vergoz V, Roussel E, Sandoz JC, Giurfa M (2007) Aversive learning
in honeybees revealed by the olfactory conditioning of the sting
extension reflex. PLoS ONE 2(3):e288
Waser NM, Chittka L, Price MV, Williams NM, Ollerton J (1996)
Generalization in pollination systems, and why it matters.
Ecology 77(4):1043–1060