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Brief Communication
Olfactory conditioning of the sting extension reflex
in honeybees: Memory dependence on trial number,
interstimulus interval, intertrial interval, and protein
synthesis
Martin Giurfa,
1,2,4
Eve Fabre,
1,2,3
Justin Flaven-Pouchon,
1,2,3
Helga Groll,
1,2,3
Barbara Oberwallner,
1,2,3
Vanina Vergoz,
1,2,3
Edith Roussel,
1,2
and Jean Christophe Sandoz
1,2
1
Universite
´de Toulouse, UPS, Centre de Recherches sur la Cognition Animale, F-31062 Toulouse Cedex 9, France;
2
CNRS, Centre de
Recherches sur la Cognition Animale, F-31062 Toulouse Cedex 9, France
Harnessed bees learn to associate an odorant with an electric shock so that afterward the odorant alone elicits the sting
extension response (SER). We studied the dependency of retention on interstimulus interval (ISI), intertrial interval (ITI),
and number of conditioning trials in the framework of olfactory SER conditioning. Forward ISIs (conditioned stimulus
[CS] before unconditioned stimulus [US]) supported higher retention than a backward one (US before CS) with an
optimum around 3 sec. Spaced trials (ITI 10 min) supported higher retention than massed trials (ITI 1 min) and led to the
formation of a late long-term memory (l-LTM) that depended on protein synthesis. Our results reaffirm olfactory SER
conditioning as a reliable tool for the study of learning and memory.
The honeybee Apis mellifera is a main invertebrate model for the
study of learning and memory as it allows combining controlled
conditioning protocols with a simultaneous access to the nervous
system in the laboratory (Menzel 1999; Giurfa 2007). The main
protocol used to this end relies on the proboscis extension reflex
(PER), the appetitive reflex exhibited by a harnessed honeybee to
a sugar reward (the unconditioned stimulus, or US) delivered to its
antennae and mouthparts. After appropriate pairing of an odorant
(the conditioned stimulus, or CS) with sucrose presentations, the
bee learns to associate odorant and sugar reward so that the
odorant alone elicits PER (Takeda 1961; Bitterman et al. 1983).
Despite the important progress made in understanding the be-
havioral, cellular, and molecular bases of this appetitive learning
(Menzel 1999; Giurfa 2007), until recently it has been impossible
to study aversive learning in bees in such a way that behavioral
records would be accompanied by access to the nervous system.
This gap has been filled by a novel conditioning protocol in which
individually harnessed bees learn to associate an initially neutral
odorant (CS) with a mild electric shock (US) (Vergoz et al. 2007).
Bees fixed individually on a metallic holder (Fig. 1A) reflexively
extend their sting (sting extension response, or SER) upon delivery
of an electric shock to the thorax (Nu
´n
˜ez et al. 1997; Balderrama
et al. 2002), thus showing a typical defensive response to poten-
tially noxious stimuli (Breed et al. 2004). After successful condi-
tioning, the odorant elicits SER, a conditioned response that can
be retrieved 1 h after conditioning (Vergoz et al. 2007). This form
of conditioning is indeed aversive as shown by the fact that bees
trained in this way and transferred to the operant context of
a Y-maze, where they can freely walk and choose between the
shock-associated odor and a non-shock-associated odor, explicitly
avoid the punished odor and choose the non-shock-associated
odor 1 h after conditioning (Carcaud et al. 2009).
Classical features of Pavlovian conditioning protocols such as
the dependence of retrieval on the interstimulus interval (ISI; the
interval between CS and US onset) and intertrial interval (ITI; the
interval between consecutive trial onset) have not been analyzed
thus far in olfactory SER conditioning. Furthermore, although
olfactory SER conditioning leads to an aversive memory that can
be retrieved either in a Pavlovian (Vergoz et al. 2007) or in an
operant framework (Carcaud et al. 2009) 1 h after conditioning, it
is unknown whether it leads to the formation of robust LTMs,
retrievable some days after conditioning. In the honeybee, one
pairing of an odorant with sucrose (i.e., one conditioning trial)
leads to an early long-term memory (e-LTM) that can be retrieved
24 to 48 h after conditioning. Three conditioning trials, on the
other hand, lead to a stable late long-term memory (l-LTM) that
can be retrieved 72 h or more after conditioning. Although the
presence of short-term memories (STM) and medium-term mem-
ories (MTM)—retrievable just after conditioning or 1 h after it,
respectively—have been shown in olfactory SER conditioning
(Vergoz et al. 2007; Carcaud et al. 2009), no evidence thus far
supports the presence of LTM after this form of learning. Here we
performed a series of experiments aimed at characterizing the
effect of variables such as ISI and ITI on learning and retention
performance in olfactory SER conditioning. We asked whether this
protocol leads to the formation of LTM and whether such a
memory is protein-synthesis dependent.
Honeybee foragers were captured when leaving the hive.
These individuals have a fully developed sting reflex (Burrell and
Smith 1994) and exhibit higher shock responsiveness and aver-
sive learning scores (Roussel et al. 2009). In all experiments, we
followed the standard criterion used in olfactory appetitive PER
conditioning, which consists of checking before conditioning and
after retention tests that the unconditioned response (SER) to the
US (the electric shock) is intact. This control ensures that bees have
3
These authors contributed equally to this work.
4
Corresponding author.
Email giurfa@cict.fr; fax 33-561-55-61-54.
Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.1603009.
16:761–765 Ó2009 Cold Spring Harbor Laboratory Press 761 Learning & Memory
ISSN 1072-0502/09; www.learnmem.org
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the potentiality to produce the conditioned response and that
absence of a response during a test is due to a memory deficit and
not to a motor problem. Only bees that did not show SER to the
shock in these conditions were discarded. This represented 5% of
the total bees conditioned and tested.
In a first experiment we studied the effect of the ISI on
retention and determined the optimal ISI for SER conditioning.
Using independent groups of bees, we studied the effect of five
different ISIs on retention. One ISI corresponded to a backward
conditioning situation as shock onset occurred 1 sec before odor
onset (ISI1); the four other ISIs corresponded to forward condi-
tioning situations as the odor always preceded the US, which
started 1, 3, 5, or 7 sec after odor onset (ISI +1, +3, +5, or +7,
respectively). Each ISI group was conditioned along 10 condition-
ing trials spaced by 5 min. Odor and shock delivery lasted 5 and
2 sec, respectively. Within each ISI group, two subgroups were
used (n=45 for each subgroup): one that experienced 1-nonanol
as the CS and one that experienced 1-hexanol as the CS. Twenty
minutes after the last conditioning trial, retention was determined
in extinction conditions (no shock delivered). Bees were then
presented with two odorants in a random succession: the CS and
a novel odorant used to determine the specificity of the memory
retrieved. Bees trained with 1-nonanol were presented with
1-hexanol as the novel odorant, while bees trained with 1-hexanol
were presented with 1-nonanol as the novel odorant.
A total of 450 bees were used in this experiment (five ISI
groups; two subgroups per ISI, one trained with 1-nonanol and the
other with 1-hexanol; 45 bees per subgroup). Performance of the
subgroups was pooled given the lack of significance for the factor
‘‘CS odor’’ (F
(1, 440)
=1.50; NS) and for the interaction between the
factors ‘‘CS odor’’ and ‘‘ISI group’’ (F
(4,440)
=0.09; NS). Retention
performance levels varied significantly depending on ISI (F
(4,445)
=
16.56; P<0.0001; Fig. 1B). They exhibited the typical bell-shaped
form of classical conditioning with asymmetry between for-
ward and backward ISIs. Indeed, no retention was found at ISI 1
(backward conditioning), while significantly higher responses
were recorded for all other ISIs (forward conditioning; Newman-
Keuls post-hoc contrasts: P<0.001 in all cases). Two ISIs, 3 and
5 sec, yielded higher retention performance, with an optimum at
3 sec. In all cases, unspecific responses to the novel odorant were
similarly low for all ISI groups (F
(1, 44)
=0.59; NS), and, in the case
of forward ISIs, they were significantly lower than responses to
the CS (F
(1,359)
=210.34; P<0.00001; Fig. 2). Thus, retention per-
formance recorded 20 min after conditioning was specific for the
CS and was clearly affected by ISI.
In a second experiment, we studied memory retention after
olfactory SER conditioning. We asked whether SER conditioning
leads to the formation of memories retrievable 1, 24, 48, and 72 h
after conditioning, the latter corresponding to l-LTM as character-
ized in olfactory PER conditioning (Menzel 1999). An indepen-
dent group of bees was used for each retention time. Two odorants,
Figure 1. (A) View of a honeybee in the experimental setup. The bee is fixed between two brass plates (E1, E2) set on a Plexiglas plate (pp), with EEG
cream smeared on the two notches (N1, N2) to ensure good contact between the plates and the bee, and a girdle (G) that clamps the thorax to restrain
mobility. The bee closes a circuit and receives a mild electric shock (7.5 V) which induces the sting extension reflex (SER). An originally neutral odorant is
delivered through a 20-ml syringe (S) placed 1 cm from the antennae. Odorant stimulation lasted 5 sec. The electric shock started 3 sec after odorant
onset and lasted 2 sec so that it ended with odorant offset. Contamination with remains of odorants used for conditioning or pheromones is avoided via
an air extractor (AE) which is on continuously. (B) The effect of the ISI on retention. Percentage of SER (+95% confidence interval) to the conditioned odor
(CS; black bars) and to a novel odor (NO; white bars) as a function of ISI (interval between CS and US onset) in retention tests performed 20 min after
conditioning. One ISI corresponded to a backward conditioning (US before CS; ISI 1) while the four others corresponded to forward conditioning (CS
before US; ISI +1, +3, +5, or +7). Letters indicate significant differences. No retention was found at ISI 1. Significant retention was found for forward ISIs
with two ISIs, +3and+5 sec, yielding higher retention performance and an optimum at +3 sec.
Figure 2. (A) Memory retention after SER differential conditioning (one
odorant associated with shock, or CS+, vs. one odorant nonassociated
with shock, or CS). Percentage of SER (+95% confidence interval) to the
CS+(black bars) and to the CS(white bars). Four groups of bees were
trained in parallel (acquisition) and tested afterward after different
retention intervals (1 h, 24 h, 48 h, and 72 h post-conditioning). Each
group was tested once. Letters indicate significant differences. All groups
remembered the discrimination learned during training. (B) Dependency
of l-LTM (72-h retention) on translation and transcription. Three groups of
bees were trained in parallel (acquisition) and tested 72 h after the last
acquisition trial and after injection of PBS, anisomycin, or actinomycin D.
Each group was tested once. Letters indicate significant differences. Only
the group injected with PBS (control) remembered the discrimination
learned during training; inhibition of transcription (actinomycin D) or
translation (anisomycin) resulted in the absence of l-LTM.
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1-hexanol and 1-nonanol, were used to condition the bees in
a differential conditioning protocol (one odor associated with
shock, or CS+, vs. another odor not associated with shock, or CS)
involving six CS+and six CStrials presented in a pseudorandom
sequence. The ISI was 3 sec (see above), and conditioning trials
were spaced by 10 min. In order to balance olfactory experiences,
two subgroups were conditioned within each retention group: one
for which 1-hexanol was the CS+, and 1-nonanol the CS, and
one for which the contingencies were inversed. In no case did we
find significant differences between the subgroup trained with
1-hexanol as CS+and the one that trained with 1-nonanol, so the
results of both subgroups were pooled (1-h group: F
(1,180)
=0.94,
NS; 24-h group: F
(1,300)
=0.55, NS; 48-h group: F
(1,345)
=0.37, NS;
72-h group: F
(1,345)
=1.77, NS).
All four groups learned to discriminate the CS+from the CS
(1-h group: F
(1,185)
=46.30, P<0.00001; 24-h group: F
(1,305)
=
44.44, P<0.00001; 48h-group: F
(1,350)
=57.44, P<0.00001; 72-h
group: F
(1,350)
=52.88, P<0.00001) and reached comparable levels
of discrimination at the end of training. Indeed, a within-group
comparison of responses to the CS+and the CSin the last (sixth)
conditioning trial yielded significant differences for all four groups
(1-h group: x
2
=17.63, P<0.0001; 24-h group: x
2
=20.90, P<
0.0001; 48-h group: x
2
=31.11, P<0.0001; 72-h group: x
2
=25.35,
P<0.0001). Neither differences in CS+responses (F
(3,238)
=1.06;
NS) nor differences in CSresponses (F
(3,238)
=1.59; NS) were
found between groups at the end of training.
After conditioning, groups were tested at different retention
intervals (Fig. 2A). To ensure survival at longer intervals (24 h,
48 h, and 72 h), bees were released from the holders and separately
caged according to their retention interval. They received 50%
sucrose solution ad libitum and were kept in an incubator in the
dark. Three hours before retention tests they were frozen and fixed
again in the holders. All groups exhibited significant retention
and responded more to the CS+than to the CS. Within-group
analysis by means of the McNemar test showed significant re-
tention performance in all cases (1-h group: x
2
=20.48, P<0.0001;
24-h group: x
2
=8.20, P<0.005; 48-h group: x
2
=6.32, P<0.02; 72-h
group: x
2
=7.61, P<0.01). Neither differences in CS+responses
(F
(3,151)
=1.75; NS) nor differences in CSresponses (F
(3,151)
=
0.60; NS) were found between groups in retention tests. These
results show, therefore, that SER conditioning leads to robust
LTMs that are retrievable even 3 d after training.
In a third experiment we asked whether the 3-d long-term
memory depends on de novo protein synthesis. The conditioning
procedure was identical to that of the previous experiment. In the
2 h following conditioning, bees were injected in the ocellar tract
either with PBS (control group), anisomycin 10
2
M (a translation
inhibitor), or actinomycin D 1.5 310
3
M (a transcription in-
hibitor). A Harvard GC 100-10 microelectrode filled with the drug
to be injected was connected to an IM 300 Narishige microinjector
and used to deliver 10 320 nl into the brain. Drugs, concentra-
tions, and injection time were chosen according to Wu
¨stenberg
et al. (1998), who showed the effectiveness of this procedure in
the study of appetitive olfactory LTM and its dependency on
protein synthesis. Each of these groups included two subgroups,
one in which 1-hexanol was the CS+and 1-nonanol the CS, and
one in which the contingencies were inversed (CS+: 1-nonanol;
CS: 1-hexanol). After injection, bees were marked to identify
them according to the treatment received and were kept in small
cages until retention tests were performed 72 h after the last
acquisition trial.
Before injection, all three groups learned to discriminate the
CS+from the CS(PBS group: F
(1,680)
=7.11, P<0.01; anisomycin
group: F
(1,600)
=9.99, P<0.01; actinomycin D group: F
(1,350)
=6.81,
P<0.05) and reached comparable levels of discrimination at the
end of training. Indeed, a within-group comparison of responses
to the CS+and the CSin the last (sixth) conditioning trial
yielded significant differences for all three groups (PBS group: x
2
=
10.81, P<0.01; anisomycin group: x
2
=9.82, P<0.01; actinomycin
D group: x
2
=13.14, P<0.001). Neither differences in CS+re-
sponses (F
(2,169)
=0.18; NS) nor differences in CSresponses
(F
(2,169)
=0.81; NS) were found between groups at the end of
training. Thus, all three groups exhibited similar acquisition
performance before drug injection.
After injection, and 72 h after conditioning, retention per-
formance varied depending on treatment (Fig. 2B). Responses to
CS+differed significantly between groups (F
(2,86)
=8.54; P<0.001)
while responses to CSremained low and did not differ (F
(2,86)
=
0.26; NS). Within-group analysis showed that retention was
significant in control bees injected with PBS (x
2
=6.86, P<0.01)
but not in bees injected either with anisomycin (x
2
=0; NS) or with
actinomycin D (x
2
=0.08; NS). Thus, both translation and tran-
scription are essential events for LTM formation and retrieval of
the odor–shock association 72 h after training.
Finally, in a fourth experiment we studied the effect of trial
number (one vs. five trials) and ITI on long-term retention. Two
single-trial groups were studied: one in which the CS preceded the
US by 3 sec (one-trial forward, or 1CT F), and one in which the US
preceded the CS by 1 sec (one-trial backward, or 1CT B). In
addition, two forward five-trial groups were studied: one in which
the ITI was 1 min (massed trials; 5CT F 1min), and one in which
the ITI was 10 min (spaced trials; 5CT F 10min). Each ITI group
experienced 1-nonanol as the CS. In retention tests, bees were
presented with the CS and with 1-hexanol as a novel odor used to
test the specificity of the memories retrieved. Retention tests were
performed 24 h and 72 h after conditioning. An independent
group of bees was used in each case. Thus, our design involved four
ITI groups (1CT B; 1CT F; 5CT F 1min, and 5CT F 10min) and two
subgroups per ITI (24-h test and 72-h test). In total eight groups,
amounting to 330 bees, were used (40 bees per group on average).
Figure 3 shows retention performance 24 h (Fig. 3A) and 72 h
after conditioning (Fig. 3B). At both times, retention varied
significantly between groups. Responses to the conditioned odor
(CS) varied between ITI groups both at 24 h (F
(3,162)
=14.37; P<
0.0001) and 72 h (F
(3,162)
=16.54; P<0.0001) while responses to
the novel odor (NO) remained low and nonsignificant both at
24 h (F
(3,162)
=0.88; NS) and 72 h (F
(3,162)
=2.42; NS). Twenty-four
hours after training, no significant retention was found in the
group conditioned with five massed trials, although performance
was marginally nonsignificant (x
2
=3.37, P=0.07). In contrast, the
group conditioned with five spaced trials exhibited significant
retention (x
2
=13.79; P<0.001), thus showing that ITI has a
significant effect per se on 24-h olfactory retention. Neither
conditioning with one backward trial (x
2
=0; NS) nor condition-
ing with one forward trial (x
2
=0.90; NS) supported significant
retention.
Seventy-two hours after training, significant retention was
again found in the group conditioned with five spaced trials (x
2
=
10.62, P<0.01). The group conditioned with five massed trials
exhibited now significant retention (x
2
=4.05, P<0.5), higher
than that of both one-trial conditioning groups, but lower than
that of the five-trial spaced group (Group 1CT B: x
2
=0.25, NS;
Group 1CT F: x
2
=0.17, NS). Thus, comparing the group condi-
tioned with one forward trial and the groups conditioned with five
forward trials, massed and spaced, clearly shows an effect of trial
number at 72 h. As for the 24-h test, significant differences
between both groups conditioned with five forward trials showed
that ITI significantly affected olfactory retention.
Our work shows, therefore, that olfactory SER conditioning
has the typical features of Pavlovian conditioning, namely a de-
pendence of retention on ISI, on ITI, and on the number of
conditioning trials. Forward ISIs supported higher retention levels
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than a backward one, and an optimal ISI was found around 3 sec.
One- and multiple-trial backward conditioning induces condi-
tioned inhibition in appetitive olfactory conditioning of PER,
which can be demonstrated if acquisition of a conditioned re-
sponse during subsequent pairings of the putative inhibitory CS
with the US is retarded (retardation of acquisition) (Hellstern et al.
1998). However, such an effect was mainly visible for an ISI of 15
sec, while no inhibitory effect was found for shorter intervals, such
as 6 sec (Hellstern et al. 1998). In our case an ISI of 1 sec was
used, so that, if conditioned inhibition also occurs in the frame-
work of olfactory SER conditioning, and if it follows similar
principles as in olfactory PER conditioning, it should be negligible.
In olfactory PER conditioning, optimal retention after a single
conditioning trial is reached when the CS precedes the US (for-
ward conditioning) by 5 to 1 sec (Menzel and Bitterman 1983). In
mechanosensory PER conditioning, the retention curve found
after one conditioning trial shows the same typical bell-shaped
form with a maximum of ;3- to 4-sec ISI (Giurfa and Malun 2004).
These coincident results suggest that, in order to learn different
kinds of stimulus associations, bees require contiguity between
events that should not exceed a few seconds.
Long-term retention was affected both by trial number and
by ITI. One conditioning trial was unable to produce either e-LTM
(24 h) or l-LTM (72 h). In contrast, five conditioning trials, massed
or spaced, yielded l-LTM (at 72 h), thus suggesting that aversive
events need to be confirmed in order to be stored as memories.
Spaced trials (ITI 10 min) resulted in better retention performance
both 24 h and 72 h after conditioning, while massed trials (ITI
1 min) yielded intermediate-level retention at 72 h but not at 24 h.
This result is intriguing as, in analogy with olfactory PER condi-
tioning, one would have expected to observe retention at 24 h but
not at 72 h after massed conditioning. However, extensive
discussion on this difference should be precluded as retention
values at 24 and 72 h are just below and above statistical
significance. Although this result remains unexplained, we veri-
fied here the classical distinction between massed and spaced trials
in terms of their efficiency to support LTM. Massed trials imply
rapid successions of affirmative information that could interfere
with each other in terms of the storage processes that each one
triggers. In appetitive PER conditioning, a higher sensitivity of the
memory trace to conflicting or affirmative information can be
found ;2–3 min after conditioning (Menzel et al. 1974; Erber et al.
1980). We have not tested the effect of an ITI of 2–3 min on
retention after SER conditioning, but we suggest that a similar
detrimental effect was found here for an ITI of 1 min.
As experiments were not performed simultaneously, two
different conditioning protocols were used in our work: absolute
(first and fourth experiments: CS+) and differential (second and
third experiments: CS+vs. CS) conditioning. This fact does not
invalidate the findings reported, as we analyzed each experiment
per se and did not compare retention levels between experiments.
It would be interesting nevertheless to compare acquisition,
discrimination, and retention resulting from these two protocols
as is done in the visual modality (Giurfa et al. 1999; Giurfa 2004).
We predict that differential conditioning will lead to better
acquisition and discrimination performance, as in the case of
achromatic patterns and color stimuli (Giurfa et al. 1999; Giurfa
2004).
Olfactory SER conditioning can induce a robust and stable
l-LTM that relies on protein synthesis as it depends on both
translation and transcription. Our results show that bees have
the capacity to remember aversive experiences long after those
experiences took place. There are multiple biological contexts in
which such capacity could be applied; for example, foragers could
avoid returning to food places in which negative experiences, or
eventually unfulfilled expectations, occurred, thus enhancing
foraging efficiency. Furthermore, aversive memories could help
in organizing defensive responses against enemies whose odors
have been previously experienced. It may thus be adaptive to
memorize and remember for long periods the smell of predators in
order to exhibit appropriate defensive responses.
Acknowledgments
We thank two anonymous reviewers for useful comments on
a previous version of the manuscript. This work was supported by
the French National Research Agency (Project ANR-BLAN08-
3_337040; INSAVEL), the French Research Council (CNRS), and
Paul-Sabatier University. H.G. was supported by the Austrian
Bundesministerium fu
¨r Bildung, Wissenschaft, und Kultur; the
Erasmus Program; and the University of Wien (KWA fellowship).
B.O. was supported by the Erasmus Program.
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Received August 22, 2009; accepted in revised form October 5, 2009.
Aversive learning in honeybees
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