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Olfactory conditioning of the sting extension reflex in honeybees: Memory dependence on trial number, interstimulus interval, intertrial interval, and protein synthesis

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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.
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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.
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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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... In classical conditioning, both stimuli are combined in a temporally organized way, and after the association, the CS is not neutral anymore and elicits the conditioned response (Pavlov 1927). In the honeybee, Pavlovian conditioning is most often used with the proboscis extension response (PER) paradigm upon appetitive association (Bitterman et al. 1983;Kuwabara 1957;Matsumoto et al. 2012;Takeda 1961), and a protocol exploiting the sting extension response upon the exposure to an unpleasant US was recently developed (Giurfa et al. 2009;Junca and Sandoz 2015;Roussel et al. 2010;Vergoz et al. 2007). Conditioned and unconditioned stimuli need to be delivered in temporal contiguity, with the US starting a few seconds later than the CS, so that the CS acquires a predictive value for the US (Bitterman et al. 1983;Szyszka et al. 2011). ...
... Traditionally, memory has been classified by how long it lasts. A single conditioning trial leads to the formation of a short-(seconds to minutes, STM) to medium-term memory (up to 1 h, MTM), whereas multiple conditioning trials lead to the formation of a long-term memory (LTM), which can be retrieved the next day (early LTM) or up to 72 h later (late LTM) Giurfa et al. 2009;Menzel 2012;Schwärzel and Müller 2006). However, this classification based on time windows was found to be unreliable, with temporal phases being shorter or longer depending on many factors, both external and internal to the animal. ...
Article
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With less than a million neurons, the western honeybee Apis mellifera is capable of complex olfactory behaviors and provides an ideal model for investigating the neurophysiology of the olfactory circuit and the basis of olfactory perception and learning. Here, we review the most fundamental aspects of honeybee's olfaction: first, we discuss which odorants dominate its environment, and how bees use them to communicate and regulate colony homeostasis; then, we describe the neuroanatomy and the neurophysiology of the olfactory circuit; finally, we explore the cellular and molecular mechanisms leading to olfactory memory formation. The vastity of histological, neurophysiological, and behavioral data collected during the last century, together with new technological advancements, including genetic tools, confirm the honeybee as an attractive research model for understanding olfactory coding and learning.
... This phenomenon, termed distributed practice effect, has long been known by psychologists and has been widely studied in both basic and applied research due to its relevance for education, therapy, and advertising (1,2). Although the distributed practice effect seems to be a fundamental principle of learning, spanning a great variety of learning tasks, study materials, and organisms (3)(4)(5)(6), its neurobiological underpinnings are still poorly understood. Recent studies demonstrated that increasing the time intervals between training episodes improves synaptic plasticity (7)(8)(9)(10)(11), supporting the hypothesis that the superiority of distributed training over massed training might depend upon enhanced memory consolidation (9). ...
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Significance Distributed training has long been known to lead to more robust memory formation as compared to massed training. Using the water maze, a well-established task for assessing memory in laboratory rodents, we found that distributed and massed training differentially engage the dorsolateral and dorsomedial striatum, and optogenetic priming of dorsolateral striatum can artificially increase the robustness of massed training to the level of distributed training. Overall, our findings demonstrate that spatial memory consolidation engages different neural substrates depending on the training regimen, identifying a therapeutic avenue for memory enhancement.
... In addition to encoding for innate odor preferences, the neural responses evoked by an odorant must also underlie how and when it is associated with other stimuli through learning 25,26 . The importance of timing between a stimulus and reward, and how it controls learning and the rate of learning is also well documented 12,18,[27][28][29][30][31] . Given that most odorants elicit spatiotemporally varying activity, and the relative timing of the reinforcing stimuli can be controlled, can any response segment be reinforced with a reward? ...
Preprint
Sensory stimuli evoke spiking neural responses that innately or after learning drive suitable behavioral outputs. How are these spiking activities intrinsically patterned to encode for innate preferences, and could the neural response organization impose constraints on learning? We examined this issue in the locust olfactory system. Using a diverse odor panel, we found that ensemble activities both during (‘ON response’) and after stimulus presentations (‘OFF response’) could be linearly mapped onto overall appetitive preference indices. Although diverse, ON and OFF response patterns generated by innately appetitive odorants were still limited to a low-dimensional subspace (a ‘neural manifold’). Similarly, innately non-appetitive odorants evoked responses that were separable yet confined to another neural manifold. Notably, only odorants that evoked neural response excursions in the appetitive manifold were conducive for learning. In sum, these results provide insights on how encoding for innate preferences can also set limits on associative learning.
... This phenomenon, termed distributed practice effect, has long been known by psychologists and has been widely studied in both basic and applied research due to its relevance for education, therapy, and advertising (1,2). Although the distributed practice effect seems to be a fundamental principle of learning, spanning a great variety of learning tasks, study materials, and organisms (3)(4)(5)(6), its neurobiological underpinnings are still poorly understood. Recent studies demonstrated that increasing the time intervals between training episodes improves synaptic plasticity (7)(8)(9)(10)(11), supporting the hypothesis that the superiority of distributed training over massed training might depend upon enhanced memory consolidation (9). ...
Preprint
Distributed training has long been known to lead to more robust memory formation as compared to massed training. Here we demonstrate that distributed and massed training differentially engage the dorsolateral and dorsomedial striatum and optogenetic priming of dorsolateral striatum can artificially increase the robustness of massed training to the level of distributed training, identifying a novel therapeutic avenue for memory enhancement.
... Tal processamento do EC, que é mediado pelos mesmos caminhos olfativos, é comum para ambas as formas de aprendizado, apetitivo e aversivo. Então, é compreensível que experimentos sobre o aprendizado em Apis mellifera mostram que a dinâmica do aprendizado olfativo apetitivo e aversivo são comparáveis, contam com avaliação perceptiva semelhante e levam a performances de retenção similares(Giurfa et al. 2009, Tedjakumala & Giurfa 2013. Como consequência, os resultados obtidos no contexto apetitivo do condicionamento REP podem ser justificadamente generalizados para o caso da polinização por engano. ...
Thesis
Deceptive pollination, a strategy common among orchids (Orchidaceae) occurs when flowers offer no reward to pollinators and draw them, nevertheless, by means of conspicuous and attractive signals, such as color and fragrance. Having variable attracting signals may improve the efficiency of deception by hindering the establishment of predictions about the absence of reward. This idea was developed by Heinrich (1975), who argued that the more dissimilar are the non-rewarding flowers of a given species, the longer it should take pollinators to learn avoiding that species. Signal variability would thus impair generalization between non-rewarding experiences. In this thesis, we tested this hypothesis by focusing on the interaction between a tropical orchid species (Ionopsis utricularioides) and its floral visitors. We designed a series of cognitive experiments to address the question of what do pollinators perceive and learn when they face variable floral signals. First, we focused on visual information and characterized the intraspecific flower color variation of the orchid. We then trained stingless bees Scaptotrigona aff. depilis (Meliponini) to visit a setup of artificial flowers that were manipulated in color and presence of sugar reward to simulate the deceptive polymorphic flowers. We found that color variability disrupted the learning process, thus resulting in an increase of the number of flowers visited until learning that all flowers lacked reward. We also focused on olfactory information and characterized the intraspecific variation of flower fragrance in the orchid. We used components of the flower odorant profile and performed olfactory conditioning experiments with the honey bee Apis mellifera, a model for the study of learning and memory. Taking advantage of the olfactory conditioning of the Proboscis Extension Response (PER), we studied if bees discriminate between floral fragrance isomers, which are common in the fragrance of many food deceptive orchids. We found that honey bees discriminate isomers under specific conditions but tend to generalize between them in most of the cases. Thus, in a pollination context, these odor signals could seldom be used as predictive cues for the presence or absence of food. We also studied if abundant components within a floral bouquet dominate minor components, a cognitive phenomenon called overshadowing. If this were the case, the more abundant odors within an orchid fragrance could be used as salient cues to be learned in association with reward or absence of reward. We found that the major component of the orchid fragrance, was overshadowed by minor components that vary between individual flowers. Overshadowing was maintained even when the concentration of the abundant component was increased relative to those of the other odorants in the mixture. Thus, bees cannot learn the predictive value of the most abundant component of the orchid fragrance, a fact that could help maintaining deceptive pollination. Taken together, our findings show that olfactory and visual variability in deceptive orchids contributes to deceptive pollination by impairing associative learning of the absence of reward. Moreover, they indicate that evaluating pollination from the perspective of pollinator cognition can lead us to a more complete understanding of insect-flower interactions, and to comprehensive analyses of complex problems in pollination studies.
... Similar to vertebrate studies, a very limited number of species largely drives our understanding of insect learning and memory. The fruit fly (Drosophila melanogaster), honey bee (Apis mellifera), and bumble bees (Bombus spp.) have become robust and influential insect model systems because they are amenable to highly controlled experimental manipulations [5,6], such as the proboscis extension reflex (PER), sting extension reflex (SER), artificial flower patch, shuttle box, and Y-maze techniques [7][8][9][10][11]. In bees, the proboscis extension reflex (PER), proposed first by [12] wild bee species. ...
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Olfactory learning and floral scents are co-adaptive traits in the plant–pollinator relationship. However, how scent relates to cognition and learning in the diverse group of Neotropical stingless bees is largely unknown. Here we evaluated the ability of Melipona eburnea to be conditioned to scent using the proboscis extension reflex (PER) protocol. Stingless bees did not show PER while harnessed but were able to be PER conditioned to scent when free-to-move in a mini-cage (fmPER). We evaluated the effect of: 1) unconditioned stimulus (US) reward, and 2) previous scent–reward associations on olfactory learning performance. When using unscented-US, PER-responses were low on day 1, but using scented-US reward the olfactory PER-response increased on day 1. On day 2 PER performance greatly increased in bees that previously had experienced the same odor and reward combination, while bees that experienced a different odor on day 2 showed poor olfactory learning. Bees showed higher olfactory PER conditioning to guava than to mango odor. The effect of the unconditioned stimulus reward was not a significant factor in the model on day 2. This indicates that olfactory learning performance can increase via either taste receptors or accumulated experience with the same odor. Our results have application in agriculture and pollination ecology.
... Tal processamento do EC, que é mediado pelos mesmos caminhos olfativos, é comum para ambas as formas de aprendizado, apetitivo e aversivo. Então, é compreensível que experimentos sobre o aprendizado em Apis mellifera mostram que a dinâmica do aprendizado olfativo apetitivo e aversivo são comparáveis, contam com avaliação perceptiva semelhante e levam a performances de retenção similares(Giurfa et al. 2009, Tedjakumala & Giurfa 2013. Como consequência, os resultados obtidos no contexto apetitivo do condicionamento REP podem ser justificadamente generalizados para o caso da polinização por engano. ...
Thesis
A polinização por engano, uma estratégia comum em orquídeas (Orchidaceae), ocorre quando as flores não apresentam recurso aos polinizadores, mas os atraem, mesmo assim, pelos seus sinais conspícuos e atrativos, como cor e fragrância. Possuir sinais atrativos variáveis pode melhorar a eficiência do engano ao atrapalhar no estabelecimento de previsões sobre a ausência de recurso. Essa ideia foi desenvolvida por Heinrich (1975) que argumentou que quanto mais diferentes as flores sem recurso de uma espécie, mais tempo os polinizadores levariam para evitá-la. A variação nos sinais atrapalharia, então, na generalização entre experiências sem recompensa. Nessa tese, testamos essa hipótese focando na interação entre uma orquídea tropical (Ionopsis utricularioides) e seus visitantes florais. Nós elaboramos uma série de experimentos cognitivos para abordar a questão do que os polinizadores percebem e aprendem quando se deparam com sinais florais variáveis. Primeiro, focamos na informação visual e caracterizamos a variação intraespecífica de cor floral dessa orquídea. Nós então treinamos abelhas sem ferrão Scaptotrigona aff. depilis (Meliponini) a visitar flores artificiais que foram manipuladas quanto à cor e presença de recompensa de açúcar, simulando as flores polimórficas de engano. Encontramos que a variação na cor floral perturba o processo de aprendizagem, resultando em um aumento no número de flores visitadas até aprenderem que todas as flores não possuíam recompensa. Nós também focamos na informação olfativa e caracterizamos a variação intraespecífica na fragrância floral dessa orquídea. Usamos compostos do perfil de odores das flores e realizamos experimentos de condicionamento olfativo em Apis mellifera, um modelo para o estudo de aprendizado e memória. Utilizando o protocolo de condicionamento olfativo de Resposta de Extensão da Probóscide (REP), estudamos se abelhas conseguem discriminar entre isômeros, que são comuns na fragrância de várias espécies de orquídeas polinizadas por engano. Encontramos que as abelhas discriminam os isômeros sob condições específicas, mas tendem a generalizar entre eles na maioria dos casos. Dessa forma, num contexto de polinização, esses sinais dificilmente seriam usados como pistas preditivas para a presença ou ausência de alimento. Nós também estudamos se compostos abundantes da fragrância floral dominam compostos minoritários, em um fenômeno cognitivo chamado overshadowing. Se esse fosse o caso, os compostos mais abundantes da fragrância da orquídea poderiam ser usados como pistas salientes a serem aprendidas em associação com presença ou ausência de recurso. Encontramos que o composto majoritário da fragrância foi dominado pelos minoritários, que variam nas flores entre indivíduos. A dominância é mantida mesmo quando a concentração do composto abundante foi aumentada em relação a dos outros compostos na mistura. Dessa forma, as abelhas não conseguem aprender o valor preditivo do composto mais abundante da fragrância da orquídea, o que ajudaria a manter a polinização por engano. Tomadas em conjunto, nossas descobertas revelam que a variabilidade olfativa e visual em orquídeas polinizadas por engano contribui para esse mecanismo ao perturbar no aprendizado associativo da ausência de recurso. Além disso, elas indicam que avaliar a polinização pela perspectiva da cognição do polinizador pode nos levar a um entendimento mais completo das interações inseto-planta e a uma análise compreensiva de problemas complexos em estudos de polinização.
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Background Bumble bees, primarily Bombus impatiens and B. terrestris , are becoming increasingly popular organisms in behavioral ecology and comparative psychology research. Despite growing use in foraging and appetitive conditioning experiments, little attention has been given to innate antipredator responses and their ability to be altered by experience. In this paper, we discuss a primarily undescribed behavior, the disturbance leg-lift response (DLR). When exposed to a presumably threatening stimulus, bumble bees often react by lifting one or multiple legs. We investigated DLR across two experiments. Methods In our first experiment, we investigated the function of DLR as a prerequisite to later conditioning research. We recorded the occurrence and sequence of DLR, biting and stinging in response to an approaching object that was either presented inside a small, clear apparatus containing a bee, or presented directly outside of the subject’s apparatus. In our second experiment, we investigated if DLR could be altered by learning and experience in a similar manner to many other well-known bee behaviors. We specifically investigated habituation learning by repeatedly presenting a mild visual stimulus to samples of captive and wild bees. Results The results of our first experiment show that DLR and other defensive behaviors occur as a looming object approaches, and that the response is greater when proximity to the object is lower. More importantly, we found that DLR usually occurs first, rarely precedes biting, and often precedes stinging. This suggests that DLR may function as a warning signal that a sting will occur. In our second experiment, we found that DLR can be altered as a function of habituation learning in both captive and wild bees, though the captive sample initially responded more. This suggests that DLR may be a suitable response for many other conditioning experiments.
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Pheromones are chemical communication signals known to elicit stereotyped behaviours and/or physiological processes in individuals of the same species, generally in relation to a specific function (e.g. mate finding in moths). However, recent research suggests that pheromones can modulate behaviours, which are not directly related to their usual function and thus potentially affect behavioural plasticity. To test this hypothesis, we studied the possible modulatory effects of pheromones on olfactory learning and memory in Agrotis ipsilon moths, which are well-established models to study sex-pheromones. To achieve this, sexually mature male moths were trained to associate an odour with either a reward (appetitive learning) or punishment (aversive learning) and olfactory memory was tested at medium- and long-term (1 h or 1.5 h, and 24 h). Our results show that male moths can learn to associate an odour with a sucrose reward, as well as a mild electric shock, and that olfactory memory persists over medium- and long-term range. Pheromones facilitated both appetitive and aversive olfactory learning: exposure to the conspecific sex-pheromone before conditioning enhanced appetitive but not aversive learning, while exposure to a sex-pheromone component of a heterospecific species (repellent) facilitated aversive but not appetitive learning. However, this effect was short-term, as medium- and long-term memory were not improved. Thus, in moths, pheromones can modulate olfactory learning and memory, indicating that they contribute to behavioural plasticity allowing optimization of the animal’s behaviour under natural conditions. This might occur through an alteration of sensitization.
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This two-part article proposes a new approach to understanding neuronal mechanisms, still unexplained despite the immense progress in neuroscience since the 1940s. The first part ("The boolean brain") first presents a brief history of the steps leading to the Convolutional Networks that now rival the performance of the human visual system. The biological plausibility of these networks is examined, leading to the paradoxical conclusion that McCulloch and Pitts' logical model was a correct approach and that it has been underestimated. A new model of neural networks, the Elective Neural Networks (ENN), is proposed on this basis, inspired by the Theory of Epigenesis by selective stabilization of synapses (Changeux et al., 1973) [1], and equipped with a logical learning mechanism by synapse elimination. Its capacity to form large-sized networks is examined, taking into account connectivity constraints, and its biological plausibility is defended, including the issue of the binary synapse. The second part ("The orthogonal brain") proposes a neuronal mechanism with an explanation of the learning curve in a classical conditioning: the proboscis extension reflex in the Apis Mellifera bee. A reinforcement learning mechanism is added to the ENN model, applying to both classical and operant conditioning. A general hypothesis on the implementation of effector control in a brain is deduced, in which no individual synapse is genetically programmed. The slide format was chosen for this paper because of its ability to represent complex dynamic phenomena.
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The understanding of the physiology of learning is dominated by two basically different hypotheses. The deterministic view, following Hebb’s (1949) concept of the memory engram, presupposes a memory groove which is built during memory formation by the adaptive change of a relatively small number of reacting sites or switch points. These so-called ’switchpoint theories’ or ‘place theories’ assume that memory involves a discrete set of cells reserved for the special function of information storage (Young 1964; Eccles 1964; Ungar 1970). The non-deterministic or statistical theory is based on Lashley’s (1950) findings which suggest that all, or nearly all, stored information is distributed throughout the whole association cortex rather than by distinct association paths or centres. The individual neuronal switch points may then be involved in the storage of many different memory traces (John 1967, 1972). The two views are similar in that they take the adaptivity of single synapses between neurones as the basic modifiable component of the nervous system (Eccles and McIntyre 1953; Eccles 1964; Ungar 1970; John 1972). They differ, however, in their conception of the gross structure of the memory system. The crucial problem, then, is to locate the stored information. The spatio-temporal pattern of activity during memory formation produces a localised change in the excitability of specific neurones. It should be possible to find such neurones using the same techniques as have been employed for the location of units in the sensory integration centres.
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Stinging behavior has been extensively studied in honey bees at the level of the individual, that is, in terms of stimuli that release stinging in adult bees, and in terms of integration of individual behavior into colony defense. Yet very little is known about the physiological basis for this behavior. Using an isolated abdominal preparation factors that influence peripheral control of the sting extension response are analyzed. Results show that:1. Electromyogram activity released by severing the ventral nerve cord changed during the first few days of adult life but not later. Abdomens from older bees (nurses, guards, foragers) showed significantly higher EMG activity than newly emerged or 24 h-old bees. 2. The reflex matured over 5–7 days after emergence as an adult. 3. Younger bees (24h) had a lower threshold for initiating sting extension than older bees. However, the threshold for initiating the full sting response, i.e., extension and venom pumping, did not differ due to age. 4. Caste status was not correlated to any of the parameters of sting extension, indicating that any effect of caste on stinging behavior must arise in more anterior ganglia and/or in the brain.
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In Pavlovian conditioning, an originally neutral stimulus (conditioned stimulus or CS) gains control over an animal's reflex after its association with a biologically relevant stimulus (unconditioned stimulus or US). As a consequence, a conditioned response is emitted by the animal upon further CS presentations. In such a situation, the subject exhibits a reflex response, so that whether the CS thereby acquires a positive or a negative value for the animal is difficult to assess. In honeybees, Apis mellifera, an odour (CS) can be associated either with sucrose solution (US) in the appetitive conditioning of the proboscis extension reflex (PER), or with an electric shock (US) in the aversive conditioning of the sting extension reflex (SER). The term ;aversive' may not apply to the latter as bees do not suppress SER as a consequence of learning but, on the contrary, start emitting SER to the CS. To determine whether the CS acquires a positive or a negative value in these conditioning forms, we compared the orientation behaviour of freely walking honeybees in an olfactory-cued Y-maze after training them with an odour-sucrose association (PER conditioning) or an odour-shock association (SER conditioning). We show that the same odours can acquire either a positive value when associated to sucrose, or a negative value when associated to an electric shock, as bees respectively approach or avoid the CS in the Y-maze. Importantly, these results clearly establish the aversive nature of SER conditioning in honeybees.
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Background: The success of social insects can be in part attributed to their division of labor, which has been explained by a response threshold model. This model posits that individuals differ in their response thresholds to task-associated stimuli, so that individuals with lower thresholds specialize in this task. This model is at odds with findings on honeybee behavior as nectar and pollen foragers exhibit different responsiveness to sucrose, with nectar foragers having higher response thresholds to sucrose concentration. Moreover, it has been suggested that sucrose responsiveness correlates with responsiveness to most if not all other stimuli. If this is the case, explaining task specialization and the origins of division of labor on the basis of differences in response thresholds is difficult. Methodology: To compare responsiveness to stimuli presenting clear-cut differences in hedonic value and behavioral contexts, we measured appetitive and aversive responsiveness in the same bees in the laboratory. We quantified proboscis extension responses to increasing sucrose concentrations and sting extension responses to electric shocks of increasing voltage. We analyzed the relationship between aversive responsiveness and aversive olfactory conditioning of the sting extension reflex, and determined how this relationship relates to division of labor. Principal findings: Sucrose and shock responsiveness measured in the same bees did not correlate, thus suggesting that they correspond to independent behavioral syndromes, a foraging and a defensive one. Bees which were more responsive to shock learned and memorized better aversive associations. Finally, guards were less responsive than nectar foragers to electric shocks, exhibiting higher tolerance to low voltage shocks. Consequently, foragers, which are more sensitive, were the ones learning and memorizing better in aversive conditioning. Conclusions: Our results constitute the first integrative study on how aversive responsiveness affects learning, memory and social organization in honeybees. We suggest that parallel behavioral modules (e.g. appetitive, aversive) coexist within each individual bee and determine its tendency to adopt a given task. This conclusion, which is at odds with a simple threshold model, should open new opportunities for exploring the division of labor in social insects.
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Ethologists have been interested for the most part in genetically based, species-typical behavior rather than in the effects of individual experience, and so was Kenneth Roeder, although he was deeply concerned with the variability of that behavior and the sources of variability. In one of his late papers (Roeder 1975), he discussed what he termed “evitability” in the evasive response of noctuid moths to the ultrasonic cries of hunting insectivorous bats, taking notice of the so-called Harvard law of animal behavior which states that, when experimental conditions are properly controlled, the animal will do just as it pleases. The paper nicely illustrates Roeder’s way of working: he would raise an interesting question on the basis of careful and critical observation of unconstrained behavior, formulate hypotheses about ecological and evolutionary determinants, and look for underlying neural mechanisms. The source of evitability in the evasive behavior of moths, Roeder suggested, might be found in some property such as trans-synaptic instability “downstream in the moth central nervous system.”
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The classical olfactory conditioned response of the honey bee, involving the extension of the proboscis on tarsal contact with sucrose solution, has been investigated. The bee showed generalization between two similar aromas but distinguished the third. The bee clearly showed experimental extinction, differentiation, and conditioned inhibition, all of which were temporary and not retained until the following day. Thus, spontaneous recovery was observed. The experiments also suggest the occurrence of a conditioned response of the second order.
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Although work in a wide variety of species and paradigms has demonstrated that long-term memory is sensitive to the blocking of protein synthesis, previous studies have suggested that the honeybee might represent an exception to this rule. Retention tested one day after training was not impaired by the inhibition of translation by cycloheximide. Using blockers of either transcription (actinomycin D) or translation (anisomycin), we present experiments that reconcile this unusual finding by testing over longer retention periods. Honeybees were conditioned to associate an odourant with a sucrose reward. Typically, this leads to stable retention over days. However, injection of either drug led to lower retention after 4 days, whereas retention after 2 or sometimes even 3 days was unaffected. This dissociates two forms of memory: a protein synthesis-independent, medium-term memory (up to 3 days) and a protein synthesis-dependent, long-term memory lasting for at least 4 days.
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Reward learning in honeybees initiates a sequence of events which leads to long-lasting memory passing through multiple phases of transient memories. The study of memory dynamics is performed at the behavioral (both natural foraging behavior and appetitive conditioning), neural circuit and molecular levels. The results of these combined efforts lead to a model which assumes five kinds of sequential memories, each characterized by a set of behavioral and mechanistic properties. It is argued that these properties, although reflecting general characteristics of step-wise memory formation, are adapted to the species-specific adaptations in natural behavior, here to foraging at scattered and unreliable food sources.
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In recognizing a pattern, honeybeesApis mellifera, may focus either on its ventral frontal part, or on the whole frontal image. We asked whether the conditioning procedure used to train the bees to a pattern determines the recognition strategy employed. Bees were trained with the same patterns presented vertically on the back walls of a Y maze. Conditioning was either absolute, that is, bees should learn to choose a rewarded pattern when there is no alternative, or differential, that is, bees should learn to choose a rewarded pattern that is paired with a different, nonrewarded one. Bees used different pattern recognition strategies depending on the conditioning procedure: absolute conditioning restricted recognition to the lower half whilst differential conditioning extended it to the whole pattern. Bees trained with absolute conditioning saw and learned the features of the upper part of the trained patterns, but assigned more weight to the lower part. Bees trained with differential conditioning learned not only the features of the reinforced stimulus in an excitatory way, but also those of the nonreinforced one in an inhibitory way. Thus, conditioning tasks that involve not only excitatory acquisition of the conditioned stimulus per se, but also discrimination of nonreinforced stimuli, result in an increase in the visual field assigned to the recognition task. Conditioning tasks that involve only excitatory acquisition of the rewarded stimulus result in a higher weighting of the lower pattern half and thus in a more reduced field assigned to the recognition task. This difference may reflect that existing between a conditioned and an incidental behavioural modification.