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First published online November 2, 2007
Journal of Experimental Biology 210, 4024-4033 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006585
Olfactory memory formation and the influence of reward pathway during appetitive learning by honey bees
1 Division of Biology, Newcastle University, Newcastle upon Tyne, NE1 7RU,
UK
2 School of Life Sciences, Arizona State University, PO Box 874501, Tempe,
AZ 85287, USA
3 Rothenbuhler Honeybee Laboratory, Ohio State University, Columbus, OH
43210, USA
Author for correspondence (e-mail:
jeri.wright{at}ncl.ac.uk)
Accepted 29 August 2007
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Key words: Apis mellifera, associative learning, memory consolidation, post-ingestive feedback, gustation
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). The strength or intensity of both the CS and the US
influence the rate of learning and the duration of the memory of the
association (Rescorla and Holland,
1982; Mackintosh,
1983). In the case of an appetitive US, changes in intensity (e.g.
the sucrose concentration in a reward) often translate directly into a
reward's nutritional value. In a natural setting, learning how to predict the
occurrence of the most nutritionally valuable reward is likely to convey
increased fitness. An important aspect of appetitive learning, therefore, is
the evaluation of food quality either via pre-ingestive mechanisms
such as gustatory receptors at the sensory periphery or using post-ingestive
mechanisms for assessing food quality after food has been consumed.
Foraging worker honey bees learn to associate floral shapes, colors and
odors with the quality and quantity of food rewards. In the laboratory,
restrained honey bees will also learn to associate such cues with food
rewards, especially floral odors (Bitterman
et al., 1983; Frings,
1944; Kuwabara,
1957; Takeda,
1961). Experiments designed to study olfactory conditioning of
honey bees have typically involved the presentation of a discrete odor CS to
the antenna followed by a sucrose solution reward (US) presented first to the
taste receptors on the antenna to elicit proboscis extension (the
unconditioned response) and then to the proboscis so that the subject can
consume the reward. Honey bees have gustatory receptors on their antennae,
proboscis (mouthparts) and tarsi; stimulation of the gustatory receptors on
the antennae produces the `proboscis extension reflex' (PER) in which a honey
bee will extend its proboscis expecting food (Kuwubara, 1957). Upon
stimulation, the probability of eliciting proboscis extension varies directly
as a function of sucrose concentration
(Braun and Bicker, 1992;
Scheiner, 2004;
Haupt and Klemt, 2005).
Sensitivity at the periphery is also modulated by a honey bee's genetic
background (Page et al.,
1998), age (Scheiner,
2004) and motivational state
(Ben-Shahar and Robinson,
2001). When honey bees are conditioned in the classical olfactory
conditioning paradigm, they are allowed to feed on the reward solution during
each trial. Consuming the reward on each trial may also provide them with the
opportunity to use post-ingestive information to assess reward quality.
It is not clear whether the consumption of the food reward is a necessary
condition of appetitive learning in honey bees. If an association between an
odor and a sucrose reward can be formed in the absence of food consumption,
this suggests that post-ingestive feedback about food quality is not necessary
for appetitive olfactory learning. When a honey bee forages, it collects
nectar in its crop and brings the nectar to its hive, where the nectar is
stored for consumption by all colony members. During foraging, a honey bee
uses the nectar it collects as food for itself, but the passage of nectar from
the crop to the midgut is optimized such that as much nectar as possible is
returned and regurgitated as food for the colony
(Blatt and Roces, 2001). Honey
bees, therefore, may not rely on post-ingestive feedback about nectar quality
since they do not forage for their own immediate benefit and may not eat most
of the food they collect. Indeed, in their definitive series of experiments,
Bitterman et al. observed that it was possible for honey bees to learn to
associate an odor stimulus with sucrose delivered to the antennae without the
subject being allowed to consume the reward
(Bitterman et al., 1983).
However, the strength and duration of the memory formed via
antennal-only or proboscis-only stimulation has not been investigated.
The purpose of the present study is to investigate the extent to which the
reward pathway experienced during olfactory learning affects the formation of
memory in honey bees. In our experiments, we examined in detail whether
stimulation of a honey bee's antennae is sufficient to allow robust
associative conditioning, short-term memory formation and long-term memory
formation. To do this, we conditioned honey bees by stimulation of the
antennae alone, stimulation of the proboscis followed by feeding or
stimulation of both the antennae and the proboscis with feeding and then
tested them for their responses to the conditioned odor immediately, 24 h and
96 h after conditioning. Furthermore, because pollen foragers are more
sensitive to sucrose than nectar foragers
(Page et al., 1998), we also
examined whether pollen foragers learned faster and had a longer memory of the
conditioned odor than nectar foragers. Finally, by feeding our subjects a
water reward, we examined whether the presence of sucrose in the ingested
reward was necessary for formation of long-term odor memory.
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Individuals were captured in small vials, placed on ice until they ceased
moving and then secured in a restraining harness by a strip of tape between
the head and the thorax, allowing free movement of the antennae and proboscis
(mouthparts). Each subject was fed 1.5 mol l–1 sucrose until
satiated and held for 
24 h before conditioning. Immediately prior to the
experiments, each honey bee was evaluated for motivation by lightly touching
one antenna with the sucrose solution without subsequent feeding. If a subject
responded by extending its proboscis (PER), it was selected for use in the
experiment.
Odors
The odor stimuli used during olfactory conditioning in our experiments were
1-hexanol and 2-octanone (98% purity; Sigma-Aldrich, St Louis, MO, USA)
diluted in hexane to 2.0 mol l–1. A 5 µl aliquot of odor
solution was placed onto a small strip of filter paper placed in a modified 1
ml tuberculin glass syringe attached to an air source that was controlled by a
2-way valve connected to a Programmable Logic Controller (Automation Direct,
Cumming, GA, USA) for precise stimulus delivery
(Wright and Smith, 2004). Odor
stimuli were counterbalanced throughout experiments and treatments.
Conditioning protocols
Three different associative conditioning regimes were used in which the
presentation of the unconditioned stimulus (US) varied: antennal-only
conditioning (AC), proboscis-only conditioning (PC) and
antennal-plus-proboscis conditioning (APC). The APC protocol is the
`classical' conditioning paradigm as described in Bitterman et al.
(Bitterman et al., 1983). For
each type of conditioning, subjects receive stimulation with an odor
conditioning stimulus (CS) that is paired with a closely timed appetitive
reward. In our experiments, an odor (CS) was presented in a discrete air pulse
for 4 s. The reward (US), a 0.4 µl droplet of a 1.5 mol
l–1 sucrose solution, was presented 3 s after the start of
the odor so that presentation of the CS and US overlapped for 1 s. In the APC
protocol, the sucrose solution was first presented to the antennae, eliciting
proboscis extension, and then to the proboscis such that the subject consumed
the entire droplet. This was performed on each trial, even if the subject
extended its proboscis in response to the odor. The AC protocol differed from
the APC protocol in that only the antennae were stimulated with sucrose; the
subject was not allowed to touch the sucrose solution with its proboscis or to
feed during conditioning. The PC protocol was different from APC in that the
antennae were not stimulated with sucrose solution to elicit proboscis
extension during conditioning. In this case, the sucrose solution was applied
directly to the proboscis such that the entire droplet was consumed on each
trial without contacting the antennae. Each subject received conditioning
trials at an inter-trial interval of 5 min. During conditioning with each
protocol, if a subject had learned to associate the CS with the US, then
proboscis extension would occur before presentation of the US. Thus, a learned
response was scored as a binary variable (response or no response). After
conditioning, each subject was tested for recall of the association either
immediately (5 min after the last acquisition trial), 24 h or 96 h after
conditioning. Each subject was tested for recall at only one time point.
During the recall tests, the odor stimuli were presented without sucrose
reinforcement at the same inter-trial interval used during conditioning. The
presence or absence of a response was recorded.
Experiment 1. Comparison of acquisition and recall in the AC, PC and APC protocols for pollen and nectar foragers
The first series of experiments examined how the conditioning protocol (AC,
PC, APC) affected the level of associative conditioning and recall for the CS.
All subjects experienced 16 acquisition trials in a pseudo-randomized sequence
(e.g. A-B-B-A-B-A-A-B-A-B-B-A-B-A-A-B, where A denotes a reinforced
conditioning trial and B denotes a trial in which another odor was experienced
without reinforcement). Eight recall test trials followed the acquisition
phase. Prior to the recall test trials, honey bees were tested for motivation
as described above; only subjects that responded to antennal stimulation were
used. During the recall tests, the reinforced odor from the A trials or the
unreinforced odor from the B trials was presented in the following sequence of
trials, A-B-B-A-B-A-A-B, and the response was recorded. No sucrose stimulation
was presented during the recall tests. Recall test trials occurred at one of
three time points after conditioning: immediately, 24 h or 96 h. The subjects
tested at either the 24 or 96 h time points were fed 1.5 mol
l–1 sucrose solution following conditioning, until satiated,
and were then held in a humidified box until the recall tests were performed.
Subjects tested at the 96 h time point were additionally fed to satiety every
24 h.
Experiment 2. Test of associative conditioning for the AC protocol
We performed a pairing control experiment with antennal stimulation to
evaluate whether conditioned responding (proboscis extension) to the odor (CS)
arose due to non-associative (sensitization) or to associative conditioning.
Subjects, exclusively pollen foragers, were assigned to one of three protocols
– forward pairing, backward pairing or unpaired – and experienced
16 acquisition trials in the pseudo-randomized sequence
A-B-B-A-B-A-A-B-A-B-B-A-B-A-A-B described before. In the forward pairing
treatment, the A trials consisted of odor (CS) followed by sucrose stimulation
(US) as described in Experiment 1. For the B trials, the subject was placed in
the conditioning arena, but no odor or sucrose was delivered. In the backward
pairing protocol, the A trials were performed such that the sucrose solution
(US) was presented to the antennae 3 s before the presentation of the 4 s odor
(CS). For the B trials, the subject was placed in the conditioning arena, but
no odor or sucrose was delivered. In the unpaired protocol, the A trials
consisted of stimulation with the odor only (CS) whereas B trials consisted of
presentation with just sucrose (US). In all three protocols, after
conditioning, two recall test trials were performed by presenting the CS
without the US and the responses were recorded.
Experiment 3. Comparison of recall after a single conditioning trial in the AP, PC, APC and unpaired protocols
This experiment was designed to examine the extent to which the memory of
the CS odor was formed after one trial of conditioning with the APC, PC or AC
protocols. Subjects received two placement trials in either the paired or
unpaired treatment. For the paired treatment, the odor (CS) was presented
approximately 3 s before the sucrose (US) (as described in Experiment 1) for
one conditioning trial. The second trial was placement of the subject in the
conditioning arena only. For the unpaired treatment, the odor (CS) was
presented on one trial and the sucrose (US) was presented on the other trial.
The order (trial 1 or trial 2) of the presentation of sucrose or odor was
randomized across trials. After conditioning, each subject was given four test
trials with the odor at one of three time points: immediately, 24 h or 96 h.
As described in Experiment 1, subjects tested at the 24 or 96 h time points
were fed to satiation every 24 h to prevent starvation.
Experiment 4. The role of sucrose consumption in memory formation
Relatively higher levels of conditioned responding by subjects in the APC
and PC protocol groups could reflect the proboscis (feeding-related)
components that were present in those protocols but not in the AC protocol. In
the APC and PC protocols, at least two processes may influence the CS–US
association. First, there are multiple mechano- and hygro-sensory components
that arise from feeding movements and water uptake during ingestion. Second,
taste receptors on the proboscis may respond to sucrose in the solution. This
experiment was designed to test whether the stimulation of the proboscis with
sucrose and/or ingestion of sucrose were necessary for the retention of CS
memory. Pollen foragers were conditioned for eight rewarded trials using one
of three protocols. Two of the protocols (APC, AC) were the same as described
in Experiment 1. In addition, a new protocol (ACW) was used in this
experiment: the antennae were stimulated with sucrose solution to elicit
proboscis extension, but a 0.4 µl droplet of water was applied to the
proboscis instead of the sucrose solution. The water was presented to the
proboscis and consumed by the subjects as in the APC protocol. Subjects were
tested for recall immediately or 24 h after training. As in the other
experiments, subjects tested at 24 h were fed to satiation after training.
Statistical analysis
For all of the behavioral experiments, the responses of subjects were
scored as binary variables. Using the SAS statistical software, we used
repeated-measures logistic regression (rpm lreg) or logistic regression (lreg)
with least-squares post hoc contrasts (lsc) for multiple comparisons
to test all hypotheses (Agresti,
1996). This method is similar to analysis of variance, in that it
allows for the construction of models to test the effects of experimental
parameters on a behavioral outcome; logistic regression was developed for
testing hypotheses when the dependent variable is scored as either a 0 or 1.
In the figures, means are reported as probabilities of responding along with
estimated standard errors of this probability
(Cox and Snell, 1989).
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21=96.4, P<0.001). In fact, all three
treatment groups reveal a pattern of responding to A relative to B that is
typical for associative differential conditioning
(Bitterman et al., 1983).
Initial response levels to odor A were low and increased rapidly to reach an
asymptote by approximately the 4th or 5th trial. The initial response to the
unreinforced odor B was initially higher than that to odor A, because the
first trial with B always followed the first trial with A. This higher initial
response was probably due to sensitization from the preceding A trial and/or
to excitatory generalization from A to B. However, the response to B never
increased beyond this and in fact remained lower than A in the 2nd–8th
trials.
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22=8.27, P=0.016). Honey bees
conditioned via the APC protocol
(Fig. 1A), in which they
experienced stimulation on both the antennae and the proboscis, had a greater
rate of acquisition than those trained with either the PC (proboscis
stimulation only) (Fig. 1B)
(lsc; APC vs PC; 21=5.39,
P=0.020) or AC (antennal stimulation only)
(Fig. 1C) (APC vs AC;
21=9.99, P=0.002) conditions. For
example, on trial 2, approximately 30% of bees responded to the odor in the
APC protocol, and this percentage increased to 40–60% on trial 3. The
corresponding percent response on the same trials was much lower in PC and AC
stimulation groups, and these differences were carried through the remaining
trials. The rate of acquisition was not significantly different for the PC and
AC protocols (AC vs PC; 21=0.60,
P=0.436).
We expected that pollen foragers would have a higher sensitivity to sucrose
and would, therefore, also have a greater ability to learn to associate an
olfactory stimulus with a food reward. Pollen foragers, however, did not
consistently display greater asymptotic levels of association of odor with a
sucrose reward than nectar foragers. Their learning abilities varied as a
function of the conditioning protocol. For the APC protocol, the asymptotic
level of acquisition was greater for the pollen than for the nectar foragers
(rpm lreg; 21=4.21, P=0.040). By
contrast, with the PC protocol, the asymptotic level of acquisition was
greater for nectar foragers than for pollen foragers (
21=5.17, P=0.020). For the AC protocol, no
difference in the final level of acquisition was observed between pollen and
nectar foragers (rpm lreg; 21=0.18,
P=0.671).
Experiment 1B. Recall of an olfactory stimulus depends on conditioning protocol, time post-conditioning and foraging phenotype
To examine how conditioning protocol affected olfactory memory formation,
we tested the ability of our subjects conditioned with each protocol (APC, PC,
AC) to recall the conditioned odor during a unreinforced test administered
immediately, 24 h or 96 h after conditioning. The response on the first test
trial to the conditioned odor was used to assess recall. We observed that the
probability that a honey bee responded during the test depended on forager
type, conditioning type and the post-conditioning time of the test
(Fig. 2) (3-way lreg;
24=12.1, P=0.017). Because the responses were
different for pollen and nectar foragers, separate analyses were performed on
each foraging phenotype. For pollen foragers, the probability of responding to
odor during the test depended on both the conditioning protocol and the time
post-conditioning (Fig. 2A)
(2-way lreg; 24=13.4, P=0.009).
Immediately after conditioning, subjects responded strongly to the conditioned
odor. However, the probability of responding during the test dropped
significantly after 24 and 96 h, with the most precipitous drop occurring in
the first 24 h on average (Fig.
2A). This decrease is consistent with decay of memory as it
consolidates through different phases. Immediately after conditioning,
subjects in the APC, PC and AC groups responded equally well to the
conditioned odor. However, 24 and 96 h after conditioning, the responses of
subjects conditioned with the AC protocol were significantly lower than those
of subjects conditioned with the APC or PC protocols. Therefore, we conclude
that the conditioning protocols produced differences in the extent to which
long-term memory was consolidated. Antennal-only stimulation supported
immediate recall, but it was less capable of supporting consolidation into
long-term memory.
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For nectar foragers (Fig.
2B), as with pollen foragers, the level of response on the first
test trial depended on the time post-conditioning (lreg;
21=31.2, P<0.001), with a decline in
response levels starting 24 h after conditioning. On average, the APC and PC
conditioned subjects also displayed a greater probability of responding to the
test odor than subjects conditioned with the AC protocol (lreg;
22=6.64, P=0.024). For example, nectar foragers
conditioned with the AC protocol responded significantly less to the
conditioned odor 24 h after conditioning than the APC and PC subjects. By 96 h
after conditioning, however, the probability of a subject responding to the
conditioned odor during the test was equal for all three conditioning
protocols.
Experiment 1C. Extinction is equal across foraging phenotype, conditioning protocol and time post-conditioning
By measuring responses during eight unreinforced recall test trials with
the odor A (reinforced CS) and odor B (unreinforced CS), we examined both the
rate of extinction of the response and the effect of time after conditioning
(immediate, 24 h or 96 h) on the rate of extinction. The response to the
reinforced CS odor (A) was significantly greater during the test trials than
the response to the unreinforced odor (B) for all treatments (rpm lreg;
21=151, P<0.001). The responses of all
subjects during the test showed extinction, as the probability of responding
decreased as a function of trial number (rpm lreg;
21=31.2, P<0.001). The rate of extinction of
the conditioned response to the CS (A trials) as a function of trial number,
however, was not significantly different for any of the conditioning protocols
measured separately at each time post-conditioning for pollen or nectar
foragers (4-way interaction, rpm lreg; 24=6.19,
P=0.186). The response on the first trial was significantly greater
than the response on all the other trials (for all three, P<0.001)
but the probability of responding on test trials 2, 3 and 4 was not
significantly different (for all, P>0.999). The largest change in
the slope of extinction during the recall test trials occurred between trials
1 and 2.
Experiment 2. Antennal-only conditioning produces associative learning
Successful discrimination of the reinforced odors (A trials) from
unreinforced odors (B trials) in Experiment 1 suggests that the responses
observed for all three types of US stimulation during conditioning were driven
by associative learning mechanisms
(Mackintosh, 1983). If the
responses during conditioning had been driven by non-associative mechanisms
such as sensitization to the sucrose stimulation, we would have expected much
higher response levels to the unreinforced odor (B trials). Previous studies
(Bitterman et al., 1983;
Sandoz et al., 2002) suggest
that antennal stimulation paired with an olfactory CS alone could produce
associative learning. We extend these studies by comparing the recall of the
CS for subjects conditioned with antennal-only forward, backward or unpaired
CS–US presentations. The most robust conditioning occurred with the
forward-paired treatment (Fig.
3) (rpm lreg; 22=7.45,
P=0.024). Subjects conditioned in the forward-paired treatment had a
significantly higher probability of responding during the recall tests than
those in either the backward-paired (P=0.024) or the unpaired
(P=0.007) groups. The levels of response for honey bees conditioned
with the backward or unpaired treatment were not significantly different
(P=0.502). The probability of responding during the test trials was
greatest on the first test trial (Fig.
3) (rpm lreg; 21=7.14,
P=0.007), and the rate of extinction over the two test trials was the
same regardless of the type of conditioning (2-way rpm lreg;
22=0.47, P=0.791). If responses during the
recall test to the unpaired or backward-paired treatments were not
significantly different from the forward-paired treatment, it is possible that
the AC protocol did not produce proboscis extension in honey bees via
associative learning of the odor (CS) with the sucrose (US). However, because
responses were greatest for the forward-paired group, we conclude that the AC
protocol produces associative conditioning between an odor CS and a sucrose
solution US applied to the antennae.
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Experiment 3. Antennal-only conditioning shows deficits in recall after one trial learning
The association formed after a single conditioning trial in olfactory
learning by honey bees is currently thought to proceed from different pathways
in the central nervous system than those that give rise to long-term memory
formed after several conditioning trials
(Eisenhardt, 2006;
Schwärzel and Müller,
2006). With this in mind, we examined how the type of conditioning
protocol influenced recall after a single conditioning trial using four
conditioning protocols: APC, PC, AC or unpaired (UNP). As before, we used the
proportion of bees responding on the first recall test trial to assess how the
conditioning protocols and the time post-conditioning affected the recall of
the CS odor (Fig. 4). As
observed in Experiment 1, the level of response to the CS odor during the test
depended upon the type of conditioning each subject received (APC, AC, PC,
UNP) (lreg; 22=39.7, P<0.001) and upon
the duration of time between conditioning and testing (lreg;
22=6.27, P=0.043). The response was greatest
for the test that took place immediately (Immediate vs 24 h,
P=0.003; Immediate vs 96 h, P=0.004), but the
response at 24 h was not significantly greater than the response at 96 h
(P=0.927).
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As before, we also examined whether the rate of extinction of conditioned
responding during the test period depended on the conditioning protocol; in
this case, we only compared the APC, AC and PC protocols. We examined the
proportion of subjects responding during four unreinforced test trials after
one forward-paired trial. The rate of extinction across all four test trials
(indicated by a significant 3-way interaction in the logistic regression
model) was the same for all three conditioning protocols (APC, PC, AC) at each
time post-conditioning (3-way rpm lreg; 26=7.36,
P=0.118). However, the number of subjects responding across all four
trials during the test decreased as a function of trial for all conditioning
types (APC, AC, PC) and for all three time points tested post-conditioning
(rpm lreg; 21=180, P=0.001). The response
on the first trial was, on average, greater than the response on every other
trial (all three lsc; P<0.001). The responses on all other trials
were not significantly different (P>0.999).
Experiment 4. Sucrose reward is necessary for long-term recall
This experiment was designed to examine whether feeding on sucrose was
necessary and/or sufficient for producing an increased ability to recall the
olfactory CS in the APC and PC protocols. We compared individuals conditioned
in the APC and AC protocols to subjects conditioned in the APW protocol, in
which the antennae were touched with a sucrose solution US but the subject was
fed water. The rate of acquisition, reflected in the difference in the slope
of the acquisition curve for each protocol, depended upon the conditioning
protocol (Fig. 5A) (2-way
interaction; rpm lreg; 22=7.80, P=0.020).
The rate was greatest for the APC protocol (APC vs AC,
P=0.052; APC vs ACW, P=0.027) and was not
significantly different for the AC and ACW protocols (P=0.841).
During the recall test trial with the conditioned odor, the level of response
was not significantly different at the immediate time point for the three
different protocols (lreg; 22=1.60,
P=0.449). However, at 24 h post-conditioning, the APC-conditioned
subjects responded with a greater probability to the conditioned odor than the
AC- or ACW-conditioned subjects (lreg; 22=9.21,
P=0.010; lsc; APC vs AC, P=0.036; APC vs
ACW, P=0.007; AC vs ACW, P=0.647).
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Formation of long-term memory: evidence for pre- vs post-ingestive mechanisms
One of the most striking results of our study is that feeding on the
reward, which includes both stimulation of the gustatory receptors on the
proboscis and ingestion of the reward solution, strongly affected long-term
memory. Subjects conditioned with an antennal-only US (AC) maintained a memory
for the association between the odor CS and sucrose US for a much shorter
period than honey bees that received proboscis stimulation (APC or PC). In a
recent study, Scheiner et al. (Scheiner et
al., 2005) observed that sensitivity to sucrose on the antenna was
correlated to sucrose sensitivity on the proboscis of honey bees. Furthermore,
they observed that, although honey bees showed higher sensitivity to
stimulation of the antennae than the proboscis, it was the concentration of
sucrose applied to the proboscis and then consumed that determined the level
of acquisition. Pre-ingestive perception of the quality of the reward,
therefore, affects acquisition differently depending on how the reward is
experienced at both the antennal and proboscis sensory pathways. Our results
confirm that information about the reward solution, as experienced at the
proboscis, strongly influences acquisition. This was especially obvious in the
experiment in which stimulation of the antennae with sucrose followed by
feeding with water (ACW) produced levels of acquisition and memory recall
equivalent to conditioning with antennal stimulation alone (AC). Our results
suggest that the mechano- and hygro-sensory stimulation of the proboscis
during feeding does not rescue recall. Taken together, these results establish
that stimulation of the proboscis, which includes both sensory perception
and/or consumption of the reward, plays a significant role in memory
retention.
Gustatory information from chemosensory receptors on the antennae and the
proboscis projects to different areas of the honey bee brain. Gustatory
sensory neurons in the proboscis project to the subesophageal ganglion
(Mitchell et al., 1999) and
connect to the mushroom bodies via the subesophageal-calycal tract
(Schröter and Menzel,
2003), whereas gustatory neurons from the antennae project to the
dorsal lobe (Suzuki, 1975) and
do not appear to project to the subesophageal ganglion or, indeed, to the
antennal lobe (Haupt, 2007).
One explanation for the differences we observed both in acquisition and in
long-term memory retention may be due to the differences in the contribution
of sensory information from each of these separate gustatory inputs. In
particular, it is likely that the mushroom bodies support a greater capacity
for associative learning and for memory consolidation of multimodal inputs. If
this is true, then the greater memory retention for the subjects conditioned
with the APC and PC protocols would be expected.
Our study also suggests that post-ingestive feedback about the quality of
the reward may influence the formation of long-term memory. In humans and
rats, glucose levels in the brain have been shown to affect learning and
memory formation and may act through a variety of mechanisms including
affecting the energy available for neurons, affecting levels of
neurotransmitters or by acting directly as a neuromodulator
(McNay and Gold, 2002). In
honey bees, post-ingestive feedback about the quality of a reward may also be
conveyed by changes in hemolymph glucose or other sugar levels following
sucrose consumption during and immediately after conditioning. In other
insects, hemolymph sugar levels have been shown to affect the probability of
feeding and gustatory sensitivity in peripheral taste cells
(Simpson et al., 1990;
Simpson and Simpson, 1992).
Satiety, and hence motivation to feed, is also mediated by hemolymph levels of
amino acids and sugars in locusts (Zanotto et al., 2002). In honey bees, the
level of satiation prior to conditioning affects single as well as multiple
trial learning (Friedrich et al.,
2004); it also affects gustatory sensitivity and expression of the
PER reflex (Pankiw et al.,
2002; Pankiw et al.,
2004). It is conceivable, therefore, that when food is absorbed
from the midgut into the hemolymph during conditioning that hemolymph sugar
levels, perhaps mediated by glucose
(Crailsheim, 1988;
Roces and Blatt, 1999;
Blatt and Roces, 2002), provide
feedback to the brain about the quality of the reward both during and after
conditioning.
Recent studies of the cAMP–PKA pathway in the brain of honey bees
have shown that, as with other invertebrates and vertebrates, this pathway is
important in the formation of long-term memory (for reviews, see
Eisenhardt, 2006;
Schwärzel and Müller,
2006). Inhibition of PKA during acquisition results in
significantly reduced levels of recall in multiple trial, but not single
trial, learning (Müller,
2000). Olfactory learning studies in satiated honey bees have
shown that satiated subjects respond at lower levels than hungry subjects
during conditioning and that the brains of satiated honey bees show
significantly lower levels of PKA activity
(Ben-Shahar and Robinson, 2001;
Friedrich et al., 2004).
Furthermore, pharmacologically increasing the low PKA activity in satiated
honey bees before conditioning rescued the ability of satiated honey bees to
form long-term memories (Friedrich et al.,
2004). Taken together, these results suggest that a feedback
mechanism may exist such that the level of satiety influences PKA activity in
the brain, which then affects both the acquisition and the formation of
memory. Our results are consistent with this model, and furthermore suggest
that the consumption of the sucrose reward provides post-ingestive feedback
that may be involved in the determination of food quality and nutritional
state.
Learning processes: acquisition, retention and extinction
Although previous studies have shown that honey bees could acquire an
association between an odor CS and a sucrose US via antennal-only
stimulation (Bitterman et al.,
1983; Sandoz et al.,
2002), our study is the first to show that the relatively high
levels of response seen during antennal conditioning do not translate into
robust retention of the association. The analysis of genetic mutants and the
use of pharmacological tools have shown that the process of acquisition is
distinct from that of recall. For example, activation of protein kinase C
(PKC) in the antennal lobes has been shown to affect memory, but not
acquisition, in the honey bee
(Grünbaum and Müller,
1998). These studies and many others (see
Schwärzel and Müller,
2006) suggest that acquisition and recall are dependent on
distinct biochemical pathways.
The ability of bees to recall an association after conditioning with
antennal-only stimulation showed marked deficits when compared to the recall
of bees conditioned with the APC or PC protocols. After a single conditioning
trial, AC-conditioned subjects exhibited a much lower response to the CS than
those conditioned with the APC protocol, even when tested immediately after
conditioning. When multiple conditioning trials were given, bees trained
via antennal-only stimulation initially showed recall levels similar
to those for APC and PC bees. This difference between single- and multi-trial
conditioning suggests that multiple training trials may compensate for the
weaker association formed by antennal-only training. However, it is clear that
multiple antennal stimulation trials cannot fully compensate for the lack of
proboscis stimulation and/or sucrose consumption, as the memory of
multiple-trial AC-conditioned bees decayed rapidly and recall was
significantly lower at 24 h after conditioning. Memories produced via
single-trial conditioning are produced by mechanisms distinct from those
producing long-term memories (reviewed in
Eisenhardt, 2006;
Schwärzel and Müller,
2006). However, the fact that memory retention is much reduced in
AC bees conditioned with single or multiple trials suggests that reward
consumption may influence multiple mechanisms underlying memory formation.
In our experiments, extinction was induced by four trials of the
conditioned odor (CS) presented without reward (US). It is presently unknown
whether extinction occurs via the same associative learning
mechanisms as excitatory conditioning or whether it is a distinct form of
learning. Current models of the process of extinction suggest that it is a
form of learning rather than the destruction of the original association
formed between the US and the CS (Bouton,
2004; Rescorla,
2004; Rescorla,
2006; Eisenhardt and Menzel,
2007). If extinction of conditioned responding proceeded from the
same physiological mechanism as excitatory learning, we might expect it to
compete with the memory of the original CS–US association. Based on this
rationale, one might expect that a strong CS–US association would show a
slower rate of extinction than a weak CS–US association. In our
experiments, therefore, extinction of conditioned responding should have been
slowest for the APC protocol. Although we observed that the APC protocol
produced a greater rate of acquisition and a longer memory than the AC or PC
protocols, the rate of extinction of the CS memory was not significantly
different for the three protocols. Because CS presentation during extinction
was the same for all three protocols, our data suggest that different
physiological mechanisms underlie acquisition and extinction in honey bees
(Eisenhardt and Menzel,
2007).
Differences between pollen and nectar foragers
In a honey bee colony, the genetic background of workers can lead to
differences in foraging behavior such that some workers focus on collecting
nectar while others collect pollen, pollen and nectar, or water
(Fewell and Page, 1993;
Page et al., 1998;
Pankiw and Page, 2000).
Foragers that specialize in collecting nectar have a reduced sensitivity to
sucrose compared to pollen, nectar and pollen, and water foragers when their
antennae or tarsi are stimulated to elicit proboscis extension
(Page et al., 1998;
Pankiw and Page, 2000). Other
factors such as age, caste, exposure to pheromones and foraging history may
also affect these thresholds (Pankiw and
Page, 1999; Pankiw and Page,
2001; Pankiw and Page,
2003; Pankiw et al.,
2001; Pankiw et al.,
2002). Pollen foragers typically exhibit faster acquisition in
associating an olfactory or tactile CS with a low concentration of sucrose
(Scheiner et al., 2001). In
addition, the ability of honey bees to associate a tactile CS and a sucrose US
correlates to each individual's sensitivity to sucrose
(Scheiner et al., 2005).
Based on these studies, we expected that, in a situation where all other
variables were similar, pollen foragers would have greater sensitivity to
sucrose and that they would perform better in all three appetitive learning
protocols than nectar foragers. Indeed, when our subjects experienced a
compound US (APC), we observed that pollen foragers achieved a higher
asymptotic learning level than nectar foragers in Experiment 1. In contrast to
our expectations, however, we observed that nectar foragers achieved a higher
level of acquisition than pollen foragers during the proboscis-only (PC)
conditioning [we failed to find a difference for the antennal-only (AC)
conditioning]. As reported in the Introduction, many variables affect a honey
bee's sensitivity to sucrose, including environmental variables, age and
genotype of the colony (Pankiw et al.,
2001). Although we collected pollen and nectar foragers using the
criteria of Page et al. (Page et al.,
1998), we may have included some water foragers as well. As water
foragers have very low sucrose response thresholds
(Pankiw and Page, 2000), it is
possible that inclusion of this group affected the average level of
acquisition observed for nectar foragers.
Although nectar foragers showed higher levels of response than pollen
foragers during acquisition for the PC conditioning, when we examined the
responses during the first recall test trial, the pollen foragers exhibited a
greater probability of responding to the CS on average than the nectar
foragers for all three time points we examined. Furthermore, 24 h after
conditioning, significant reductions in the level of response were observed
for nectar foragers conditioned with the APC or PC protocol when they were
compared to the immediate test period, whereas pollen foragers did not show
such reductions in recall ability at the 24 h time point. These results
suggest that, as well as having differences in sucrose sensitivity and
acquisition, pollen and nectar foragers may differ in their abilities to form
or consolidate long-term memories. Recently, studies on lines of honey bees
selected for high or low levels of foraging for pollen have shown that these
strains differ in the amount of PKA and PKC present in the central brain
(Humphries et al., 2003). PKA
and PKC play important roles in sensory processing and the formation of memory
in honey bees (Grünbaum and
Müller, 1998; Friedrich
et al., 2004; Müller,
2000). PKA has been previously shown to correlate with long-term
gustatory responsiveness such that honey bees sensitive to sucrose also had
high levels of PKA in their brains
(Scheiner et al., 2003).
Differences in the levels of PKA or PKC between pollen and nectar foragers
may, therefore, at least partially explain the differences in learning that we
observed.
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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